# Patent

## Title
Segmented micro RNA mimetics

## Abstract

This invention relates generally to segmented oligonucleotides capable of modulating gene expression. Specifically, the instant invention relates to segmented microRNA (miRNA) oligonucleotides, including segmented miRNA precursors and segmented pre-microRNAs. The invention also relates to compositions comprising such segmented oligonucleotides, as well as to methods of making and using such oligonucleotides for diagnosis and treatment of diseases associated or causally linked to aberrant levels or activities of gene expression, including aberrant levels of coding and/or non-coding RNA.

## Background
PRIORITY APPLICATIONS
This application claims the benefit of U.S. Patent Application No. 61/236,486 filed Aug. 24, 2009.


TECHNICAL FIELD
This invention relates generally to segmented oligonucleotides capable of modulating gene expression. Specifically, the instant invention relates to segmented microRNA (miRNA) mimetic oligonucleotides, including segmented miRNA precursors and segmented pre-microRNAs. The invention also relates to compositions comprising such segmented oligonucleotides, as well as to methods of making and using such oligonucleotides for diagnosis and treatment of diseases associated or causally linked to aberrant levels or activities of gene expression, including aberrant levels of coding and/or non-coding RNA.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The sequence listing of the present application is being submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “SIRMIS00131USPCT-SEQTXT-07MAY2013.txt”, creation date of May 7, 2013 and a size of 210 KB. This sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.
BACKGROUND
Segmented oligonucleotides based on short interfering RNA (siRNA) have been evaluated for RNA interference (RNAi) activity. Leuschner et al., (2006 EMBO 7:314) described an RNA-induced silencing complex which has a discontinued passenger (or sense) strand and a 2′-O-methyl modified nucleotide at position 9 of the passenger strand (5′ to 3′), the natural cleavage site. Bramsen et al., (2007 Nucleic Acids Res. 35:5886) described an RNAi-active siRNA molecule comprising an internally segmented passenger strand, where the nick or gap is not necessarily located at the natural cleavage site, stabilized with locked nucleic acid (LNA) modifications at a number of positions. See also, e.g., Wengel et al., PCT Publication WO 2007/107162 A2; Quay et al., PCT Publication WO 2008/049078. None of Leuschner, Bramsen, Wengel and Quay described RNAi-active molecules having discontinued guide (or antisense) strands. In fact, Bramsen and Wengel indicated that duplexes designed to contain discontinuities in the guide strands completely eliminated silencing of the target.
The mechanistic difference between miRNA-mediated RNAi and siRNA-mediated RNAi can make certain modifications and/or designs suitable for one but not the other. Thus, there remains a heightened interest in formulating new and advantageous design features suitable for miRNA mimetics.
SUMMARY OF THE INVENTION
The specification describes certain segmented double-stranded miRNA mimetics having at least one non-contiguous (or discontinuous) strand comprising a miRNA sequence, which can be introduced or applied to cells, tissues, and/or organisms to mediate RNAi. These molecules are referred to herein as segmented miRNA mimetics, which comprise a guide strand and a passenger strand. The guide strand comprises at least two contiguous stretches of nucleotides separated by a discontinuity. The passenger strand can be fully contiguous, or alternatively can also comprise at least two contiguous stretches of nucleotides separated by a discontinuity. Segmented miRNA mimetics of the invention therefore have at least one non-contiguous guide strand comprising one or more miRNA sequences, or a portion thereof, including the seed sequence of such miRNA sequences. Non-limiting examples of miRNA sequences are those selected from the miRBase as of the filing date of the present invention; see for example Griffiths-Jones (2006) miRBase: the microRNA sequence database. Methods in Molecular Biology 342: 129-138 and miRBase release 13.0; http://micrirna.sanger.ac.uk/. A segmented miRNA of the invention therefore can include one or more miRNA sequences selected from SEQ ID NOs: 1-1090 of Table I, including portions thereof, such as the seed sequences.
A segmented miRNA mimetic of the invention comprises at least one discontinuity in the guide strand, and optionally at least one discontinuity in the passenger strand that can be the same or different as the discontinuity in the guide strand. Such discontinuities include nicks, gaps, substitutions, and/or insertions. Segmented miRNA mimetic can comprise mixtures of different discontinuities in one or both strands.
A segmented miRNA mimetic of the invention comprises about 12 to about 26 nucleotides in each strand, and further comprises about 10 to about 26 base pairs between the strands. Thus, a prototypical segmented miRNA mimetic of the invention generally comprises two strands having complementarity to form a duplex, each strand having between about 12 to about 26 nucleotides, wherein the guide strand comprises any of SEQ ID NOs: 1-1090 or a portion thereof, and wherein the guide strand further comprises at least on discontinuity.
Segmented miRNA mimetics of the invention can be administered to a cell, a tissue or an organism to supplement or increase the levels of their corresponding endogenous miRNAs and hence potentiate RNAi activity against their corresponding miRNAs targets. Because each endogenous miRNA typically has multiple targets, an exogenously introduced segmented miRNA mimetic of the invention does not necessarily share the same number, identity or type of targets with its corresponding endogenous miRNA. However, the exogenously-introduced segmented miRNA mimetic exerts activity on at least one (i.e., one or more or all) of the targets of its corresponding endogenous miRNA.
A segmented miRNA mimetic can be chemically modified at the nucleic acid base, phosphodiester backbone, or sugar to achieve, for instance, increased stability and/or reduced immunogenicity, and other pharmaceutically desirable attributes, including properties that would allow for enhanced delivery or lower toxicity. Methods of chemically modifying oligonucleotides to achieve such ends are known in the art. For instance, numerous such methods are set forth in McSwiggen, et al., U.S. Publication No. 2006/0211642.
In a further aspect, the specification provides a composition comprising one or more (i.e., in the number of individual molecules and/or in types) segmented miRNA mimetics in a pharmaceutically acceptable carrier or diluent. In another aspect, the specification provides a method of introducing or applying one or more segmented miRNA mimetics to cells (regardless of whether the RNAi or other gene modulation process takes place inside the cells, outside the cells, or on the cell-membrane), tissues, organisms, or reconstituted in vitro systems, to increase the levels of corresponding endogenous miRNAs. Embodiments of the invention include methods of modulating gene expression, biologic pathways, or physiologic pathways in cells, cultures, tissues, or organisms such as subjects or patients, comprising administering one or more segmented miRNA mimetics of the invention in an amount that is sufficient to modulate the expression of one or more genes that are regulated by the corresponding endogenous miRNAs. In a specific embodiment, more than one type of segmented miRNA mimetic is administered. For example, a number of different segmented miRNA mimetics of the invention can be administered concurrently, in sequence, or in an ordered progression.
In certain embodiments, administration of the composition(s) can be enteral or parenteral. In certain aspects, enteral administration is oral. In further aspects, parenteral administration is intralesional, intravascular, intracranial, intrapleural, intratumoral, intraperitoneal, intramuscular, intralymphatic, intraglandular, subcutaneous, topical, intracronchial, intratracheal, intranasal, inhaled, or instilled. Compositions of the invention can be administered regionally or locally, and not necessarily directed into a lesion.
Embodiments of the invention can include obtaining or assessing a gene expression profile or miRNA profile of a target cell, tissue, or organism prior to selecting the mode of treatment, by, for example, administration of one or more segmented miRNA mimetics. In certain aspects of the invention, one or more segmented miRNA mimetics can modulate a single gene. In a further aspect, one or more genes in one or more genetic, cellular, or biologic/physiologic pathways can be modulated by a single segmented miRNA mimetic or a complement thereof, alone or in combination with other miRNAs or mimetics, or with other nucleic acid-based gene modulators, such as siRNAs, antisense molecules, ribozyme molecules, and the like.
A further aspect of the invention is directed to a method of modulating a cellular pathway comprising administering to the cell an amount of a segmented miRNA mimetic, alone or in combination with other miRNAs, mimetics, siRNAs, or other suitable nucleic-acid based or non-nucleic acid based agents capable of modulating one or more relevant genes in the same or associated pathways. In a related aspect, the invention is directed to methods of modulating a cellular pathway comprising administering to the cell a segmented miRNA mimetic in an amount sufficient to modulate the gene expression, function, status, or state of a cellular pathway, in particular a pathway that is known to include one or more genes associated with the corresponding endogenous miRNA. Modulation of a cellular pathway includes, but is not limited to, modulating the expression of one or more genes associated with the pathway. Modulation of a gene includes inhibiting its function, also called “down-regulate a gene,” or providing an agonist to augment its functional, also called “up-regulating a gene.” What is modulated is either the expression level or activity of a gene or its related gene product or protein.
Compositions and methods comprising a segmented miRNA mimetic are also useful for treating diseases or disorders associated with aberrant expression levels or activity of one or more corresponding miRNA targets. These diseases and/or disorders include, for example, hyperproliferative disorders (e.g., cancer), inflammatory conditions (e.g., arthritis), respiratory diseases, pulmonary diseases, cardiovascular diseases, autoimmune diseases, allergic disorders, neurologic diseases, infectious diseases (e.g., viral infections), renal diseases, transplant rejections, or any other conditions that respond to such modulation.
Still a further embodiment includes methods of treating a patient with a pathological condition comprising one or more steps: (a) administering to the patient an isolated or a synthetic segmented miRNA mimetic of the invention in an amount sufficient to modulate the expression of a cellular pathway; and (b) administering a second therapy, wherein the modulation of the cellular pathway in (a) sensitizes the patient to the second therapy. A cellular pathway can include, but is not limited to, one or more pathways that are known to be associated with known miRNAs listed in the miRBase as of the date of filing of the instant application. A second therapy can include administration of one or more miRNAs or mimetics targeting the same or different mRNAs, or one or more other therapeutic nucleic acids. A second therapy can also be one selected from other standard therapies, such as chemotherapy, radiation therapy, drug therapy, immunotherapy, and the like.
The invention also features a kit or article of manufacture comprising one or more segmented miRNA mimetics, typically in a pharmaceutical composition, and instructions for administering the composition to treat a pathological condition. Optionally, the kit or article of manufacture can contain one or more other pharmaceutical compositions or agents and instructions for their use in conjunction with the pharmaceutical composition comprising the segmented miRNA mimetics.
In yet a further aspect of the invention, one or more segmented miRNA mimetics of the invention can be included in a kit or article of manufacture for assessment or diagnosing of a pathological condition or the risk of developing a pathological condition.
It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments can be combined.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1-6 illustrate examples of different structural features of segmented miRNA mimetics. It will be apparent to the skilled person in the art that the various structural features illustrated in these figures can be combined. For example, a particular pattern of the overhangs can be combined with a molecule comprising one or more discontinuities at particular positions, which can or can not be linked, by phosphodiester bonds or non-phosphodiester connectors. Therefore, these figures should be interpreted as merely representative but non-limiting, such that, for example, the nature and position of the discontinuities can be changed and additional features, such as, for example, overhangs and nucleotide analogs, can be introduced to the same molecules.
FIG. 1 illustrates the basic structural features of a segmented miRNA mimetic. A shows 3 representative segmented miRNA mimetics, comprising nicks or gaps in the guide (the lower strand), or in both the passenger (upper strand) and the guide strands. Each box represents a nucleotide (including a nucleotide analog), or a non-nucleotide substitute moiety. Each line connecting the boxes represents a phosphodiester bond or non-phosphodiester connector. Distinct contiguous stretches of nucleotides in a given strand are colored/shaded differently to facilitate the identification of the nick or gap positions. B shows a representative conformation of a segmented miRNA mimetic, wherein the passenger strand is uninterrupted and wherein the guide strand comprises a gap of 1 nucleotide. A similar motif comprising a nick instead of a gap is contemplated by the instant invention. C shows a representative segmented miRNA mimetic wherein each of the passenger strand and the guide strand comprises a nick. Some of the discontinuities in this figure are shown as nicks. However, those skilled in the art would appreciate that gaps can be present at similar positions, and other types of discontinuities such as substitutions and insertions are also contemplated. Moreover, those skilled in the art would appreciate that a gap can be a stretch of 1 to about 10 contiguous vacant nucleotide positions.
FIG. 2A illustrates various overhangs and blunt ends can be utilized in the design of a segmented miRNA mimetic of the invention. The discontinuities are shown in this figure as nicks or gaps of a single nucleotide, but other kinds of discontinuities, including larger gaps of one or more (e.g., up to 10) nucleotides, as well as substitutions and/or insertions are also contemplated. FIG. 2B illustrates a representative segmented miRNA mimetic wherein one set of the terminal ends are connected by a linker.
FIG. 3 illustrates that one or more discontinuities (nicks or gaps are shown, but also substitutions and insertions) can be present in the guide strand, or in both the passenger and the guide strands.
FIG. 4 illustrates that the sizes or positions of the discontinuities on the guide strand, or both the passenger and guide strands can vary. The molecules are named according to the starting and ending positions of the discontinuities from the 5′-end.
FIG. 5 illustrates that one or more suitable non-nucleotide substitutions or insertions can be used to connect a pair of neighboring contiguous stretches of nucleotides in the passenger strand, the guide strand, or both the passenger and guide strands.
FIG. 6 illustrates certain segmented miRNA mimetics comprising inverted abasic modifications at the internal ends. The molecules are named according to the starting and ending positions of the discontinuities.
FIGS. 7-20 show experimental data obtained from the examples herein.
FIG. 7 illustrates RNAi activity against an endogenous miR-124 target, CD164. RNAi activity of various segmented miRNA mimetics derived from miR-124 (“segmented miR-124”) was measured. Levels of knockdown achieved by the segmented miR-124 constructs and by the corresponding non-segmented miR-124 mimetic, comprising the endogenous mature miR-124 sequence as its guide strand, were determined. The segmented miR-124 constructs can comprise one or more locked nucleic acid (“LNA”) nucleotides in one or both strands, each represented by an underlined nucleotide in Table III herein, although the LNAs are not necessarily placed at the underlined nucleotide positions.
FIG. 8 illustrates RNAi activity against another endogenous miR-124 target, VAMP3. RNAi activity of various segmented miR-124 mimetics was measured. Levels of knockdown achieved by the segmented miR-124 constructs and by the corresponding non-segmented miR-124 mimetic, comprising the endogenous mature miR-124 sequence as its guide strand, were determined. The segmented miR-124 constructs can comprise one or more LNA nucleotides in one or both strands, each represented by an underlined nucleotide in Table IV herein, although the LNAs are not necessarily placed at the underlined nucleotide positions.
FIG. 9A illustrates RNAi activity of various segmented miR-124 mimetics (i.e., of Table V) against CD164. Levels of knockdown achieved by the segmented miR-124 constructs and by the corresponding non-segmented miR-124 mimetic, comprising the endogenous mature miR-124 sequence as its guide strand, were determined. Knockdown or inhibition, or the lack thereof, by segmented miR-34 constructs, which were designed based on human miR-34, is presented as a negative control.
FIG. 9B illustrates RNAi activity against an endogenous miR-34 target, TK1, of various segmented miRNA mimetics derived from miR-34 (“segmented miR-34”) (i.e., molecules of Table V). Levels of knockdown achieved by the segmented miR-34 constructs and by the corresponding non-segmented miR-34 mimetic, comprising the endogenous mature miR-34 sequence as its guide strand, were determined. Knockdown or inhibition, or the lack thereof, by segmented miR-124 constructs, is presented as a negative control.
FIG. 10A illustrates RNAi-mediated activity against CD164 by segmented miR-124 constructs comprising one or more inverted abasic modified internal ends (i.e., molecules of Table VI). FIG. 10B illustrates RNAi-mediated activity against VAMP3 by segmented miR-124 constructs comprising one or more inverted abasic modified internal ends.
FIG. 11A illustrates RNAi-mediated activity against CD164 by segmented miRNA-124 constructs comprising abasic substitutions (i.e., molecules of Table VII). FIG. 11B illustrates RNAi-mediated activity against VAMP3 by segmented miR-124 constructs comprising abasic substitutions.
FIG. 12 demonstrates that miR-124 activity tolerates segmentation of the passenger or guide strands. FIG. 12A includes schematic illustrations of miR-124 duplex designs, wherein the top strand represents the passenger strand and the bottom strand represents the guide strand. The gray-shade change indicates the site of a break in the strand backbone. The dark gray circle in the G10Cy3.12/P schematic represents a 5′-Cy3 label, and the light gray circle in the G10i.12/P schematic indicates a 5′ inverted abasic nucleotide. FIG. 12B illustrates the dose-dependent response of CD164, a known miR-124 target, to various concentrations of the designs shown in FIG. 12A in HCT-116 cells as measured by RT-qPCR. UC3 corresponds to the negative control oligomer. EC50s for each curve are as follows: G/P, 0.12 nM; G/P10.12, 0.29 nm; G10.12/P, 0.22 nM; G10.11/P, 0.53 nM; G10i.12/P, 0.21 nM; G10Cy3.12/P, 0.45 nm.
FIG. 13 provides a microarray analysis showing seed-based activity from a segmented guide strand. FIG. 13A is a graphic illustration of the microarray signature 24 hours after transfection of 10 nM G10.12/P, plotted as expression ratio (relative to mock transfection) versus fluorescence intensity. Significantly down regulated probes (P<1e-6) are seen as gray data points below the black data points and unregulated probes are seen as gray data points above the black data points. Hypergeometric analysis of the hexamer content of the down regulated UTRs showed that the most significantly enriched hexamer (P<1e-20) was GCCTTA, corresponding to positions 2-7 of the transfected miR-124. FIG. 13B provides a comparison of gene expression data from G10.12/P transfected cells and G/P transfected cells. Expression ratio of the G/P transfection versus the mock transfection is plotted on the x-axis and the G10.12/P expression ratio versus mock transfection on the y-axis. The weighted correlation coefficient was calculated as 0.9, illustrating the similar effects of both RNA complexes on gene expression.
FIG. 14 illustrates that the activity of an unsegmented miR-124 guide strand is enhanced by additional complementarity beyond the seed sequence, but activity of a segmented guide strand is not. FIG. 14A is a schematic of miR-124 target sites that were duplicated and inserted into dual luciferase reporter vectors. FIG. 14B illustrates that G/P miR-124 exhibits suppression of activity from a reporter with a seed sequence match, which is enhanced by reporters containing additional complementarity to the 3′ end of the guide strand (2×7a3p) or full-length complementarity. The EC50s for the curves: 2×7a, 0.39 nM; 2×7a3p, 0.08 nM; 2×FL, 0.09 nM. The EC50s for the curves are: 2×7a, 0.41 nM; 2×7a3p, 0.06 nM; 2×FL, 0.09 nM. FIG. 14C illustrates that segmentation of the passenger strand preserves the trends seen in FIG. 14B. FIG. 14D shows that activity from reporters with 3′ complementarity is identical to that observed from the seed-only reporter when the guide strand is segmented. The EC50s for the curves are: 2×7a, 0.89 nM; 2×7a3p, 0.61 nM; 2×FL, 0.80 nM.
FIG. 15 illustrates RNAi activity mediated by a segmented miR-124 precursor. A 58-mer miR-124 precursor was designed and transfected into HCT-116 cells at varying concentrations. FIG. 15A is a miR-124 precursor schematic. FIG. 15B is a graph showing CD164 knockdown as measured by RT-qPCR. Activity of the unsegmented precursor is comparable to that of the mature miRNA, while activity of the segmented precursor is reduced but is significantly above background. The EC50s for the curves are: G/P, 0.14 nM; hairpin precursor, 0.31 nM; segmented precursor, 1.48 nM.
FIG. 16 illustrates that a segmented guide strand exhibits decreased knockdown for targets containing 3′ supplementary pairing. Microarray data shown in FIG. 13B was further analyzed, specifically to analyze 1057 downregulated genes that contain TargetScan seed matches. The TargetScan 3′ pairing score was then calculated for these genes, and the downregulation of the top quartile of 3′ scores (containing a high degree of 3′ pairing) was compared with the downregulation of the bottom quartile of 3′ scores. The cumulative distribution of the difference in downregulation (between G/P and G10.10/P) is plotted for the top and bottom quartiles. A Kolmogorov-Smirnov test (p=0.02) shows that the top quartile of genes shows less knockdown in G10.10/P versus G/P, as compared with the bottom quartile.
FIG. 17A illustrates knockdown of CD164 expression by segmented miR-124 mimetics comprising deletions and c3 or c6 substitutions, while FIG. 17B illustrates knockdown of VAMP3 expression by these segmented microRNAs.
FIG. 18A illustrates knockdown of CD164 expression by segmented miR-124 comprising c3 substitutions, while FIG. 18B illustrates knockdown of VAMP3 expression by these segmented microRNAs.
FIG. 19A illustrates knockdown of CD164 expression by segmented miR-124 comprising c6 substitutions, while FIG. 19B illustrates knockdown of VAMP3 expression by these segmented microRNAs.
FIG. 20A illustrates knockdown of CD164 expression by segmented miR-124 comprising c3 and c6 insertions, while FIG. 20B illustrates knockdown of VAMP3 expression by these segmented microRNAs.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.
“About” as used herein indicates that a value includes the standard deviation of error for the device or method being employed to determine the value.
“Analog” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a compound that is structurally similar to a parent compound (e.g., a nucleotide), but differs in composition (e.g., one or more atom(s) or functional group(s) is/are different, added, or removed). The analog can have different chemical or physical properties than the original parent compound and can have improved biological or chemical activity. For example, the analog can be more hydrophilic or it can have altered activity of the parent compound. The analog can be a naturally or non-naturally occurring (e.g., chemically-modified or recombinant) variant of the original parent compound. An example of an RNA analog is an RNA molecule comprising a nucleotide analog. An example of a nucleotide analog is a nucleotide that is chemically modified at the sugar, base or nucleoside, as is generally known in the art.
The term “aptamer” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule wherein the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art (see, e.g., Gold et al, 1995 Annu. Rev. Biochem. 64:163; Brody and Gold, 2000 J. Biotechnol. 74:5; Sun, 2000 Curr. Opin. MoI. Ther. 2: 100; Kusser, J. 2000 Biotechnol. 74:21; Hermann and Patel, 2000 Science 257:820; and Jayasena, 1999 Clinical Chem. 45:1628).
As described herein, a “base pair” can be formed between two nucleotides, a nucleotide and a modified nucleotide, two modified nucleotides, a nucleotide and a nucleotide analog, two nucleotide analogs, a nucleotide and a non-nucleotide substitute moiety, or two non-nucleotide substitute moieties. In a specific embodiment, a non-nucleotide substitute can comprise any chemical moiety that is capable of associating with a component of the cellular RNAi machinery, such as, for example, the PAZ domain, the PIWI domain, and/or other Argonaute protein domains associated with the RISC. Non-traditional Watson-Crick base pairs are also understood as “non-canonical base pairs,” which is meant any non-Watson Crick base pair, such as mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions. Non-limiting examples of such non-canonical base pairs include, but are not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC 4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA N1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+ carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H—N3, GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.
The term “biodegradable” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.
The term “biodegradable linker” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, connecting a biologically active molecule to a segmented miRNA mimetic of the invention or to either the passenger and/or guide strands of a segmented miRNA mimetic of the invention. The biodegradable linker can be attached to a segmented miRNA mimetic of the invention at one or more of the terminal ends, internal ends, or any other nucleotide positions that is not vacant. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.
The term “biologically active molecule” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active molecules either alone or in combination with other molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, guide nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siRNA, miRNA mimetics, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.
By “capable of participating in RNAi against endogenous RNA targets of their corresponding naturally-occurring miRNAs” is meant that, when RNAi activity is measured by a suitable in vivo or in vitro assay or method, a segmented miRNA mimetic molecule of the invention demonstrates at least 5% or more of the knockdown effect against a target of its corresponding naturally-occurring miRNA as compared to the knockdown effect achieved by a non-segmented miRNA mimetic molecule directed to the same target under same experimental conditions. Preferably, a segmented miRNA mimetic molecule of the invention is capable of achieving 25% or more, 35% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or even 100% or more (i.e., equal or more potent RNAi activity) knockdown of the target than a non-segmented miRNA mimetic against the same target.
The term “cap structure” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to chemical modifications, which have been incorporated into the ends of oligonucleotide (see, for example, Matulic-Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications can protect certain nucleic acid molecules from exonuclease degradation, and can impart certain advantages in delivery and/or cellular localization.
As used herein, the term “complementary” (or “complementarity”) refers to its meaning as is generally accepted in the art. The term generally refers to nucleic acid sequences that are capable of base-pairing according to the standard Watson-Crick complementarity rules, that is purines will base pair with pyrimidines to form combinations: guanine paired with cytosine (G:C); and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Base-pairing according to the Standard Waston-Crick complementarity rules can include base pairs formed between modified or nucleotide analogs. Aside from forming hydrogen bond(s) with each other according to the traditional Waston-Crick rules, a nucleic acid sequence can form other non-traditional types of base pairing with another nucleic acid sequence, and as such, the two nucleic acid sequences can also be called “complementary.” As used herein, the term “complementary” thus encompasses any “base-pairing,” which can be by hydrogen bonds or by any other interactions, of nucleotides, modified nucleotides, analogs, and/or non-nucleotides that provide sufficient binding free energy between the strands to allow the relevant function of the segmented miRNA mimetic, e.g., RNAi activity, to proceed. Determination of binding free energies for nucleic acid molecules is known in the art (see, e.g., Turner et al., 1987 CSH Symp. Quant. Biol. LII:123; Frier et al., 1986 Proc. Nat. Acad. Sci. USA 83:9373; Turner et al., 1987 J. Am. Chem. Soc. 109:3783).
A percent complementarity indicates the percentage of contiguous residues in a first nucleic acid molecule that can form hydrogen bonds (e.g., in Watson-Crick base-pairing) with a second nucleic acid sequence. For example, a first nucleic acid molecule can have 10 nucleotides and a second nucleic acid molecule can have 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules, which can or can not form a contiguous double-stranded region, represents 50%, 60%, 70%, 80%, 90%, or 100% complementarity, respectively. Complementarity can be found between two regions of a same nucleic acid molecule, such as, for example, in a hairpin loop or a stem loop structure. In other embodiments, complementarity can be found between two different nucleic acid molecules, such as, for example, in a segmented miRNA mimetic of the invention comprising distinct and separate passenger and guide strands.
In keeping with the usual practice by those of ordinary skill in the art, when the passenger strand and guide strand of the corresponding non-segmented miRNA are aligned on paper, (with the passenger strand arranged from 5′ to 3′ (left to right) and the guide strand arranged from 3′ to 5′ (left to right)) such that the each pair of complementary (base-pairing) nucleobases are located at directly opposite positions in the passenger and guide strand, the relative positions of the base-pairing nucleotides are termed “complementary nucleotide positions.” It is often helpful to mark the position of the nucleotides in the non-segmented miRNA mimetic and use those positions to mark nicks, gaps, substitutions, or insertions introduced into a corresponding segmented mimetic construct. Typically the first nucleotide position at the 5′-end of the passenger strand of a non-segmented duplex miRNA mimetic is position 1 of passenger strand, the nucleotide immediately adjacent to it is position 2, and so on and so forth Likewise, the first nucleotide position at the 5′-end of the guide strand of the non-segmented duplex miRNA mimetic is position 1 of the guide strand, the nucleotide immediately adjacent to it is position 2, and so on and so forth.
By “a contiguous stretch of nucleotides” or “a contiguous stretch of nucleotide positions” is meant a continuous series of at least 2 nucleotides or at least two nucleotide positions. For example, a contiguous stretch of nucleotides can refer to an unsegmented or uninterrupted oligonucleotide of 2 to 20 nucleotides in length. When referring to a contiguous stretch of nucleotides, the bonds connecting the nucleotides within the stretch can be phosphodiester bonds or non-phosphodiester linkages. A gap comprising a contiguous stretch of nucleotide positions can refer to a gap occupying, for example, from 1 to 10 or more nucleotide positions.
A segmented miRNA mimetic of the invention provided to a cell is typically designed based on the sequence of a naturally-occurring miRNA in the cell. As such, the naturally-occurring miRNA in the cell is referred to herein as “the corresponding miRNA.” A segmented miRNA mimetic of the invention provided to a cell is also understood to target one or more target mRNAs that are also targeted by the corresponding naturally-occurring miRNA. As such, each RNA targeted by the corresponding naturally-occurring miRNA is referred to as “the corresponding miRNA target.” It is contemplated that a segmented miRNA molecule introduced to a cell is not necessarily or does not necessarily comprise a nucleic acid sequence that is identical, essentially homologous, or even substantially homologous to a naturally-occurring miRNA, but the segmented miRNA is capable of either becoming or functioning as a naturally-occurring miRNA under appropriate conditions.
The term “discontinuity” as used herein refers to a non-contiguous segment of the nucleotide sequence of the guide strand, passenger strand or both the passenger and guide strands of a segmented micro RNA mimetic. A discontinuity can include one or more nicks, gaps, substitutions or insertions. The discontinuity can comprise, for example, from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more unoccupied or vacant nucleotide positions in the guide strand, the passenger strand, or both the guide and passenger strands. For example, a nick will comprise 0 unoccupied or vacant nucleotide positions, whereas a gap will comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10 or more) vacant or unoccupied nucleotide positions. Likewise, the discontinuity can comprise, for example, from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more nucleotide positions that are occupied or replaced by a non-nucleotide moiety in the guide strand, the passenger strand, or both the guide and passenger strands. For example, an insertion will comprise a non-nucleotide moiety that can occupy 0 nucleotide positions, whereas a substitution will comprise a non-nucleotide moiety that occupies one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10 or more) otherwise vacant nucleotide positions.
As used herein, “endogenous” refers to its meaning as is generally accepted in the art. The term generally refers to any material from or produced inside an organism, cell, tissue or system. As used herein, an “endogenous miRNA” is a naturally-occurring miRNA in a cell, tissue, organism, including a mammal, such as, for example, a human. “Exogenous” generally refers to any material introduced from or produced outside an organism, cell, tissue or system.
The term “expression” refers to its meaning as is generally accepted in the art. The term generally refers to the transcription and/or translation of a particular nucleotide sequence, for example when driven by its promoter.
The term “gap” as used herein refers to a contiguous stretch of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more), internal (as opposed to “terminal”) vacant, unoccupied or “unfilled” nucleotide positions in one or both strands of a segmented miRNA mimetic of the invention. The gap can be present in the guide strand, the passenger strand, or in both guide and passenger strands of a segmented microRNA mimetic of the invention.
The term “gene” as used herein, especially in the context of “target gene” for an RNAi agent, refers to the meaning as is generally accepted in the art. The term generally refers to a nucleic acid (e.g., “target DNA” or “target RNA”) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide. The target gene can also include a UTR (i.e., untranslated region) or non-coding region of the nucleic acid sequence. A gene or target gene can also encode a functional RNA (fRNA) or non-coding RNA (ncRNA) as are generally known in the art, such as endogenous antisense RNA, small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof, or any other regulatory RNA or precursor thereof. Such non-coding RNAs can serve as target nucleic acid molecules for RNA interference in modulating the activity of fRNA or ncRNA involved in functional or regulatory cellular processes. Aberrant fRNA or ncRNA activity leading to disease can therefore be modulated by the RNAi agents of the invention. RNAi agents targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of a subject, organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, methylation etc.). A target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. A cell containing a target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts. For a review, see for example Snyder and Gerstein, 2003, Science 300:258-260. In one aspect of the present invention, a segmented miRNA mimetic is capable of exerting regulatory effects on multiple target genes. Also, at least one of these target genes, but typically more than one target genes, can be shared between a segmented miRNA mimetic of the invention and its corresponding endogenous miRNA.
As used herein, “gene silencing” refers to a partial or complete loss-of-function through targeted inhibition of an endogenous miRNA target in a cell. As such, the term is used interchangeably with RNAi, “knockdown,” “inhibition,” “down-regulation,” or “reduction” of expression of a miRNA target gene. Depending on the circumstances and biological problem to be addressed, it can sometimes be preferable to increase expression of one or more related genes, which is termed “up-regulation” herein. Alternatively, it might be desirable to reduce or increase gene expression as much as possible or only to a certain extent.
By “guide strand” of a segmented miRNA of the invention is meant two or more distinct contiguous stretches of nucleotides at least one of which is substantially homologous or identical to the whole or a part of a sequence of a corresponding naturally-occurring miRNA, such as one selected from the miRBase, and for example, such as one selected from SEQ ID NOs: 1-1090 of Table I herein. The nucleotides within each contiguous stretch can be connected by traditional phosphodiester bonds and/or non-phosphodiester connectors. In addition, the guide strand of a segmented miRNA mimetic can comprise two or more distinct stretches of nucleotides that are capable of forming base pairs with the nucleotides or residues at the complementary nucleotide positions of the passenger strand.
As used herein, the term “homologous” (or “homology”) refers to its meaning as is generally accepted in the art. The term generally refers to the number of nucleotides of the subject nucleic acid sequence that has been matched to identical nucleotides of a reference nucleic acid sequence, typically by a sequence analysis program or by visual inspection. For example, nucleic acid sequences can be compared using computer programs that align the similar sequences of nucleic acids and therefore define the differences. Exemplary computer programs includes the BLAST program (NCBI) and parameters used therein, as well as the DNAstar system (Madison, Wis.), which can be used to align sequence fragments. Equivalent alignments and assessments can also be obtained through the use of any standard alignment software.
As used herein, the terms “including” (and any form thereof, such as “includes” and “include), “comprising” (and any form thereof, such as “comprise” and “comprises”), “having” (and any form thereof, such as “has” or “have”), or “containing” (and any form thereof, such as “contains” or “contain”) are inclusive and open-ended and do not exclude additional, un-recited elements or method steps.
The term “insertion” as used herein refers to a discontinuity wherein one or more non-nucleotide moieties are incorporated into the guide strand and/or passenger strand, while preserving the base pairs in the guide and passenger strands. Examples of such non-nucleotide moieties are provided herein and others are provided as is generally known to those of skill in the art.
The term “internal ends” refers to the ultimate nucleotides of the contiguous stretches of nucleotides on either side of a gap or a nick. Gaps or nicks do not have “terminal ends” for the purpose of this disclosure.
As used herein, the term “internally unpaired nucleotides” refers to nucleotides, which do not form base pairs with nucleotides at the complementary nucleotide positions in the opposite strand according to the standard Waston-Crick base-pairing rules. The term “internally unpaired nucleotides” also refers to nucleotide analogs or non-nucleotide residues that do not form hydrogen bonds or base pairs with the nucleotides, nucleotide analogs, or non-nucleotide residues at the complementary nucleotide positions in the opposite strand.
In certain embodiments, a segmented miRNA of the invention can be isolated. As used herein, an “isolated” oligonucleotide is nucleic acid molecule that exists in a physical form differing from any nucleic acid molecules of identical sequence as found in nature. “Isolated” does not require, although it does not prohibit, that the nucleic acid be physically removed from its native environment. For example, a nucleic acid can be said to be “isolated” when it includes nucleotides and/or internucleotide bonds not found in nature. A nucleic acid can be said to be “isolated” when it exists at a purity not found in nature, where purity can be adjudged with respect to the presence of nucleic acids of other sequences, with respect to the presence of proteins, with respect to the presence of lipids, or with respect to the presence of any other component of a biological cell, or when the nucleic acid lacks sequence that flanks an otherwise identical sequence in an organism's genome, or when the nucleic acid possesses sequence not identically present in nature. In aspects of the invention, a segmented miRNA is isolated by virtue of its having been synthesized in vitro. It will be understood, however, that isolated nucleic acids can be subsequently mixed or pooled together.
As used herein, the term “locked nucleic acid” (LNA) refers to its meaning as is generally accepted in the art. The term generally refers to a structure of the general Formula I:




where X and Y are independently selected from the group consisting of —O—, —S—, —N(H)—, —N(R)—, —CH2—, or —CH— (if part of a double bond), —CH2—O—, CH2—S—, CH2—N(H)—, —CH2—N(R)—, —CH2—CH2—, and CH2—CH-(if part of a double bond), —CH═CH—, where R is selected from hydrogen and C1-4-alkyl; Z and Z* are independently selected from an internucleotide linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleobase; and the asymmetric groups can be found in either orientation.

The 4 chiral centers of Formula I, as shown, are in a fixed configuration. But their configurations are not necessary fixed. Also comprised in the invention are compounds of the generally Formula I, wherein the chiral centers are found in different configurations, such as those represented in Formula II (below). Thus each chiral center in Formula I can exist in either R or S configuration. The definition of R (rectus) and S (sininster) are described in the IUPAC 1974 Recommendations, Section E, Fundamental Stereochemistry: The rules can be found in Pure Appl. Chem. 45, 13-30 (1976) and In “Nomenclature of Organic Chemistry” pergamon, New York, 1979.
LNA compounds can include an activation group for —OH, —SH, and —NH(RH), respectively. Such activation groups are, for example, selected from optionally substituted O-phosphoramidite, optionally substituted O-phosphortriester, optionally substituted O-phosphordiester, optionally substituted H-phosphonate, and optionally substituted O-phosphonate.
B constitutes a natural or non-natural nucleobase and selected among adenine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine, guanine, thymine, uracil, 5-bromouracil, 5-propynyluracil, 5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine, and 2-chloro-6-aminopurine.
Preferably, the Locked Nucleic Acid (LNA) used in a segmented miRNA mimetic of the invention comprises at least one nucleotide comprises a Locked Nucleic Acid (LNA) unit according any of the Formulas II:




wherein Y is —O—, —S—, —NH—, or N(RH); Z and Z* are independently selected among an internucleotide linkage, a terminal group or a protecting group; and B constitutes a natural or non-natural nucleobase. These exemplary LNA monomers and others, as well as their preparation are described in WO 99/14226 and subsequent applications, WO 00/56746, WO 00/56748, WO 00/66604, WO 00/125248, WO 02/28875, WO 2002/094250 and WO 2003/006475, the disclosure of all of which are incorporated herein by reference.

As used herein, the term “mimetic” refers to its meaning as is generally accepted in the art. The term generally refers to a molecule that is structurally different from the reference molecule (e.g., the corresponding naturally-existing molecule or the corresponding non-segmented mimetic molecule) but is capable of performing one or more or all of the biological, physiological, and/or chemical functions that are within the capabilities of the references molecule. The mimetic and the reference molecule do not have to be functional equivalents but the mimetic should be able to perform one or more functions, and exhibiting at least 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of the reference molecule, as measured and compared using assays or parameters that are suitable to represent the shared function(s). As used herein, a segmented miRNA molecule is a miRNA mimetic when the former shares at least one function with its corresponding endogenous miRNA. A miRNA mimetic can be a synthetic RNA duplex, such as a segmented miRNA duplex of the invention, a vector-encoded hairpin molecule, or other suitable structures designed based on a corresponding naturally-occurring endogenous miRNA.
The term, “miRNA” or “microRNA” refers to its meaning as is generally accepted in the art. The term generally refers to an endogenous short RNA molecule, which can be isolated or synthetic, which is found in eukaryotes and is involved in RNA-based gene regulation. A representative set of known endogenous miRNA species is described in the publicly available miRBase sequence database as described in Griffith-Jones et al., Nucleic Acids Research, 2004, 32:D109-D111 and Griffith-Jones et al., Nucleic Acids Research, 2006, 34:D 140-D144, accessible on the World Wide Web at the Wellcome Trust Sanger Institute website. A more selected set of miRNA species are included in Table I herein. Each mature miRNA is partially complementary to one or more messenger RNA (mRNA) molecules, which are also called “miRNA targets,” thereby regulating the expression of genes associated with the miRNA targets.
The term “nick” as used herein refers to a break in an internucleotide linkage in one or both strands of a segmented miRNA mimetic of the invention.
The term “non-nucleotide” refers to its meaning as is generally accepted in the art. The term generally refers to any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, such as for example but not limitation abasic moieties, alkyl moieties, polymers such as PEG, peptides, sterols, peptide nucleic acids, and the like.
The term “nucleotide” refers to its meaning as is generally accepted in the art. The term generally refers to compounds that comprise a nucleobase, a sugar, and an internucleoside linkage, e.g., a phosphate group such as a phosphodiester. The base can be a natural bases (standard), modified bases, or a base analog, as are well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Additionally, the nucleotides can be unmodified or modified at the sugar, internucleoside linkage, and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and others; see, for example, U.S. application Ser. No. 12/064,014).
The term “parenteral,” refers to its meaning as is generally accepted in the art. The term generally includes subcutaneous, intravenous, intramuscular, intraarterial, intraabdominal, intraperitoneal, intraarticular, intraocular or retrobulbar, intraaural, intrathecal, intracavitary, intracelial, intraspinal, intrapulmonary or transpulmonary, intrasynovial, and intraurethral injection or infusion techniques.
By “passenger strand” of a segmented miRNA of the invention is meant one or more distinct nucleic acid sequences or contiguous stretches of nucleotides capable of forming base pairs (including traditional base pairs and non-traditional base pairs) to one or more non-overlapping contiguous stretches of nucleotides in the guide strand. The nucleotides within each contiguous stretch can be connected by traditional phosphodiester bonds and/or non-phosphodiester connectors. In addition, the passenger strand of a segmented miRNA can comprise one or more nucleic acid sequences having at least substantial homology, or at least essential homology, or even perfect homology to a RNA sequence that is a target of a corresponding naturally-occurring miRNA, such as one selected from the miRBase, and for example, one selected from Table I herein.
The terms “patient,” “subject,” “individual” refer to their ordinary meanings as are generally accepted in the art. The terms generally refer to any animal or cells or tissues thereof whether in vitro or in situ, amendable to the methods described herein. They typically refer to an organism, which is a donor or recipient of explanted cells or the cells themselves. They also refer to an organism to which the segmented miRNAs of this disclosure can be administered. In certain non-limiting embodiments, the patient, subject or individual is a mammal or a mammalian cell. In other non-limiting embodiments, the patient, subject or individual is a human or a human cell.
The term “phospholipid” refers to its meaning as is generally accepted in the art. The term generally refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.
The term “perfect complementarity” (or “perfectly complement”) as used herein refers to complete (100%) complementarity within a contiguous region of double-stranded nucleic acid, such as, for example, between a hexamer or heptamer seed sequence of a miRNA and its complementary sequence in a target mRNA. “Perfectly complementary” can also mean that all the contiguous residues of a first nucleic acid sequence form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence. For example, 2 or more perfectly complementary nucleic acid strands can have the same number of nucleotides (i.e., have the same length and form one double-stranded region with or without an overhang), or have a different number of nucleotides (e.g., one strand can be shorter but fully contained within a second strand). “Perfect complements” can be formed between modified nucleotides and nucleotide analogs.
The term “perfect homology” (or “perfectly homologous”) as used herein refers to complete (100%) homology or “identity” between a reference sequence and a subject nucleic acid sequence. When there is a perfect homology, the reference and the subject sequences are the same.
The term “phosphorothioate” refers to its meaning as is generally accepted in the art. The term generally refers a sulphur substituted internucleotide phosphate linkage, but can also refer to internucleotide linkages selected from the group consisting of: —O—P(O)2—O—, —O—P(O,S)—O—, —O—P(S)2—O—, —S—P(O)2—O—, —S—P(O,S)—O—, —S—P(S)2—O—, —O—P(O)2—S—, —OP(O,S)—S—, —S—P(O)2—S—, —O—PO(RH)—O—, O—PO(OCH3)—O—, —O—PO(NRH)—O—, —O—PO(OCH2CH2S—R)—O—, —O—PO(BH3)—O—, —O—PO(NHRH)—O—, —O—P(O)2—NRH—, —NRH—P(O)2—O—, —NRH—CO—O—, —NRH—CO—NRH—, and/or the internucleotide linkage can be selected form the group consisting of: —O—CO—O—, —O—CO—NRH—, —NRH—CO—CH2—, —O—CH2—CO—NRH—, —O—CH2—CH2—NRH—, —CO—NRH—CH2—, —CH2—NRH—CO—, —O—CH2—CH2—S—, —S—CH2—CH2—O—, —S—CH2—CH2—S—, —CH2—SO2—CH2—, —CH2—CO—NRH—, —O—CH2—CH2—NRH—CO—, —CH2—NCH3—O—CH2—, where RH is selected from hydrogen and C1-4-alkyl, Suitably, in some embodiments, sulphur (S) containing internucleotide linkages as provided above can be preferred. Moreover, a segmented miRNA mimetic of the invention can comprise other linkages or mixtures of different linkages—for example, both phosphate or phosphorothioate linkages, or just phosphate linkages, or other linkages as described herein.
The terms “polynucleotide” and “oligonucleotide” refer to their meanings as are generally accepted in the art. The terms generally refers to a chain of nucleotides. “Nucleic acids” or “nucleic acid molecules” are polymers of nucleotides. Thus, “nucleic acids” and “polynucleotides” or “oligonucleotides” are interchangeable herein. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric nucleotides. The monomeric nucleotides can be further hydrolyzed into nucleosides.
The term “protecting group” refers to its meaning as is generally accepted in the art. Protection groups of hydroxy substituents comprises substituted trityl, such as 4,4′-dimethoxytrityloxy (DMT), 4-monomethoxytrityloxy (MMT), and trityloxy, optionally substituted 9-(9-phenyl)xanthenyloxy (pixyl), optionally substituted methoxytetrahydro-pyranyloxy (mthp), silyloxy such as trimethylsilyloxy (TMS), triisopropylsilyloxy (TIPS) tert-butyldimethylsilyloxy (TBDMS), triethylsilyloxy, and phenyldimethylsilyloxy, tert-butylethers, acetals (including two hydroxy groups), acyloxy such as acetyl or halogen substituted acetyls.
The term “purine” refers to its meaning as is generally accepted in the art. The term generally refers to conventional purine nucleotides, including those with standard purine bases adenine and guanine. In addition, the term “purine” is contemplated to embrace nucleotides with natural non-standard purine bases or acids, such as N2-methylguanine, inosine, 2,6-diaminopurine and the like, as well as chemically modified bases or “universal bases,” which can be used to substitute for standard urines herein.
The term “pyrimidine” refers to its meaning as is generally accepted in the art. The term generally refers to conventional pyrimidine nucleotides, including those with standard pyrimidine bases uracil, thymidine and cytosine. In addition, the term pyrimidine is contemplated to embrace nucleotides with natural non-standard pyrimidine bases or acids, such as 5-methyluracil, 2-thio-5-methyluracil, 4-thiouracil, pseudouracil, dihydrouracil, orotate, 5-methylcytosine, or the like, as well as a chemically modified bases or “universal bases,” which can be used to substitute for a standard pyrimidine within the nucleic acid molecules of this disclosure.
The term “RNA” refers to its meaning as is generally accepted in the art. The term generally refers to a molecule comprising at least one ribofuranoside residue, such as a ribonucleotide. The term “ribonucleotide” means a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety. The term refers to a double-stranded RNA, a single-stranded RNA, an isolated RNA such as a partially purified RNA, an essentially pure RNA, a synthetic RNA, a recombinantly produced RNA, or an altered RNA that differs from a naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides therein. Such alterations can include addition of non-nucleotide material, for example, at one or more non-terminal nucleotides of an RNA molecule. As such, nucleotides in the segmented miRNA mimetics of the invention can comprise non-standard nucleotides, such as non-naturally occurring nucleotides, chemically synthesized and/or modified nucleotides, or deoxynucleotides. The altered RNAs are referred to as “RNA analogs” or “analogs of naturally-occurring RNA” containing standard nucleotides (i.e., adenine, cytidine, guanidine, thymidine and uridine), or generally as “modified RNA”.
As used herein, the phrase “RNA interference” (also called “RNAi” herein) refers to its meaning as is generally accepted in the art. The term generally refers to the biological process of inhibiting, decreasing, or down-regulating gene expression in a cell, and which is mediated by short interfering nucleic acid molecules (e.g., siRNAs, miRNAs, shRNAs), see for example Zamore and Haley, 2005, Science 309:1519-1524; Vaughn and Martienssen, 2005, Science 309:1525-1526; Zamore et al., 2000, Cell 101:25-33; Bass, 2001, Nature 411:428-429; Elbashir et al., 2001, Nature 411:494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, 2002, Science 297:1818-1819; Volpe et al., 2002, Science 297:1833-1837; Jenuwein, 2002, Science 297:2215-2218; and Hall et al., 2002, Science 297:2232-2237; Hutvagner and Zamore, 2002, Science 297:2056-60; McManus et al., 2002, RNA 8:842-850; Reinhart et al., 2002, Gene & Dev. 16:1616-1626; and Reinhart & Bartel, 2002, Science 297:1831). Additionally, the term “RNA interference” (or “RNAi”) is meant to be equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, or epigenetics. For example, segmented microRNA mimetics of the invention can be used to epigenetically silence genes at either the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic modulation of gene expression by segmented microRNA mimetics of the invention can result from modification of chromatin structure or methylation patterns to alter gene expression (see, for example, Verdel et al., 2004, Science 303:672-676; Pal-Bhadra et al., 2004, Science 303:669-672; Allshire, 2002, Science 297:1818-1819; Volpe et al., 2002, Science 297:1833-1837; Jenuwein, 2002, Science 297:2215-2218; and Hall et al., 2002, Science 297:2232-2237). In another non-limiting example, modulation of gene expression by segmented microRNA mimetics of the invention can result from cleavage of RNA (either coding or non-coding RNA) via RISC, or via translational inhibition, as is known in the art or modulation can result from transcriptional inhibition (see for example Janowski et al., 2005, Nature Chemical Biology 1:216-222).
The term “RNA profile” or “gene expression profile” refers to a set of data regarding the expression pattern for one or more gene or genetic marker in the sample (e.g., a plurality of nucleic acid probes that identify one or more markers). In some embodiments, it can be useful to know whether a cell expresses a particular miRNA endogenously or whether such expression is affected under particular conditions or when it is in a particular disease state. Thus in some embodiments of the invention, methods include assaying a cell or a sample containing a cell for the presence of one or more marker genes or mRNA or other analyte indicative of the expression level of a gene of interest. Consequently in some embodiments, methods include a step of generating an RNA profile for a sample.
As used herein, the term “seed sequence” refers to at least 6 consecutive nucleotides within any of nucleotide positions 1 to 10 of the 5′-end of a naturally-occurring mature miRNA, such as one selected from those listed in miRBase (http://www.mirbase.org/) as of the filing date of the present application, and for example, such as one selected from those listed in Table I, wherein the seed sequence nucleotides of positions 1 to 8 are capitalized. See, e.g., Brennecke et al., 2005, PLOS Biol. 3(3):e85. In a naturally-occurring miRNA, the seed sequence typically determines the target mRNA sequence to which the miRNA can bind and provide gene regulation. As such, multiple naturally-occurring miRNAs can share a seed sequence, or share substantial homology in the seed sequences, and these miRNAs are members of the same miRNA family.
The term “segmented miRNA mimetic” (or “segmented miRNA,” interchangeably) as used herein refers to a miRNA mimetic molecule comprising at least one discontinuity in the guide strand that is capable of modulating the expression of a target gene that is also regulated by a corresponding naturally-occurring miRNA, such as one selected from the miRBase as of the filing date of the present application, and for example, such as one selected from SEQ ID NOs: 1-1090 of Table I herein. The discontinuity comprises one or more nicks, gaps, substitutions, or insertions. In one aspect, a segmented miRNA mimetic of the invention will mediate gene silencing via an RNAi mechanism such as RISC mediated cleavage, translational inhibition, or epigenetic silencing as is known in the art. A segmented miRNA of the invention can comprise one or more or all ribonucleotides. Segmented miRNAs of the invention can also comprise nucleotide and non-nucleotide analogs as described herein and as otherwise known in the art.
A segmented miRNA mimetic of the invention is said to be “double-stranded” if the molecule has an overall double-stranded conformation. Each of the “strands” is not necessarily continuous, but rather can comprise one or more distinct contiguous stretches of nucleotides, separated by non-contiguous segments (i.e. gaps, nicks, substitutions, insertions). The strand (including the one or more contiguous stretches of nucleotides) that comprises or comprises essentially of a sequence of a corresponding miRNA target is termed the “passenger strand of the segmented miRNA.” The strand (including the one or more contiguous stretches of nucleotides) that comprises or comprises essentially of at least a portion (e.g., a stretch of about 5 to about 8 nucleotides within the seed sequence) of a corresponding endogenous miRNA is termed the “guide strand of the segmented miRNA.” Moreover, a guide strand comprising one or more discontinuities (i.e., gaps, nicks, substitutions, insertions) can form a double-stranded RNA complex even if it is hybridized to a passenger strand that also comprises one or more discontinuities (i.e., gaps, nicks, substitutions, insertions). When both strands comprise discontinuities, the discontinuities can, in certain embodiments, be arranged in such relative positions with each other that the segmented miRNA mimetic of the invention maintains a generally double-stranded conformation, thereby allowing its recognition by the cellular RNAi machinery. Linkers can be introduced and various other stabilizing modifications can be applied to confer added thermodynamic stability. In a specific embodiment, linkers or stabilizing modifications are introduced to a molecule comprising structurally overlapping discontinuities, where otherwise the double-stranded molecule would have broken into two double-stranded sections if the contiguous stretches adjacent to the overlapping gaps are not connected.
Each segmented miRNA mimetic of the invention can have a corresponding non-segmented double-stranded miRNA mimetic, where the non-segmented mimetic comprises all of the contiguous stretches of nucleotides of the segmented mimetic, and where the non-segmented mimetic and the segmented mimetic share the same RNA target(s) with their corresponding endogenous miRNA. Essentially, the segmented miRNA mimetic is designed based on the corresponding non-segmented miRNA mimetic by deleting certain internal phosphodiester backbone linkages and/or internal nucleotides (i.e., by placing nicks or gaps) or substituting such nicks or gaps with one or more non-nucleotide moieties.
It is contemplated that multiple segmented miRNAs, having respective multiple corresponding miRNAs, can be applied to a cell. In particular embodiments, two or more segmented miRNAs are introduced to a cell. A combination of multiple segmented miRNAs can act as one or more points of regulation in cellular pathways within the cell, which has aberrant phenotype(s) (i.e., that the cell is a “targeted cell”), and that such combination can have increased efficacy for correcting the aberrant phenotype(s) of the targeted cell. If the targeted cell is mixed with normal cells, it is contemplated that the segmented miRNAs added to the collection of cells, while providing improved efficacy to correct the aberrant phenotypes of the targeted cell, have minimal adverse effect on the normal cells.
The term “siRNA” (also “short interfering RNA” or “small interfering RNA”) is given its ordinary meaning as is recognized in the art.
A double-stranded nucleic acid molecule can have strands that are not perfectly complementary, but merely “substantially complementary.” By “substantially complementary” it is meant that the nucleic acid sequence of the first strand is at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to the nucleic acid sequence of the second strand. In certain embodiments, complementary nucleic acid molecules can have wrongly paired bases—that is, bases that cannot form a traditional Waston-Crick base pair (i.e., forming a hydrogen bond) or other non-traditional types of base pair (i.e., “mismatched” bases, formed or held together by non-traditional forces that are not hydrogen bonds). Non-traditional Waston-Crick base pairs are also understood as “non-canonical base pairs,” which is meant any non-Watson Crick base pair, such as mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions. Non-limiting examples of such non-canonical base pairs include, but are not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC 4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA N1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+ carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H—N3, GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.
As used herein, the term “substantially homologous” (or “substantial homology”) is meant that the subject sequence shares at least 25% (e.g., at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) homologous nucleotides with the nucleotides of the same nucleotide positions in a reference sequence. By “essentially homologous” (or “essential homology’) it is meant that, a first part of a subject sequence having a number of consecutive nucleotides is identical to a first part of a reference sequence having the same number or consecutive nucleotides, whereas the rest of the subject sequence, which does not overlap with the first part of the subject sequence, is substantially homologous to the rest of the reference sequence, which does not overlap with the first part of the reference sequence. For example, as used herein, the term “essentially homologous” with regard to miRNA sequences, can refer to the contiguous stretch from the 5′-terminal of the guide strand of a segmented miRNA mimetic of the invention comprising a sequence that is essentially homologous to a sequence, including the seed sequence, of a corresponding naturally-occurring miRNA. For example, the first contiguous stretch from the 5′-terminal of the guide strand can comprise a 6 to 7-nucleotide stretch within that is perfectly complementary to a 6 to 7-nucleotide stretch of the seed sequence, where the rest of the nucleotides (including nucleotide analogs) in the contiguous stretch can be at least 50% homologous to the rest of the corresponding endogenous mature miRNA sequence. The comparison of sequences and determination of percent homology and/or identity between two sequences can be accomplished using a mathematical algorithm of Karlin and Altschul (1990, PNAS 87:2264-2268), modified as in Karlin and Altschul (1993, PNAS 90:5873-5877) or by visual inspection.
As used herein, the term “substitute non-nucleotide moieties” refers to chemical moieties that are capable of substituting one or more nucleotides in a segmented miRNA mimetic of the invention. The substitute non-nucleotide moieties can allow for non-traditional base-pairing (i.e., not forming traditional hydrogen bonds) between the strands and contribute to the binding free energy. In certain embodiments, the substitute non-nucleotide moieties of the instant disclosure are those that are capable of associating or otherwise interacting with one or more components of the cellular RNAi machinery, including, for example, the PAZ domain, the PIWI domain and/or other Argonaute protein domains associated with the RISC.
The term “substitution” as used herein refers to a discontinuity in which one or more nucleotide(s) of the otherwise continuous nucleotide sequence of the guide strand and/or passenger strand is replaced with one or more non-nucleotide moieties. Examples of such non-nucleotide moieties are provided herein and others are provided as is generally known to those of skill in the art.
The segmented miRNAs of the invention are typically synthetic. The term “synthetic” as used herein generally refers to nucleic acid molecules that are not produced naturally in a cell. In certain aspects, the chemical structure of a synthetic nucleic acid molecule can deviate from a naturally-occurring nucleic acid molecule. On the other hand, a synthesized segmented miRNA can encompass all or part of a naturally-occurring miRNA sequence or a component thereof. Moreover, it is contemplated that, in a specific embodiment, a synthetic segmented miRNA mimetic administered to a cell can subsequently be altered or processed by the cellular components such that its post-processing structure or sequence can be identical to the whole or a part of a naturally-occurring miRNA. The difference between a synthetic miRNA mimetic and its corresponding endogenous miRNA, including miRNA precursors and complements, can comprise missing internal (i.e., at non-terminal positions) phosphordiester bonds, or missing internal nucleotides, altered types of nucleotides, altered internucleotide connectors or linkages, or chemically modified nucleotides. In certain aspects, a synthetic segmented miRNA of the invention is an RNA or an RNA analog.
The phrases “target site,” “target sequence,” and “target region,” as used herein, refer to their meanings as generally accepted in the art. The terms generally refer to a sequence within a target nucleic acid molecule (e.g., target RNA) that is “targeted,” e.g., for cleavage mediated by an RNAi molecule that contains a sequence within its guide/antisense region that is partially, substantially, or perfectly complementary to that target sequence. A “target site” for a miRNA mimetic molecule of the present invention refers to a nucleic acid sequence that is partially, substantially, or perfectly complementary to the guide strand of the miRNA mimetic. The target site can be within a coding or a non-coding (i.e., untranslated) region of a target RNA. The target site can be the target site for an endogenous miRNA for which the segmented miRNA molecule is a mimetic, in which case the “target site” can also be referred to as a “miRNA target site” or a “corresponding miRNA target site.”
Linkers connecting the terminal ends of a segmented miRNA mimetic of the invention are referred to as “terminal linkers” herein.
The term “therapeutic” refers to its meaning as is generally accepted in the art. The term generally refers to a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state. In the instant application, the disease state is particularly referred to as one associated with aberrant biological pathways regulated by miRNAs, such as those listed in the miRBase at the time of filing of this application, and especially those listed in Table I herein. The term “treatment” as used herein is meant to include therapeutic treatment as well as prophylactic, or suppressive measures for diseases or disorders. Thus, for example, the term “treatment” includes the administration of an agent prior to or following the onset of a disease or disorder thereby preventing or removing all signs of the disease or disorder. As another example, administration of the agent after clinical manifestation of the disease to combat the symptoms of the diseases is also comprised by the term “treatment.”
As used herein, the term “therapeutically effective amount” refers to its meaning as is generally accepted in the art. The term can refer to an amount of a segmented miRNA that is sufficient to result in a decrease in severity of disease symptoms, an increase in frequency or duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease, in the subject (e.g., a mammal or human) to which it is administered. One of ordinary skill in the art would be able to determine such therapeutically effective amounts based on such factors such as the subject's size, the severity of symptoms, and the particular composition or route of administration selected. For example, a therapeutically effective amount of a segmented miRNA of the invention, individually, in combination, or in conjunction with other drugs, can be used or administered at a therapeutically effective amount to a subject or by administering to a particular cells under conditions suitable for treatment, to, for example, decrease tumor size, or otherwise ameliorate symptoms associated with a particular disorder in the subject.
As used herein, “terminals” or “terminal ends” refers to the ultimate ends at the first 5′-nucleotide or the first 3′-nucleotide of a given strand. Substitutions of such terminal ends can be selected independently from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto, Prot-S—, Act-S—, C1-6-alkylthio, amino, Prot-N(RH)—, Act-N(RH)—, mono- or di(C1-6-alkyl)amino, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkyl, optionally substituted C2-6-alkenyl, optionally substituted C2-6-alkenyloxy, optionally substituted C2-6-alkynyl, optionally substituted C2-6-alkynyloxy, monophosphate, monothiophosphate, diphosphate, dithiophosphate triphosphate, trithiophosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH2—, Act-O—CH2—, aminomethyl, Prot-N(RH)—CH2—, Act-N(RH)—CH2—, carboxy methyl, sulphonomethyl, where Prot is a protection group for —OH, —SH, and —NH(RH), respectively, Act is an activation group for —OH, —SH, and —NH(RH), respectively, and RH is selected from hydrogen and C1-6-alkyl.
Linkers connecting the terminal ends of a segmented miRNA mimetic of the invention are referred to as “terminal linkers” herein.
The term “universal base” refers to its meaning as is generally accepted in the art. The term generally refers to nucleotide base analogs that form base pairs with each of the standard DNA/RNA bases with little discrimination among them, and is recognized by intracellular enzymes. See, e.g., Loakes et al., J. Mol. Bio. 1997, 270:426-435. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carbozamides, and nitroazole derivatives such as 3′-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art. See, e.g., Loakes, 2001 Nucleic Acids Res. 29:2437.
A “vector” refers to its meaning as is generally accepted in the art. The term generally refers to a replicon, such as a plasmid, phagemid, cosmid, baculovirus, bacmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), as well as other bacterial, yeast, or viral vectors, to which another nucleic acid segment can be operatively inserted so as to bring about replication or expression of the inserted segment. “Expression vector” refers to a vector comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
Any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range, and when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
Other objects, features and advantages of the present invention will become apparent from the detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description.
A Segmented miRNA of the Invention
The instant disclosure provides a segmented miRNA mimetic molecule (segmented miRNA mimetic) that is double-stranded comprising a passenger strand and a distinct guide strand, wherein at least the guide strand includes one of more discontinuities and wherein the passenger strand and the guide strand each independently comprises, in sum, about 12 to about 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides, and the mimetic molecule comprises, in sum, about 10 to about 26 (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) base pairs. In one aspect, a segmented miRNA of the invention comprises 2 or more (e.g., 2, 3, or 4) distinct contiguous stretches of nucleotides in the guide strand. A “contiguous stretch of nucleotides” can comprise as little as 2 nucleotides, according to the present invention. In another aspect, a segmented miRNA of the invention comprises 2 or more (e.g., from 2, 3, 4, 5, 6, 7, 8, 9, or 10) or more distinct contiguous stretches of nucleotides in each of the passenger strand and the guide strand. The distinct contiguous stretches of nucleotides are arranged such that their complementary sequences located on the opposite strand or within a corresponding miRNA target sequence do not overlap. Within each of the contiguous stretches of nucleotides, the nucleotides are connected by phosphodiester bonds and/or non-phosphodiester connectors. The distinct contiguous stretches of nucleotides in a given strand are arranged from 5′- to 3′-, and the each pair of neighboring stretches can be separated by a nick, gap, substitution, or insertion.
In one embodiment, a first discontinuity in the passenger strand and a second discontinuity in the guide strand of a segmented miRNA mimetic do not overlap provided that RNAi activity against one or more miRNA targets is maintained.
In another embodiment, a first discontinuity in the passenger strand and a second discontinuity in the guide strand of a segmented miRNA mimetic partially overlap provided that RNAi activity against one or more miRNA targets is maintained.
In another embodiment, a first discontinuity in the passenger strand and a second discontinuity in the guide strand of a segmented miRNA mimetic overlap completely provided that RNAi activity against one or more miRNA targets is maintained. For example, overlapping nicks or gaps can result in a miRNA mimetic molecule that is no longer able to associate into duplex form. One of skill in the art will readily appreciate that such designs are to be avoided in order to maintain miRNA medicated RNAi activity.
In one embodiment, a first nick in the passenger strand and a second nick in the guide strand of a segmented miRNA mimetic do not overlap.
In one embodiment, a first gap in the passenger strand and a second gap in the guide strand of a segmented miRNA mimetic do not overlap by at least one complementary nucleotide position.
In one aspect, a segmented miRNA mimetic molecule of the invention can be represented or depicted by Formula III:




wherein the molecule comprises a passenger strand and a guide strand, where each line shown in the Formula and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2” and “X3” represent the number of nucleotide positions in each stretch, “G/N” represents a discontinuity in the guide strand, “Y1” represents a number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present or absent, and each of “Z1” and “Z2” represents the number of overhanging nucleotides.

With reference to Formula III, in one embodiment, X1 is an integer from about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X2 is an integer from about 2 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X3 is an integer from about 6 to about 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y1 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X2, X3 and Y1 is an integer from about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide strand is a nick. In certain embodiments the discontinuity in the guide strand is a gap. In certain embodiments the discontinuity in the guide strand is a substitution. In certain embodiments the discontinuity in the guide strand is an insertion. In one embodiment, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, i.e., wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In a further embodiment, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strand, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).
With reference to Formula III, in one embodiment, X1 is an integer from about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X2 is an integer from about 2 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X3 is an integer from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y1 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X2, X3 and Y1 is an integer from about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide strand is a nick. In certain embodiments the discontinuity in the guide strand is a gap. In certain embodiments the discontinuity in the guide strand is a substitution. In certain embodiments the discontinuity in the guide strand is an insertion. In one embodiment, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, i.e., wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In a further embodiment, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strand, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).
In another aspect, a segmented miRNA mimetic molecule of the invention can be represented or depicted by Formula IV:




wherein the molecule comprises a passenger strand and a guide strand, where each line in the Formula and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2,” “X3” and “X4” represents a number of nucleotide positions in each stretch, “P/N” represents a discontinuity in the passenger strand, “G/N” represents a discontinuity in the guide strand, “P/N” represents a discontinuity in the passenger strand, each of “Y1” and “Y2” represents a number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present or absent, and each of “Z1” and “Z2” represents the number of overhanging nucleotides.

With reference to Formula IV, in one embodiment, X1 and X2 are integers independently from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25), Y1 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X1, X2 and Y1 is an integer from about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X3 is an integer from about 2 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X4 is an integer from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y2 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X3, X4 and Y2 is about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide and/or passenger strand is a nick. In certain embodiments the discontinuity in the guide and/or passenger strand is a gap. In certain embodiments the discontinuity in the guide and/or passenger strand is a substitution. In certain embodiments the discontinuity in the guide and/or passenger strand is an insertion. In certain embodiments, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, i.e. wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strands, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).
With reference to Formula IV, in one embodiment, X1 and X2 are integers independently from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25), Y1 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X1, X2 and Y1 is an integer from about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X3 is an integer from about 2 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X4 is an integer from about 6 to about 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y2 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X3, X4 and Y2 is about 16 to about 26 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide and/or passenger strand is a nick. In certain embodiments the discontinuity in the guide and/or passenger strand is a gap. In certain embodiments the discontinuity in the guide and/or passenger strand is a substitution. In certain embodiments the discontinuity in the guide and/or passenger strand is an insertion. In certain embodiments, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, i.e. wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strands, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).
In yet another aspect, a segmented miRNA mimetic molecule of the invention can be represented or depicted by Formula V:




wherein the molecule comprises a passenger strand and a guide strand, where each line in the Formula and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2,” “X3” and “X4” represents the number of nucleotide positions in each stretch, each “G/N” represents a discontinuity in the guide strand, each of “Y1” and “Y2” represents the number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present or absent, and each of “Z1” and “Z2” represents the number of overhanging nucleotides; wherein X1 is an integer from about 12 to about 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X2 and X3 are each independently an integer from about 1 to about 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17), X4 is an integer from about 6 to about 22 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23), Y1 and Y2 are each independently an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X2, X3, X4, Y1 and Y2 is an integer from about 10 to about 26 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide and/or passenger strand is a nick. In certain embodiments the discontinuity in the guide and/or passenger strand is a gap. In certain embodiments the discontinuity in the guide and/or passenger strand is a substitution. In certain embodiments the discontinuity in the guide and/or passenger strand is an insertion. In certain embodiments, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strands, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).

In a further aspect, a segmented miRNA mimetic molecule of the invention can be represented or depicted by Formula VI:




wherein the molecule comprises a passenger strand and a guide strand, where each line in the Formula and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2,” “X3,” “X4” and “X5” represents the number of nucleotide positions in each stretch, “P/N” represents a discontinuity in the passenger strand, “G/N” represents a discontinuity in the guide strand, each “P/N” represents a discontinuity in the passenger strand, each of “Y1,” “Y2” and “Y3” represents the number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present or absent, and each of “Z1” and “Z2” represents the number of overhanging nucleotides; wherein X1, X2, and X3 are each independently an integer from about 2 to about 22 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23), Y1 and Y2 are each independently an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X1, X2, X3, Y1 and Y2 is an integer from about 12 to about 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X4 is an integer from about 1 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21), X5 is an integer from about 6 to about 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y3 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X4, X5 and Y3 is an integer from about 10 to about 26 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide and/or passenger strand is a nick. In certain embodiments the discontinuity in the guide and/or passenger strand is a gap. In certain embodiments the discontinuity in the guide and/or passenger strand is a substitution. In certain embodiments the discontinuity in the guide and/or passenger strand is an insertion. In certain embodiments, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strands, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).

In a further aspect, a segmented miRNA mimetic molecule of the invention can be represented or depicted by Formula VII:




wherein the molecule comprises a passenger strand and a guide strand, where each line in the Formula and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2,” “X3,” “X4,” “X5” and “X6” represents the number of nucleotide positions in each stretch, each “P/N” represents a discontinuity in the passenger strand, each “G/N” represents a discontinuity in the guide strand, each of “Y1,” “Y2,” “Y3” and “Y4” represents the number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present or absent, and each of “Z1” and “Z2” represents the number of overhanging nucleotides; wherein X1, X2, and X3 are each independently an integer from about 2 to about 22 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23), Y1 and Y2 are each independently an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X1, X2, X3, Y1 and Y2 is an integer from about 12 to about 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X4 and X5 are each independently an integer from about 1 to about 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17), X6 is an integer from about 7 to about 22 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23), Y3 and Y4 are independently an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X4, X5, X6, Y3 and Y4 is about 10 to about 26 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide and/or passenger strand is a nick. In certain embodiments the discontinuity in the guide and/or passenger strand is a gap. In certain embodiments the discontinuity in the guide and/or passenger strand is a substitution. In certain embodiments the discontinuity in the guide and/or passenger strand is an insertion. In certain embodiments, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strands, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).

In a further aspect, a segmented miRNA mimetic molecule of the invention can be represented or depicted by Formula VIII:




wherein the molecule comprises a passenger strand and a guide strand, where each line in the Formula and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2,” “X3” and “X4” represents the number of nucleotide positions in each stretch, “P/N” represents a discontinuity in the passenger strand, each “G/N” represents a discontinuity in the guide strand, “P/N” represents a discontinuity in the passenger strand, each of “Y1” and “Y2” represents the number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present or absent, and each of “Z1” and “Z2” represents the number of overhanging nucleotides; wherein X1 and X2 are each independently an integer from about 2 to about 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25), Y1 is an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X1, X2 and Y1 is an integer from about 12 to about 26 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27), X3 and X4 are each independently an integer from about 1 to about 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17), X5 is an integer from about 6 to about 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), Y2 and Y3 are each independently an integer from 0 to about 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), provided that the sum of X3, X4, X5, Y2 and Y3 is an integer from about 10 to about 26 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27). In certain embodiments the discontinuity in the guide and/or passenger strand is a nick. In certain embodiments the discontinuity in the guide and/or passenger strand is a gap. In certain embodiments the discontinuity in the guide and/or passenger strand is a substitution. In certain embodiments the discontinuity in the guide and/or passenger strand is an insertion. In certain embodiments, there is no 3′-terminal overhanging nucleotides present (i.e., blunt-ended) in the passenger strand, in the guide strand, or in either strand, wherein Z1, Z2, or both Z1 and Z2 are 0. In another embodiment, there are one or more 3′-terminal overhanging nucleotides present in the passenger strand, wherein Z1 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In certain embodiments, there are one or more 3′-terminal overhanging nucleotides present in the guide strand, wherein Z2 is about 1 to about 5 (e.g., 1, 2, 3, 4, or 5). In yet another embodiment, there are one or more 3′-terminal overhanging nucleotides present in both the passenger strand and the guide strands, wherein Z1 and Z2 are independently about 1 to about 5 (e.g., 1, 2, 3, 4, or 5).

At least one of the 2 or more contiguous stretches of nucleotides in the guide strand of a segmented miRNA mimetic molecule of Formula III, IV, V, VI, VII, or VIII comprises a sequence that is substantially, essentially or perfectly homologous (e.g., at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% homologous) to a nucleotide sequence of a naturally-occurring endogenous miRNA, such as one selected from miRBase as of the filing date of the present invention, and for example, one selected from SEQ ID NOs: 1-1090 of Table I herein. In certain embodiments, the first contiguous stretch of nucleotides from the 5′-end of the guide strand comprises at least 6 (e.g., 6, 7, or 8) consecutive nucleotides that are identical (or perfectly homologous) to a 6, 7, or 8-nucleotide sequence within the seed sequence of a naturally-occurring miRNA, such as one selected from Table I (wherein the seed sequence nucleotides are capitalized). In another embodiment, at least 2 of the 2 or more contiguous stretches of nucleotides in the guide strand of a segmented miRNA mimetic of Formula III-VIII comprise sequences that are substantially, essentially, or perfectly homologous (at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% homologous) to non-overlapping regions of a naturally-occurring endogenous miRNA. In yet another embodiment, all of the contiguous stretches of nucleotides in the guide strand comprise sequences that are substantially, essentially, or perfectly homologous (e.g., at least at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% homologous) to non-overlapping regions of a naturally-occurring endogenous miRNA. Each of the 1 or more contiguous stretches of nucleotides in the passenger strand of a segmented miRNA mimetic molecule of Formula III, IV, V, VI, VII, or VIII comprises a sequence that is substantially or perfectly complementary (e.g., at least 25, 20, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% complementary) to a non-overlapping region of a naturally-occurring endogenous miRNA, such as one selected from miRBase as of the filing date of the present invention, and for example, one selected from SEQ ID NOs: 1-1090 of Table I herein.
At least one of the 2 or more contiguous stretches of nucleotides in the guide strand of a segmented miRNA mimetic molecule of Formula III, IV, V, VI, VII, or VIII comprises sequence having 6 or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) contiguous nucleotides of a naturally-occurring endogenous miRNA, such as one selected from miRBase as of the filing date of the present invention, and for example, one selected from SEQ ID NOs: 1-1090 of Table I herein. In certain embodiments, the first contiguous stretch of nucleotides from the 5′-end of the guide strand comprises at least 6 (e.g., 6, 7, or 8) consecutive nucleotides of a seed sequence of a naturally-occurring miRNA, such as one selected from Table I (wherein the seed sequence nucleotides are capitalized). In another embodiment, at least 2 of the 2 or more contiguous stretches of nucleotides in the guide strand of a segmented miRNA mimetic of Formula III-VIII comprise sequence having 6 or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) contiguous nucleotides non-overlapping regions of a naturally-occurring endogenous miRNA. In yet another embodiment, all of the contiguous stretches of nucleotides in the guide strand comprises sequence having 6 or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) contiguous nucleotides to non-overlapping regions of a naturally-occurring endogenous miRNA. Each of the 1 or more contiguous stretches of nucleotides in the passenger strand of a segmented miRNA mimetic molecule of Formula III, IV, V, VI, VII, or VIII comprises a sequence capable of forming 2 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26) base pairs to a non-overlapping region of a naturally-occurring endogenous miRNA, such as one selected from miRBase as of the filing date of the present invention, and for example, one selected from SEQ ID NOs: 1-1090 of Table I herein.
In one aspect, a segmented miRNA mimetic of the invention comprises two separate strands, a guide strand and a separate passenger strand, wherein the strands are not connected to each other at either the 5′ or the 3′ terminal ends by a linker. Linkers connecting the terminal ends of a segmented miRNA mimetic of the invention are referred to as “terminal linkers” herein. In another aspect, one or both terminal ends of a segmented miRNA mimetic molecule can be connected or linked together by a terminal nucleotide and/or non-nucleotide linker. In certain embodiments, either or both ends of the passenger strand and the guide strand of a segmented miRNA mimetic of the invention can be covalently linked by a terminal nucleotide and/or non-nucleotide linker as described herein and known in the art.
One or more substitutions or insertions can be present in the absence of any terminal linkers, as described above. Alternatively, one or more substitutions or insertions can be present in a given molecule with one or more terminal linkers.
One or more or all of nucleotides of each of the contiguous stretches of nucleotides can be ribonucleotides, modified ribonucleotides, or suitable nucleotide analogs. Incorporation of nucleotide analogs, such as various known sugar, base, and backbone modifications, and LNA monomer units into disrupted strands can significantly enhance serum stability and prolong target knockdown or expression regulatory effects. The segmented miRNA mimetic molecules of the present invention can functionally accommodate and are compatible with various chemical modifications, in various combinations and juxtapositions, and to varying degrees. For example, from one to all (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52) of the ribonucleotides of the segmented miRNA mimetics of the invention can be modified. The improved properties conferred by the functionally compatible chemical modifications to the sugar, base and/or backbone, or by including suitable nucleotide analog residues, are of particular importance for application of these segmented miRNA mimetic molecules in vivo, for example, for use as a therapeutic agent or as a functional genomic tool.
In certain embodiments, a segmented miRNA mimetic molecule of the present invention can comprise a 3′-terminal overhang in its passenger strand, guide strand, or both the passenger and guide strands. The “overhang” nucleotides are unpaired and single stranded regions located at the terminal ends of an otherwise generally double-stranded nucleic acid molecule. An exemplary segmented miRNA mimetic of the invention can comprise a 3′-terminal overhang of 1 to 5 nucleotides (e.g., 1, 2, 3, 4, or 5 nucleotides) in the passenger strand, the guide strand, or both the passenger and the guide strands. In alternative embodiments, a segmented miRNA mimetic of the present invention can be blunt-ended (i.e., comprising no terminal overhang nucleotides) at either or both terminal ends.
In a further aspect, the segmented miRNA mimetics of the invention, according to any of the embodiments herein, are capable of participating in RNAi against endogenous RNA targets of their corresponding naturally-occurring miRNAs. The inhibition of the miRNA target can be achieved via the standard miRNA-specific interference mechanism. For example, the inhibition of the miRNA target can be by interaction (e.g., base-paring, binding, etc.) with the untranslated mRNA region, with which the corresponding endogenous miRNA interacts, which effectuates the translational regulation of one or more downstream genes. Or, the inhibition of the miRNA target can be achieved via an siRNA-like interference mechanism wherein the binding of the miRNA target by the guide strand of the segmented miRNA mimetic results in the cleavage of the untranslated miRNA target.
Modified Segmented miRNA Mimetics
The introduction of modified nucleotide analogs into segmented miRNA mimetic molecules of the invention provides a tool for overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules (i.e., having standard nucleotides) that are exogenously delivered. In certain embodiments, the use of modified segmented miRNA mimetics of this disclosure can enable achievement of a given therapeutic effect at a lower dose since these molecules can be designed to have an increased melting temperature or half-life in a subject or biological samples (e.g., serum). Furthermore, certain modifications can be used to improve the bioavailability of segmented miRNA mimetics by targeting particular cells or tissues or improving cellular uptake of the segmented miRNA mimetics. Therefore, even if the activity of a segmented miRNA mimetic of this disclosure is somewhat reduced (e.g., by less than about 20%, or 30%, or even 40%) as compared to an unmodified segmented miRNA mimetic of the same structure, the overall activity of the modified segmented miRNA mimetic can be greater than that of its native counterpart due to improved stability or delivery of the molecule. Modified segmented miRNA mimetics can also minimize the possibility of activating an interferon response in, for example, humans.
In certain embodiments, segmented miRNA mimetics of the invention comprise ribonucleotides at about 1 or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26) of the nucleotide positions in one strand, in each strand, or any combination thereof.
In related embodiments, a segmented miRNA mimetic according to the instant disclosure comprises one or more natural or synthetic non-standard nucleotides. In related embodiments, the non-standard nucleotide is one or more deoxyuridine, L- or D-locked nucleic acid (LNA) molecule (e.g., a 5-methyluridine LNA) or substituted LNA (e.g., having a pyrene), or a universal-binding nucleotide, or a G clamp, or any combination thereof. In certain embodiments, the universal-binding nucleotide can be C-phenyl, C-naphthyl, inosine, azole carboxamide, 1-β-D-ribofuranosyl-4-nitro indole, 1-β-D-ribofuranosyl-5-nitroindole, 1-β-D-ribofuranosyl-6-nitroindole, or 1-β-D-ribofuranosyl-3-nitropyrrole.
Modified nucleotides, which can be present in either or both the passenger and the guide strands of a segmented miRNA mimetic of the invention, comprise modified nucleotide analogs having characteristics similar to natural or standard ribonucleotides. For example, this disclosure features segmented miRNA mimetics comprising nucleotides having a Northern conformation (see, e.g., Northern pseudorotation cycle, Saenger, Springer-Verlag ed., 1984), which are known to potentially impart resistant to nuclease degradation while maintaining the capacity to mediate RNAi, at least when applied to siRNA molecules. Exemplary nucleotides having a Northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-methoxyethyl (MOE) nucleotides, 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, 5-methyluridines, or 2′-O-methyl nucleotides). In any of these embodiments, one or more substituted or modified nucleotides can be a G clamp (e.g., a cytosine analog that forms an additional hydrogen bond to guanine, such as 9-(aminoethoxy)phenoxazine). See, e.g., Lin and Mateucci, 1998 J. Am. Chem. Soc. 720:8531).
In certain embodiments, a segmented miRNA mimetic of the invention comprises an overhang of 1 to 5 nucleotides. The overhang can comprise one or more 2′-O-alkyl modifications or locked nucleic acid (LNAs) as described herein or otherwise known in the art. In certain embodiments, a segmented miRNA mimetic of the invention can comprise one or more 3′-end 2′-O-alkyl or LNA at one or more of the internal terminals. A 2′-O-alkyl or LNA can also be present at positions that are not in the gaps, near the nicks or at the terminal ends of a segmented miRNA mimetic. In any of the embodiments of segmented miRNA mimetics, the 3′-terminal overhangs, if present, can comprise chemically-modified nucleotides that are modified at a nucleic acid sugar, base, or backbone. In any of the embodiments of segmented miRNA mimetics, the 3′-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. In any of the embodiments of segmented miRNA mimetics, the 3′-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.
In certain embodiments, the 5′-terminal end of the passenger strand or guide strand of a segmented miRNA mimetic of the invention is phosphorylated. In any of the embodiments of segmented miRNA mimetics described herein, the segmented miRNA can further comprise a terminal phosphate group, such as a 5′-phosphate (see Martinez et al., 2002 Cell 110:563; Schwarz et al., 2002 Mole. Cell 70:537) or a 5′3′-diphosphate.
In certain embodiments, a segmented miRNA mimetic comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) 2′-sugar substitutions in one strand or independently each strand, such as a 2′-deoxy, 2′-O-2-methoxyethyl, 2′-O-methoxyethyl, 2′-O-methyl, 2′-halogen (e.g., 2′-fluoro), 2′-O-allyl, or the like, or any combination thereof. In still further embodiments, a segmented miRNA mimetic comprises a terminal cap substituent at one or more terminal ends, internal ends, or both, of the passenger strand and/or the guide strands, such as, for example, an alkyl, abasic, deoxy abasic, glyceryl, dinucleotide, acyclic nucleotide, inverted deoxynucleotide moiety, or any combination thereof. In certain embodiments, at least one or more 5′-terminal-end or 5′-internal-end ribonucleotides of the passenger strand have 2′-sugar substitutions. In certain other embodiments, at least one or more 5′-terminal-end or 5′-internal-end ribonucleotides of the guide strand have 2′-sugar substitutions. In certain embodiments, at least one or more 5′-terminal-end or 5′-internal-end ribonucleotides of the passenger strand and the guide strand have 2′-sugar substitutions.
In other embodiments, a segmented miRNA mimetic comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) substitutions in the sugar in one strand or independently each strand, including any combination of ribosyl, 2′-deoxyribosyl, a tetrofuranosyl (e.g., L-α-threofuranosyl), a hexopyranosyl (e.g., β-allopyranosyl, β-altropyranosyl and β-glucopyranosyl), a pentopyranosyl (e.g., β-ribopyranosyl, α-lyxopyranosyl, β-xylopyranosyl and α-arabinopyranosyl), a carbocyclic analog, a pyranose, a furanose, a morpholino, or analogs or derivatives thereof.
In yet other embodiments, a segmented miRNA mimetic comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) modified internucleoside linkages in one strand or independently each strand, such as independently a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate, 3′-alkylene phosphonate, 5′-alkylene phosphonate, chiral phosphonate, phosphonoacetate, thiophosphonoacetate, phosphinate, phosphoramidate, 3′-amino phosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, boranophosphate linkage, or any combination thereof.
A modified internucleotide linkage, as described herein, can be present in one or more strands of a segmented miRNA mimetic, for example, in the passenger strand, the guide strand, or in both strands. A segmented miRNA mimetic can comprise one or more modified internucleotide linkages at the 3′-terminal end, the 5′-terminal end, or both of the 3′-terminal and 5′-terminal ends of the passenger strand, the guide strand, or both strands. In certain embodiments, a segmented miRNA mimetic of the invention has one modified internucleotide linkage at the 3′-terminal end, such as a phosphorothioate linkage. An exemplary segmented miRNA mimetic comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages in either strand. Another exemplary segmented miRNA mimetic comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages in both strands. A further exemplary segmented miRNA mimetic comprises about 1 to about 5 or more consecutive phosphorothioate internucleotide linkages at for example, the 5′-terminal end of its passenger strand, the 5′-terminal end of its guide strand, both the 5′-terminal ends of both strands, or for example, at one or more of the 5′-internal ends. In yet another exemplary segmented miRNA mimetic, there can be one or more pyrimidine phosphorothioate internucleotide linkages in the passenger strand and/or the guide strand. In a further exemplary segmented miRNA mimetic, there can be one or more purine phosphorothioate internucleotide linkages in the passenger strand and/or the guide strand.
Many exemplary modified nucleotide bases or analogs thereof useful in segmented miRNA mimetics of the instant disclosure include 5-methylcytosine; 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl, 2-propyl, or other alkyl derivatives of adenine and guanine; 8-substituted adenines and guanines (e.g., 8-aza, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, or the like); 7-methyl, 7-deaza, and 3-deaza adenines and guanines; 2-thiouracil; 2-thiothymine; 2-thiocytosine; 5-methyl, 5-propynyl, 5-halo (e.g., 5-bromo or 5-fluoro), 5-trifluoromethyl, or other 5-substituted uracils and cytosines; and 6-azouracil. Further useful nucleotide bases can be found in Kurreck, 2003 Eur. J. Biochem. 270:1628; Herdewijn, 2000 Guide Nucleic Acid Develop. 10:297; Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990; U.S. Pat. No. 3,687,808, and similar references, all of which are incorporated by reference herein.
Certain modified nucleotide base moieties are also contemplated. These include 5-substituted pyrimidines; 6-azapyrimidines; and N-2, N-6, or O-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine). Further, for example, 5-methyluridine and 5-methylcytosine substitutions are known to increase nucleic acid duplex stability, which can be combined with 2′-sugar modifications (e.g., 2′-O-methyl or 2′-methoxyethyl) or internucleoside linkages (e.g., phosphorothioate) that provide the desired nuclease resistance to the modified or substituted segmented miRNA mimetics.
In certain embodiments, at least one pyrimidine of a segmented miRNA mimetic of the invention is a locked nucleic acid (LNA) in the form of a bicyclic sugar. In a related embodiment, the LNA comprises a base substitution, such as a 5-methyluridine LNA or 2-thio-5-methyluridine LNA. In certain embodiments, a ribose of the pyrimidine nucleoside or the internucleoside linkage can be optionally modified.
In any of these embodiments, one or more modified nucleotides can be a G clamp (e.g., a cytosine analog that forms an additional hydrogen bond to guanine, such as 9-(aminoethoxy) phenoxazine). See, e.g., Lin and Mateucci, 1998 Nucleic Acids Res. 19:3111).
In addition, the terminal structure of segmented miRNA mimetics of this disclosure can comprise a stem-loop structure in which an end of one strand (e.g., the guide strand) of a segmented miRNA mimetic is connected by a linker nucleic acid, e.g., a linker RNA to an end of the opposite strand (e.g., the passenger strand). When linker segments are employed, there is no particular limitation in the length of the linker as long as it does not hinder pairing of the stem portion. For example, for stable pairing of the stem portion, the linker portion can have a clover-leaf tRNA structure. Even if the linker has a length that would hinder pairing of the stem portion, it is possible, for example, to construct the linker portion to include introns so that the introns are excised during processing of a precursor miRNA mimetic into a mature miRNA mimetic, thereby allowing pairing of the stem portion. In the case of a stem-loop dsRNA, either end (head or tail) of a segmented miRNA mimetic with no loop structure can comprise a low molecular weight RNA, for example, a natural RNA molecule such as a tRNA, rRNA or viral RNA, or an artificial RNA molecule.
A segmented miRNA mimetic of the invention can be constructed such that it takes on an overall circular structure, wherein the entire molecule is about 10 to about 60 nucleotides in length having from about 5 to about 26 base pairs (e.g., about 19 to about 21) wherein the circular oligonucleotide forms a dumbbell shaped structure having about 10 to about 26 base pairs and two loops. In certain embodiments, a circular segmented miRNA mimetic contains two loop motifs, wherein one or both loop portions are biodegradable.
In another aspect of this disclosure, the segmented miRNA mimetic structures of the invention and their potential of allowing more suitable types of chemical modification and allowing modification to a higher extent can be used to reduce interferon activation when a segmented miRNA mimetic is contacted with a biological sample, for example, when it is introduced into a eukaryotic cell. A segmented miRNA mimetic of the invention comprises at least 6 ends, including terminal and internal ends, as compared to its traditional non-segmented duplex miRNA mimetic counterpart, which comprises 4 ends. These ends can conveniently be used for tethering functional chemical groups to enhance, for example, lipophilic and other properties associated with cellular delivery. For instance, it is possible to tether bulky groups like cholesterol to the 5′-ends of each of the contiguous stretches of nucleotides without losing RNAi activity.
Moreover, because the yield of synthesis is usually higher for shorter RNA strands, the cost of large-scale synthesis in connection with therapeutic application can be substantially reduced using the segmented miRNA mimetics of the present invention.
In any of the embodiments described herein, a segmented miRNA mimetic can include multiple types of modifications in combination. For example, a segmented miRNA mimetic having at least one ribothymidine or 2-thioribothymidine can further comprise at least one LNA, 2′-methoxy, 2′-fluoro, 2′-deoxy, phosphorothioate linkage, an inverted base terminal cap, or any combination thereof. In certain exemplary embodiments, a segmented miRNA mimetic can comprise one or more or all uridines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substituted with 2′-O-methyl uridine and have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more LNA substitutions. In other exemplary embodiments, a segmented miRNA mimetic can comprise from one or more or all uridines substituted with 2′-O-methyl uridine and have up to about 25% phosphorothioate substitutions. In still other exemplary embodiments, a segmented miRNA mimetic can comprise one or more or all uridines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substituted with 2′-O-methyl uridine and have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2′-deoxy-2′-fluoro substitutions.
Within certain aspects, the present disclosure also provides segmented miRNA molecules comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base nucleotides. In certain aspects, a segmented miRNA mimetic disclosed herein can include about 1 to about 10 universal base nucleotides, so long as the resulting segmented miRNA mimetic remains capable of modulating one or more of its endogenous miRNA targets.
Suitable modifications can also include one or more suitable conjugates attached to, typically the ends, including the terminal ends and internal ends, of a segmented miRNA mimetic of the invention. The conjugate can be attached via a covalent attachment. In some cases, the conjugate can be linked to the segmented miRNA mimetic via a biodegradable linker, attached the 3′-end, 5′ end, or both ends of the passenger strand, the guide strand and/or the internal ends of each of the contiguous stretches of nucleotides. The conjugate molecule can facilitate the delivery of the double-stranded oligonucleotide molecule into a biological system, such as a cell. The conjugate can also be a polyethylene glycol (PEG), human serum albumin, or a ligand for a cellular receptor that can facilitate cellular uptake. However, as explained above, the endogenous miRNA and siRNA paths of biogenesis and machineries are distinct, featuring different components or participants, therefore conjugates or other modifications in this class that are suitable for an exogenously introduced siRNA molecule can still be unsuitable for an exogenously introduced miRNA mimetic molecule such as a segmented miRNA mimetic of the invention.
Substitutions and Insertions
Various non-nucleotide moieties as are provided herein or otherwise known in the art can be used as substitutions and/or insertions in the segmented miRNA mimetics of the invention provided that RNAi activity against one or more miRNA targets is maintained.
In one aspect of the invention, substitutions and/or insertions in the segmented mimetic miRNAs of the invention can comprise one or more alkyl moieties, e.g., any C1-C20, and preferably a C1, C2, C3, C4, C5, C6, C7, C8, C9 or C10 alykl moiety. The alykl moiety can be straight chain, branched, aliphatic or aromatic. The alykl moiety can be substituted or unsubstituted. In certain embodiments the alkyl moieties are C3 and/or C6.
Segmented mimetic miRNAs of the present invention can comprise substitutions or insertions that incorporate one or more small molecules, lipids or lipophiles, terpenes, phospholipids, antibodies, toxins, cholesterol, a protein binding agent (e.g., a ligand for a cellular receptor that can facilitate cellular uptake), a vitamin, negatively charged polymers and other polymers, for example proteins (e.g., human serum albumin), peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, and those described in, for example, U.S. Patent Publication No. 2005/0196781, and U.S. Patent Publication No. 2006/0293271, the disclosures of which are incorporated herein by reference. Substitutions and insertions can include alkyl chains optionally substituted with a functional group. For example, the alkyl chain can be substituted with a moiety that binds specifically to a target molecule of interest.
Substitutions and insertions of the present invention can further comprise a polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g., polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, 1990, Nucleic Acids Res. 18:6353; Seela and Kaiser, 1987, Nucleic Acids Res. 15:3113; Cload and Schepartz, 1991, J. Am. Chem. Soc. 113:6324; Richardson and Schepartz, 1991, J. Am. Chem. Soc. 113:5109; Ma et al., 1993, Nucleic Acids Res. 27:2585; Ma et al., 1993, Biochemistry 32:1751; Durand et al., 1990, Nucleic Acids Res. 18:6353; McCurdy et al., 1991, Nucleosides & Nucleotides 70:287; Jaschke et al., 1993, Tetrahedron Lett. 34:301; Ono et al., 1991, Biochemistry 30:9914; and others. A chemical moiety that provides additional functionality (e.g., specifically binds to a target molecule of interest or facilitates/enhances cellular delivery of the molecule) to the miRNA mimetic can be a part of the substitution or insertion or covalently attached or linked thereto. For example, the additional functional group can impart therapeutic activity to miRNA mimetic of the invention by assisting in transferring the RNAi molecule compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of RNAi molecules of the invention.
Substitutions and insertions of the present invention can aid in delivery and/or localization of RNAi molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). For example, the conjugate member can be naproxen, nitroindole (or another conjugate that contributes to stacking interactions), folate, ibuprofen, or a C5 pyrimidine linker. The conjugate member can be a glyceride lipid conjugate (e.g., a dialkyl glyceride derivatives), vitamin E conjugates, or thio-cholesterols. The conjugate molecule can alternatively be a peptide that functions, when conjugated to a miRNA mimetic, to facilitate delivery of the molecule into a target cell, or otherwise enhance delivery, stability, or activity of the molecule when contacted with a biological sample. Exemplary peptide conjugate members for use within these aspects of this disclosure, include peptides PN27, PN28, PN29, PN58, PN61, PN73, PN158, PN159, PN173, PN182, PN202, PN204, PN250, PN361, PN365, PN404, PN453, and PN509 as described, for example, in U.S. Patent Application Publication Nos. 2006/0040882 and 2006/0014289, and U.S. Provisional Patent Application No. 60/939,578, which are all incorporated herein by reference.
A substitution or insertion can comprise a moiety that specifically binds to a target molecule. The target molecule can be any molecule of interest. For example, the target molecule can be a ligand-binding domain of a protein, thereby preventing or competing with the interaction of the naturally-occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art (see, e.g., Gold et al, 1995, Annu. Rev. Biochem. 64:163; Brody and Gold, 2000, J. Biotechnol. 74:5; Sun, 2000, Curr. Opin. MoI. Ther. 2:100; Kusser, J., 2000, Biotechnol. 74:21; Hermann and Patel, 2000, Science 257:820; and Jayasena, 1999, Clinical Chem. 45:1628).
The substitution or insertion can provide the ability to administer the segmented miRNA mimetic to specific cell types, such as hepatocytes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262:4429) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). Binding of such glycoproteins or synthetic glycoconjugates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell 22: 611; Connolly et al., 1982, J. Biol. Chem. 257:939). Lee and Lee (1987, Glycoconjugate J. 4:317) obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem. 24: 1388). The use of galactose and galactosamine based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment of liver disease. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of bioconjugates of this disclosure.
Terminal Analogs, Modifications, Linkers, and Conjugates
In one embodiment, a miRNA mimetic of the invention comprises one or more terminal nucleotide analogs, non-nucleotide analogs, nucleotide linkers, non-nucleotide linkers, caps, conjugates and the like as are generally known in the art, at the 5′-end, 3′-end, or both 5′- and 3′-ends of the passenger strand, or alternately at the 3′-end of the guide strand.
In certain embodiments, the invention features a nucleic acid linker that covalently attaches on strand to the other. A nucleotide linker can be a nucleic acid aptamer. A non-nucleotide linker can be an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g., polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, 1990 Nucleic Acids Res. 18:6353; Seela and Kaiser, 1987 Nucleic Acids Res. 15:3113; Cload and Schepartz, 1991 J. Am. Chem. Soc. 113:6324; Richardson and Schepartz, 1991 J. Am. Chem. Soc. 113:5109; Ma et al., 1993 Nucleic Acids Res. 27:2585; Ma et al., 1993 Biochemistry 32:1751; Durand et al., 1990 Nucleic Acids Res. 18:6353; McCurdy et al., 1991 Nucleosides & Nucleotides 70:287; Jaschke et al., 1993 Tetrahedron Lett. 34:301; Ono et al., 1991 Biochemistry 30:9914; and others.
In another embodiment, a conjugate molecule can be optionally attached to a segmented miRNA mimetic or an analog thereof. For example, such conjugate molecules can be polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that can, for example, mediate cellular uptake. The conjugate molecule can be attached at one or more of the terminal ends and/or one or more of the internal ends. Examples of specific conjugate molecules contemplated by the instant disclosure are described in, for example, U.S. Patent Publication No. 2005/0196781 A1, and U.S. Patent Publication No. 2006/0293271 A1, the disclosures of which are incorporated herein by reference.
In a certain aspect, the invention features conjugates and/or complexes of segmented miRNA mimetics of the invention. Such conjugates and/or complexes can be used to facilitate delivery of one or more segmented miRNA mimetics to a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of segmented miRNA mimetics of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates described herein can be attached to biologically active segmented miRNA mimetics via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.
A person of skill in the art can screen segmented miRNA mimetics of this disclosure having various conjugates to determine which of the segmented miRNA-conjugate complexes possess improved properties (e.g., pharmacokinetic profile, bioavailability, stability) while maintaining the ability to mediate RNAi in, for example, an animal model as described herein or generally known in the art.
In another aspect, a segmented miRNA mimetic of the invention comprises one or more 5′- and/or a 3′-cap structures, for example at the terminal ends of the passenger strand, guide strand, both strands, or any of the internal ends of the contiguous stretches of nucleotides. In non-limiting examples: a suitable 5′-cap can be one selected from the group comprising inverted abasic residue (moiety); LNA; 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.
In another non-limiting example, a suitable 3′-cap can be selected from a group comprising, LNA; 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties. For more details, see Beaucage and Iyer, 1993, Tetrahedron 49:1925, which is incorporated by reference herein.
Making microRNA Mimetics of the Invention
Exemplary molecules of the instant disclosure can be recombinantly produced (e.g., isolated), chemically synthesized, or a combination thereof. Oligonucleotides or individual contiguous stretches of nucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example, as described in Caruthers et al., 1992 Methods in Enzymol. 211:3; Thompson et al, PCT Publication No. WO 99/54459, Wincott et al., 1995 Nucleic Acids Res. 23:2677; Wincott et al., 1997 Methods MoI. Bio. 74:59; Brennan et al., 1998 Biotechnoh Bioeng. 67:33; and Brennan, U.S. Pat. No. 6,001,311. Synthesis of RNA, including certain segmented miRNA mimetics thereof of this disclosure, can be made using the procedure as described in Usman et al., 1987 J. Am. Chem. Soc. 109:7845; Scaringe et al., 1990 Nucleic Acids Res. 18:5433; and Wincott et al., 1995 Nucleic Acids Res. 23:2677; Wincott et al., 1997 Methods MoI. Bio. 74:59. In certain embodiments, segmented miRNA mimetics of the present disclosure can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992 Science 256:9923; Draper et al., PCT Publication No. WO 93/23569; Shabarova et al., 1991 Nucleic Acids Res. 19:4247; Bellon et al., 1997 Nucleosides & Nucleotides 16:951; Bellon et al., 1997 Bioconjugate Chem. 8:204), or by hybridization following synthesis or deprotection. In certain embodiments, a segmented miRNA mimetic of this disclosure can be made as single or multiple transcription products expressed by a polynucleotide vector encoding one or more contiguous stretches of RNAs and directing their expression within host cells. In all of the embodiments herein, the double-stranded portion of a final transcription product to be expressed within the target cell can be, for example, about 10 to about 26 bp, about 12 to about 25 bp, or about 14 to about 22 bp long.
Methods for Designing a Segmented miRNA Mimetic
As described herein, a segmented miRNA mimetic can be designed based on a corresponding non-segmented duplex miRNA mimetic molecule, which is in turn designed based on known endogenous miRNA molecules, such as those listed in the miRBase as of the filing date of the present application, and for example, SEQ ID Nos: 1-1090 in Table I. The segmented miRNA mimetic is then characterized as described below and in the Examples herein.
Specifically, any segmented miRNA mimetic of the invention can be designed by introducing one or more discontinuities of the invention (nicks, gaps, substitutions, and/or insertions) into the passenger strand, the guide strand, or both the passenger and the guide strands. The discontinuity can be introduced at the 5′-end of any of position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, and/or position 26 of the guide strand. The discontinuity can be introduced at the 5′-end of any of position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, and/or position 26 of the passenger strand.
In certain embodiments, a method is provided wherein one or more genes, which are known to be regulated by an endogenous miRNA, are selected to indicate the RNAi potency of a segmented miRNA mimetic. The RNAi activity of a given segmented miRNA mimetic can be measured using known methods, such as those described generally in Fire et al., PCT Publication No. WO99/32619. In some embodiments, the instant specification provides methods for selecting more efficacious segmented miRNA mimetic designs by using one or more reporter gene constructs comprising a constitutive promoter, such as a cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter, operably fused to, and capable of altering the expression of one or more reporter genes, such as a luciferase, chloramphenicol (CAT), or β-galactosidase, which, in turn, is operably fused in-frame with a segmented miRNA mimetic. These reporter gene expression constructs can be co-transfected with one or more segmented miRNA mimetics and the control (corresponding) non-segmented miRNA mimetics. The capacity of a given segmented miRNA mimetic to mediate RNAi of a target mRNA can be determined by comparing the measured reporter gene activity in cells transfected with the segmented miRNA mimetic and the activity in cells transfected with a negative control (i.e., in cells not transfected with the segmented miRNA mimetic) and a positive control (i.e., in cells transfected with the corresponding non-segmented duplex miRNA mimetic). The segmented miRNA mimetics having at least 20% or more, preferably at least 40% or more, or 60% or more, or 80% or more, of the activity of their corresponding non-segmented duplex miRNA mimetics are selected.
Certain embodiments disclosed herein also provide methods for selecting one or more segmented miRNA mimetics based on their predicted stability. A theoretical melting curve can be prepared for each of the segmented miRNA mimetic designs such that those with high theoretical melting curves, and therefore higher duplex stability and corresponding lower cytotoxic effects, would be selected. Alternatively, stability of a segmented miRNA mimetic can be determined empirically and those with higher measured melting temperatures would be selected.
Compositions and Methods of Use
As set forth herein, segmented miRNA mimetics of the invention are miRNA mimetics that are designed to supplement or take the place of corresponding naturally-occurring miRNAs, the reduced or otherwise unsuitably low levels of which have been associated with pathological or diseased conditions. A segmented miRNA mimetic of the invention is therefore preferably capable of participating in the cellular RNAi pathway or otherwise capable of modulating the same or related pathway(s). A segmented miRNA mimetic of the invention can be introduced to a cell, tissue, organism, an in vitro, or an in vivo system to mediate RNAi against an endogenous RNA target of its corresponding naturally-occurring miRNA. As such, a segmented miRNA mimetic can regulate a number of genes, for example, downstream from its RNA target, whose expression levels are associated with or otherwise regulated by the corresponding naturally-occurring miRNA. Because aberrant expression levels of certain naturally-occurring miRNAs have been implicated in various human ailments, including, but are not limited to, hyperproliferative, angiogenic, or inflammatory diseases, states, or adverse conditions, the segmented miRNA mimetics of the present invention can offer valuable therapeutic opportunities. In this context, a segmented miRNA mimetic of this disclosure can regulate (e.g., knockdown or up-regulate) expression of one or more downstream genes of its corresponding endogenous miRNA, such that prevention, alleviation, or reduction of the severity or recurrence of one or more associated disease symptoms can be achieved. Alternatively, for various distinct disease models in which expression of one or more target mRNAs are not necessarily reduced or at a lower-than-normal level as a consequence or sequel of diseases or other adverse conditions, introducing exogenous miRNA mimetics, such as one or more segmented miRNA mimetics of the invention, can nonetheless result in a therapeutic result by affecting the expression levels of genes associated with the disease pathway. The segmented miRNA mimetics of the invention thus are useful reagents, which can be in methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.
In certain embodiments, aqueous suspensions containing one or more segmented miRNA mimetics of the invention can be prepared in admixture with suitable excipients, such as suspending agents or dispersing or wetting agents. Exemplary suspending agents include sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia. Representative dispersing or wetting agents include naturally-occurring phosphatides (e.g., lecithin), condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). In certain embodiments, the aqueous suspensions can optionally contain one or more preservatives (e.g., ethyl or w-propyl-p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents, or one or more sweetening agents (e.g., sucrose, saccharin). In additional embodiments, dispersible powders and granules suitable for preparation of an aqueous suspension comprising one or more segmented miRNA mimetics of the invention can be prepared by the addition of water with the segmented miRNA mimetics in admixture with a dispersing or wetting agent, suspending agent and optionally one or more preservative, coloring agent, flavoring agent, or sweetening agent. The present disclosure also includes segmented miRNA mimetic compositions prepared for storage or administration that include a pharmaceutically effective amount of a desired compound in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., A. R. Gennaro edit., 21st Edition, 2005. In certain embodiments, pharmaceutical compositions of this disclosure can optionally include preservatives, antioxidants, stabilizers, dyes, flavoring agents, or any combination thereof. Exemplary preservatives include sodium benzoate, esters of p-hydroxybenzoic acid, and sorbic acid.
The segmented miRNA mimetic compositions of the instant disclosure can be effectively employed as pharmaceutically-acceptable formulations. Pharmaceutically-acceptable formulations prevent, alter the occurrence or severity of, or treat (alleviate one or more symptom(s) to a detectable or measurable extent) a disease state or other adverse condition in a subject. A pharmaceutically acceptable formulation includes salts of the above compounds, for example, acid addition salts, such as salts of hydrochloric acid, hydrobromic acid, acetic acid, or benzene sulfonic acid. A pharmaceutical composition or formulation refers to a composition or formulation in a form suitable for administration into a cell, or a subject such as a human (e.g., systemic administration). The formulations of the present disclosure, having an amount of segmented miRNA mimetic sufficient to treat or prevent a disorder associated with target gene expression are, for example, suitable for topical (e.g., creams, ointments, skin patches, eye drops, ear drops) application or administration. Other routes of administration include oral, parenteral, sublingual, bladder washout, vaginal, rectal, enteric, suppository, nasal, and inhalation. The pharmaceutical compositions of the present disclosure are formulated to allow the segmented miRNA mimetic contained therein to be bioavailable upon administration to a subject.
In certain embodiments, a segmented miRNA of this disclosure can be formulated as oily suspensions or emulsions (e.g., oil-in-water) by suspending the segmented miRNA mimetic in, for example, a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil) or a mineral oil (e.g., liquid paraffin). Suitable emulsifying agents can be naturally-occurring gums (e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides (e.g., soy bean, lecithin, esters or partial esters derived from fatty acids and hexitol), anhydrides (e.g., sorbitan monooleate), or condensation products of partial esters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). In certain embodiments, the oily suspensions or emulsions can optionally contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. In related embodiments, sweetening agents and flavoring agents can optionally be added to provide palatable oral preparations. In yet other embodiments, these compositions can be preserved by the optionally adding an anti-oxidant, such as ascorbic acid.
In certain embodiments, a segmented miRNA mimetic can be formulated as syrups and elixirs with sweetening agents (e.g., glycerol, propylene glycol, sorbitol, glucose or sucrose). Such formulations can also contain a demulcent, preservative, flavoring, coloring agent, or any combination thereof. In other embodiments, pharmaceutical compositions comprising a segmented miRNA mimetic can be in the form of a sterile, injectable aqueous or oleaginous suspension. The sterile injectable preparation can also be a sterile, injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., as a solution in 1,3-butanediol). Among the exemplary acceptable vehicles and solvents useful in the compositions of this disclosure is water, Ringer's solution, or isotonic sodium chloride solution. In addition, sterile, fixed oils can be employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of parenteral formulations.
Within certain embodiments of this disclosure, pharmaceutical compositions and methods are provided that feature the presence or administration of one or more segmented miRNA mimetics, combined, complexed, or conjugated with a polypeptide, optionally formulated with a pharmaceutically-acceptable carrier, such as a diluent, stabilizer, buffer, or the like. The negatively charged segmented miRNA mimetics can be administered to a patient by any standard means, with or without stabilizers, buffers, or the like, to form a composition suitable for treatment. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present disclosure can also be formulated and used as a tablet, capsule or elixir for oral administration, suppository for rectal administration, sterile solution, or suspension for injectable administration, either with or without other compounds known in the art. Thus, a segmented miRNA mimetic of the present disclosure can be administered in any form, such as nasally, transdermally, parenterally, or by local injection.
In accordance with this disclosure herein, a segmented miRNA mimetic (optionally substituted or modified or conjugated), compositions thereof, and methods for inhibiting expression of one or more corresponding target mRNAs in a cell or organism are provided. In certain embodiments, this disclosure provides methods and segmented miRNA mimetic compositions for treating a subject, including a human cell, tissue or individual, having a disease or at risk of developing a disease caused by or associated with the aberrant levels of its corresponding naturally-occurring miRNA. In a certain embodiment, the method includes administering a segmented miRNA mimetic or a pharmaceutical composition containing the segmented miRNA mimetic to a cell or an organism, such as a mammal, such that the level of its corresponding naturally-occurring miRNA within the cell or the organism is increased. Subjects (e.g., mammalian, human) amendable for treatment using the segmented miRNA mimetics (optionally substituted or modified or conjugated), compositions thereof, and methods of the present disclosure include those suffering from one or more disease or condition mediated, at least in part, by an aberrant expression level of its corresponding naturally-occurring miRNA, or which are amenable to treatment by replenishing or increasing the level of RNAi mediated by the corresponding miRNA, including a hyperproliferative (e.g., cancer), angiogenic, metabolic, or inflammatory (e.g., arthritis) disease or disorder or condition.
Compositions and methods disclosed herein are useful in the treatment of a wide variety of target viruses, including retrovirus, such as human immunodeficiency virus (HIV), Hepatitis C Virus, Hepatitis B Virus, Coronavirus, as well as respiratory viruses, including human Respiratory Syncytial Virus, human Metapneumovirus, human Parainfluenza virus Rhinovirus and Influenza virus.
In other examples, the compositions and methods of this disclosure are useful as therapeutic tools to treat or prevent symptoms of, for example, hyperproliferative disorders. Exemplary hyperproliferative disorders include neoplasms, carcinomas, sarcomas, tumors, or cancer. More exemplary hyperproliferative disorders include oral cancer, throat cancer, laryngeal cancer, esophageal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, gastrointestinal tract cancer, gastrointestinal stromal tumors (GIST), small intestine cancer, colon cancer, rectal cancer, colorectal cancer, anal cancer, pancreatic cancer, breast cancer, cervical cancer, uterine cancer, vulvar cancer, vaginal cancer, urinary tract cancer, bladder cancer, kidney cancer, adrenocortical cancer, islet cell carcinoma, gallbladder cancer, stomach cancer, prostate cancer, ovarian cancer, endometrial cancer, trophoblastic tumor, testicular cancer, penial cancer, bone cancer, osteosarcoma, liver cancer, extrahepatic bile duct cancer, skin cancer, basal cell carcinoma (BCC), lung cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), brain cancer, melanoma, Kaposi's sarcoma, eye cancer, head and neck cancer, squamous cell carcinoma of head and neck, tymoma, thymic carcinoma, thyroid cancer, parathyroid cancer, Hippel-Lindau syndrome, leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, hairy cell leukemia, lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, T-cell lymphoma, multiple myeloma, malignant pleural mesothelioma, Barrett's adenocarcinoma, Wilm's tumor, or the like. In other examples, the compositions and methods of this disclosure are useful as therapeutic tools to regulate expression of one or more target gene to treat or prevent symptoms of, for example, inflammatory disorders. Exemplary inflammatory disorders include diabetes mellitus, rheumatoid arthritis, pannus growth in inflamed synovial lining, collagen-induced arthritis, spondylarthritis, ankylosing spondylitis, multiple sclerosis, encephalomyelitis, inflammatory bowel disease, Chron's disease, psoriasis or psoriatic arthritis, myasthenia gravis, systemic lupus erythematosis, graft-versus-host disease, atherosclerosis, and allergies.
Other exemplary disorders that can be treated with segmented miRNA mimetics, compositions and methods of the instant disclosure include metabolic disorders, cardiac disease, pulmonary disease, neovascularization, ischemic disorders, age-related macular degeneration, diabetic retinopathy, glomerulonephritis, diabetes, asthma, chronic obstructive pulmonary disease, chronic bronchitis, lymphangiogenesis, and atherosclerosis.
Within additional aspects, combination formulations and methods are provided comprising an effective amount of one or more segmented miRNA mimetics in combination with one or more secondary or adjunctive active agents that are formulated together or administered coordinately with the segmented miRNA mimetics of the invention to control one or more target gene-associated disease or condition as described herein. Useful adjunctive therapeutic agents in these combinatorial formulations and coordinate treatment methods include, for example, enzymatic nucleic acid molecules, allosteric nucleic acid molecules, guide, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules and other organic or inorganic compounds including metals, salts and ions, and other drugs and active agents indicated for treating one or more target gene-associated disease or condition, including chemotherapeutic agents used to treat cancer, steroids, non-steroidal anti-inflammatory drugs (NSAIDs), or the like. Exemplary chemotherapeutic agents include alkylating agents (e.g., cisplatin, oxaliplatin, carboplatin, busulfan, nitrosoureas, nitrogen mustards, uramustine, temozolomide), antimetabolites (e.g., aminopterin, methotrexate, mercaptopurine, fluorouracil, cytarabine), taxanes (e.g., paclitaxel, docetaxel), anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idaruicin, mitoxantrone, valrubicin), bleomycin, mytomycin, actinomycin, hydroxyurea, topoisomerase inhibitors (e.g., camptothecin, topotecan, irinotecan, etoposide, teniposide), monoclonal antibodies (e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab, panitumumab, rituximab, tositumomab, trastuzumab), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), cyclophosphamide, prednisone, leucovorin, oxaliplatin. To practice the coordinate administration methods of this disclosure, a segmented miRNA mimetic is administered simultaneously or sequentially in a coordinated treatment protocol with one or more secondary or adjunctive therapeutic agents described herein or known in the art. The coordinate administration can be done in either order, and there can be a time period while only one or both (or all) active therapeutic agents, individually or collectively, exert their biological activities. A distinguishing aspect of all such coordinate treatment methods is that the segmented miRNA mimetic present in a composition elicits some favorable clinical response, which can or can not be in conjunction with a secondary clinical response provided by the secondary therapeutic agent. For example, the coordinate administration of a segmented miRNA mimetic with a secondary therapeutic agent as contemplated herein can yield an enhanced (e.g., synergistic) therapeutic response beyond the therapeutic response elicited by either or both the purified segmented miRNA mimetic and the secondary therapeutic agent alone.
In another embodiment, a segmented miRNA mimetic of this disclosure can include a conjugate member on one or more of the nucleotides, at the terminal positions or the internal positions. The conjugate member can be, for example, a lipophile, a terpene, a protein binding agent, a vitamin, a carbohydrate, or a peptide. For example, the conjugate member can be naproxen, nitroindole (or another conjugate that contributes to stacking interactions), folate, ibuprofen, or a C5 pyrimidine linker. In other embodiments, the conjugate member is a glyceride lipid conjugate (e.g., a dialkyl glyceride derivatives), vitamin E conjugates, or thio-cholesterols. Additional conjugate members include peptides that function, when conjugated to a modified segmented miRNA mimetic, to facilitate delivery of the mimetic into a target cell, or otherwise enhance delivery, stability, or activity of the mimetic when contacted with a biological sample. Exemplary peptide conjugate members for use within these aspects of this disclosure, include peptides PN27, PN28, PN29, PN58, PN61, PN73, PN158, PN159, PN173, PN182, PN202, PN204, PN250, PN361, PN365, PN404, PN453, and PN509 are described, for example, in U.S. Patent Application Publication Nos. 2006/0040882 and 2006/0014289, and U.S. Provisional Patent Application No. 60/939,578, which are all incorporated herein by reference. In certain embodiments, when peptide conjugate partners are used to enhance delivery of one or more segmented miRNA mimetics of this disclosure, the resulting formulations and methods will often exhibit further reduction of an interferon response in target cells as compared to a segmented miRNA mimetic delivered in combination with alternate delivery vehicles, such as lipid delivery vehicles (e.g., LipofectamineTM). In still another embodiment, a segmented miRNA mimetic of the invention can be conjugated to a polypeptide and admixed with one or more non-cationic lipids or a combination of a non-cationic lipid and a cationic lipid to form a composition that enhances intracellular delivery of the segmented miRNA mimetic as compared to delivery resulting from contacting the target cells with a naked segmented miRNA mimetic without the lipids. In more detailed aspects of this disclosure, the mixture, complex or conjugate comprising a segmented miRNA mimetic and a polypeptide can be optionally combined with (e.g., admixed or complexed with) a cationic lipid, such as LipofectineTM. To produce these compositions comprised of a polypeptide, a segmented miRNA mimetic and a cationic lipid, the segmented miRNA mimetic and the polypeptide can be mixed together first in a suitable medium such as a cell culture medium, after which the cationic lipid is added to the mixture to form an segmented miRNA mimetic/delivery peptide/cationic lipid composition. Optionally, the peptide and cationic lipid can be mixed together first in a suitable medium such as a cell culture medium, followed by the addition of the segmented miRNA mimetic to form the segmented miRNA mimetic/delivery peptide/cationic lipid composition.
This disclosure also features the use of segmented miRNA mimetic compositions comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations can offer increased accumulation of drugs in target tissues (Lasic et al., 1995 Chem. Rev., 95:2601; Ishiwata et al., 1995 Chem. Pharm. Bull. 43:1005). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., 1995 Science 267:1215; Oku et al., 1995 Biochim. Biophys. Acta 1238:86). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of nucleic acid molecules as compared to conventional cationic liposomes, which are known to accumulate in tissues of the mononuclear phagocytic system (MPS) (Liu et al., 1995 J. Biol. Chem. 42:24864; Choi et al., PCT Publication No. WO 96/10391; Ansell et al., PCT Publication No. WO 96/10390; Holland et al., PCT Publication No. WO 96/10392). Long-circulating liposomes can also provide additional protection from nuclease degradation as compared to cationic liposomes in theory due to avoiding accumulation in metabolically aggressive MPS tissues, such as the liver and spleen. In a certain embodiment, this disclosure provides compositions suitable for administering segmented miRNA mimetics of this disclosure to specific cell types, such as hepatocytes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987 J. Biol. Chem. 262:4429) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). Binding of such glycoproteins or synthetic glycoconjugates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980 Cell 22: 611; Connolly et al., 1982 J. Biol. Chem. 257:939). Lee and Lee (1987 Glycoconjugate J. 4:317) obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981 J. Med. Chem. 24: 1388). The use of galactose and galactosamine based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment of liver disease. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of bioconjugates of this disclosure.
The present disclosure also features a method for preparing segmented miRNA mimetic nanoparticles. A first solution containing melamine derivatives is dissolved in an organic solvent such as dimethyl sulfoxide, or dimethyl formamide to which an acid such as HCl has been added. The concentration of HCl would be about 3.3 moles of HCl for every mole of the melamine derivative. The first solution is then mixed with a second solution, which includes a nucleic acid dissolved or suspended in a polar or hydrophilic solvent (e.g., an aqueous buffer solution containing, for instance, ethylenediaminetraacetic acid (EDTA), or tris(hydroxymethyl)aminomethane (TRIS), or combinations thereof. The mixture forms a first emulsion. The mixing can be done using any standard technique such as, for example, sonication, vortexing, or in a micro fluidizer. The resultant nucleic acid particles can be purified and the organic solvent removed using size-exclusion chromatography or dialysis or both. The complexed nucleic acid nanoparticles can then be mixed with an aqueous solution containing either polyarginine or a Gln-Asn polymer, or both, in an aqueous solution. A preferred molecular weight of each polymer is about 5000 to about 15,000 Daltons. This forms a solution containing nanoparticles of nucleic acid complexed with the melamine derivative and the polyarginine and the Gln-Asn polymers. The mixing steps are carried out in a manner that minimizes shearing of the nucleic acid while producing nanoparticles on average smaller than about 200 nanometers in diameter. It is believed that the polyarginine complexes with the negative charge of the phosphate groups within the minor groove of the nucleic acid, and the polyarginine wraps around the trimeric nucleic acid complex. At either terminus of the polyarginine other moieties, such as the TAT polypeptide, mannose or galactose, can be covalently bound to the polymer to direct binding of the nucleic acid complex to specific tissues, such as to the liver when galactose is used. While not being bound to theory, it is believed that the Gln-Asn polymer complexes with the nucleic acid complex within the major groove of the nucleic acid through hydrogen bonding with the bases of the nucleic acid. The polyarginine and the Gln-Asn polymer should be present at a concentration of 2 moles per every mole of nucleic acid having 20 base pairs. The concentration should be increased proportionally for a nucleic acid having more than 20 base pairs. For example, if the nucleic acid has 25 base pairs, the concentration of the polymers should be 2.5-3 moles per mole of double-stranded nucleic acid. The resultant nanoparticles can be purified by standard means such as size exclusion chromatography followed by dialysis. The purified complexed nanoparticles can then be lyophilized using techniques well known in the art. In certain embodiments of the present disclosure provides nanoparticles less than 100 nanometers (nm) comprising a segmented miRNA mimetic.
A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of subject being treated, the physical characteristics of the specific subject under consideration for treatment, concurrent medication, and other factors that those skilled in the medical arts will recognize. For example, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients can be administered depending on the potency of a segmented miRNA mimetic of this disclosure.
Dosage levels in the order of about 0.1 mg to about 140 mg per kilogram of body weight per day can be useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.
It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy. Following administration of a segmented miRNA mimetic composition according to the formulations and methods of this disclosure, test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the disease or disorder being treated, as compared to placebo-treated or other suitable control subjects.
Nucleic acid molecules and polypeptides can be administered to cells or organisms by a variety of methods known to those of skill in the art, including administration of formulations that comprise a miRNA mimetic and/or a polypeptide alone, or formulations that further comprise one or more additional components, such as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, buffer, stabilizer, preservative, or the like. In certain embodiments, a segmented miRNA mimetic of the invention, and/or the polypeptide can be encapsulated in liposomes, administered by iontophoresis, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (see, e.g., PCT Publication No. WO 00/53722). Alternatively, a nucleic acid/peptide/vehicle combination can be locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of this disclosure, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies, such as those described in Conroy et al, 1999 Clin. Cancer Res. 5:2330; and PCT Publication No. WO 99/31262.
A segmented miRNA mimetic of the invention can also be administered in the form of suppositories, for example, for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.
For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
Further methods for delivery of nucleic acid molecules, such as a segmented miRNA mimetic of this invention, have been described in, for example, Boado et al., 1998 J. Pharm. ScL, 87:1308; Tyler et al., 1999 FEBS Lett. 421:2m; Pardridge et al., 1995 Proc. Nat'l Acad. ScL USA 92:5592; Boado, 1995 Adv. Drug Delivery Rev. 15:73; Aldrian-Herrada et al. 1998 Nucleic Acids Res. 26:4910; Tyler et al., 1999 Proc. Nat'l Acad. Sci. USA 96:7053; Akhtar et al., 1992 Trends Cell Bio. 2:139; “Delivery Strategies for Guide Oligonucleotide Therapeutics,” ed. Akhtar, 1995, Maurer et al., 1999 MoI. Membr. Biol. 16:129; Lee et al., 2000 ACS Symp. Ser., 752:184. In addition to in vivo and therapeutic applications, a skilled person in the art will appreciate that the segmented miRNA mimetics of the present disclosure are useful in a wide variety of in vitro applications, such as in scientific and commercial research (e.g., elucidation of physiological pathways, drug discovery and development), and medical and veterinary diagnostics.
All U.S. patents, U.S. patent publications, U.S. patent applications, foreign patents, foreign patent applications, non-patent publications, figures, and websites referred to in this specification are expressly incorporated herein by reference, in their entirety.
Table I lists certain endogenous mammalian and viral miRNA sequences, wherein the seed sequences, confirmed or projected, are capitalized. All miRNA sequences in Table I are derived from humans and are shown in 5′ to 3′ orientation. Other miRNA sequences of the present invention can be found in the miRBase, the content of which is incorporated by reference herein.










TABLE I 






miRBase

SEQ ID


miRNA name
number
Sequence
NO


















hsa-let-7a
MIMAT0000062
UGAGGUAGuagguuguauaguu
1


 


hsa-let-7a*
MIMAT0004481
CUAUACAAucuacugucuuuc
2


 


hsa-let-7a-2*
MIMAT0010195
CUGUACAGccuccuagcuuucc
3


 


hsa-let-7b
MIMAT0000063
UGAGGUAGuagguugugugguu
4


 


hsa-let-7b*
MIMAT0004482
CUAUACAAccuacugccuuccc
5


 


hsa-let-7c
MIMAT0000064
UGAGGUAGuagguuguaugguu
6


 


hsa-let-7c*
MIMAT0004483
UAGAGUUAcacccugggaguua
7


 


hsa-let-7d
MIMAT0000065
AGAGGUAGuagguugcauaguu
8


 


hsa-let-7d*
MIMAT0004484
CUAUACGAccugcugccuuucu
9


 


hsa-let-7e
MIMAT0000066
UGAGGUAGgagguuguauaguu
10


 


hsa-let-7e*
MIMAT0004485
CUAUACGGccuccuagcuuucc
11


 


hsa-let-7f
MIMAT0000067
UGAGGUAGuagauuguauaguu
12


 


hsa-let-7f-1*
MIMAT0004486
CUAUACAAucuauugccuuccc
13


 


hsa-let-7f-2*
MIMAT0004487
CUAUACAGucuacugucuuucc
14


 


hsa-miR-15a
MIMAT0000068
UAGCAGCAcauaaugguuugug
15


 


hsa-miR-15a*
MIMAT0004488
CAGGCCAUauugugcugccuca
16


 


hsa-miR-16
MIMAT0000069
UAGCAGCAcguaaauauuggcg
17


 


hsa-miR-16-1*
MIMAT0004489
CCAGUAUUaacugugcugcuga
18


 


hsa-miR-17
MIMAT0000070
CAAAGUGCuuacagugcagguag
19


 


hsa-miR-17*
MIMAT0000071
ACUGCAGUgaaggcacuuguag
20


 


hsa-miR-18a
MIMAT0000072
UAAGGUGCaucuagugcagauag
21


 


hsa-miR-18a*
MIMAT0002891
ACUGCCCUaagugcuccuucugg
22


 


hsa-miR-19a*
MIMAT0004490
AGUUUUGCauaguugcacuaca
23


 


hsa-miR-19a
MIMAT0000073
UGUGCAAAucuaugcaaaacuga
24


 


hsa-miR-19b-1*
MIMAT0004491
AGUUUUGCagguuugcauccagc
25


 


hsa-miR-19b
MIMAT0000074
UGUGCAAAuccaugcaaaacuga
26


 


hsa-miR-19b-2*
MIMAT0004492
AGUUUUGCagguuugcauuuca
27


 


hsa-miR-20a
MIMAT0000075
UAAAGUGCuuauagugcagguag
28


 


hsa-miR-20a*
MIMAT0004493
ACUGCAUUaugagcacuuaaag
29


 


hsa-miR-21
MIMAT0000076
UAGCUUAUcagacugauguuga
30


 


hsa-miR-21*
MIMAT0004494
CAACACCAgucgaugggcugu
31


 


hsa-miR-22*
MIMAT0004495
AGUUCUUCaguggcaagcuuua
32


 


hsa-miR-22
MIMAT0000077
AAGCUGCCaguugaagaacugu
33


 


hsa-miR-23a*
MIMAT0004496
GGGGUUCCuggggaugggauuu
34


 


hsa-miR-23a
MIMAT0000078
AUCACAUUgccagggauuucc
35


 


hsa-miR-24-1*
MIMAT0000079
UGCCUACUgagcugauaucagu
36


 


hsa-miR-24
MIMAT0000080
UGGCUCAGuucagcaggaacag
37


 


hsa-miR-24-2*
MIMAT0004497
UGCCUACUgagcugaaacacag
38


 


hsa-miR-25*
MIMAT0004498
AGGCGGAGacuugggcaauug
39


 


hsa-miR-25
MIMAT0000081
CAUUGCACuugucucggucuga
40


 


hsa-miR-26a
MIMAT0000082
UUCAAGUAauccaggauaggcu
41


 


hsa-miR-26a-1*
MIMAT0004499
CCUAUUCUugguuacuugcacg
42


 


hsa-miR-26b
MIMAT0000083
UUCAAGUAauucaggauaggu
43


 


hsa-miR-26b*
MIMAT0004500
CCUGUUCUccauuacuuggcuc
44


 


hsa-miR-27a*
MIMAT0004501
AGGGCUUAgcugcuugugagca
45


 


hsa-miR-27a
MIMAT0000084
UUCACAGUggcuaaguuccgc
46


 


hsa-miR-28-5p
MIMAT0000085
AAGGAGCUcacagucuauugag
47


 


hsa-miR-28-3p
MIMAT0004502
CACUAGAUugugagcuccugga
48


 


hsa-miR-29a*
MIMAT0004503
ACUGAUUUcuuuugguguucag
49


 


hsa-miR-29a
MIMAT0000086
UAGCACCAucugaaaucgguua
50


 


hsa-miR-30a
MIMAT0000087
UGUAAACAuccucgacuggaag
51


 


hsa-miR-30a*
MIMAT0000088
CUUUCAGUcggauguuugcagc
52


 


hsa-miR-31
MIMAT0000089
AGGCAAGAugcuggcauagcu
53


 


hsa-miR-31*
MIMAT0004504
UGCUAUGCcaacauauugccau
54


 


hsa-miR-32
MIMAT0000090
UAUUGCACauuacuaaguugca
55


 


hsa-miR-32*
MIMAT0004505
CAAUUUAGugugugugauauuu
56


 


hsa-miR-33a
MIMAT0000091
GUGCAUUGuaguugcauugca
57


 


hsa-miR-33a*
MIMAT0004506
CAAUGUUUccacagugcaucac
58


 


hsa-miR-92a-1*
MIMAT0004507
AGGUUGGGaucgguugcaaugcu
59


 


hsa-miR-92a
MIMAT0000092
UAUUGCACuugucccggccugu
60


 


hsa-miR-92a-2*
MIMAT0004508
GGGUGGGGauuuguugcauuac
61


 


hsa-miR-93
MIMAT0000093
CAAAGUGCuguucgugcagguag
62


 


hsa-miR-93*
MIMAT0004509
ACUGCUGAgcuagcacuucccg
63


 


hsa-miR-95
MIMAT0000094
UUCAACGGguauuuauugagca
64


 


hsa-miR-96
MIMAT0000095
UUUGGCACuagcacauuuuugcu
65


 


hsa-miR-96*
MIMAT0004510
AAUCAUGUgcagugccaauaug
66


 


hsa-miR-98
MIMAT0000096
UGAGGUAGuaaguuguauuguu
67


 


hsa-miR-99a
MIMAT0000097
AACCCGUAgauccgaucuugug
68


 


hsa-miR-99a*
MIMAT0004511
CAAGCUCGcuucuaugggucug
69


 


hsa-miR-100
MIMAT0000098
AACCCGUAgauccgaacuugug
70


 


hsa-miR-100*
MIMAT0004512
CAAGCUUGuaucuauagguaug
71


 


hsa-miR-101*
MIMAT0004513
CAGUUAUCacagugcugaugcu
72


 


hsa-miR-101
MIMAT0000099
UACAGUACugugauaacugaa
73


 


hsa-miR-29b-1*
MIMAT0004514
GCUGGUUUcauauggugguuuaga
74


 


hsa-miR-29b
MIMAT0000100
UAGCACCAuuugaaaucaguguu
75


 


hsa-miR-29b-2*
MIMAT0004515
CUGGUUUCacaugguggcuuag
76


 


hsa-miR-103-2*
MIMAT0009196
AGCUUCUUuacagugcugccuug
77


 


hsa-miR-103
MIMAT0000101
AGCAGCAUuguacagggcuauga
78


 


hsa-miR-105
MIMAT0000102
UCAAAUGCucagacuccuguggu
79


 


hsa-miR-105*
MIMAT0004516
ACGGAUGUuugagcaugugcua
80


 


hsa-miR-106a
MIMAT0000103
AAAAGUGCuuacagugcagguag
81


 


hsa-miR-106a*
MIMAT0004517
CUGCAAUGuaagcacuucuuac
82


 


hsa-miR-107
MIMAT0000104
AGCAGCAUuguacagggcuauca
83


 


hsa-miR-16-2*
MIMAT0004518
CCAAUAUUacugugcugcuuua
84


 


hsa-miR-192
MIMAT0000222
CUGACCUAugaauugacagcc
85


 


hsa-miR-192*
MIMAT0004543
CUGCCAAUuccauaggucacag
86


 


hsa-miR-196a
MIMAT0000226
UAGGUAGUuucauguuguuggg
87


 


hsa-miR-197
MIMAT0000227
UUCACCACcuucuccacccagc
88


 


hsa-miR-198
MIMAT0000228
GGUCCAGAggggagauagguuc
89


 


hsa-miR-199a-5p
MIMAT0000231
CCCAGUGUucagacuaccuguuc
90


 


hsa-miR-199a-3p
MIMAT0000232
ACAGUAGUcugcacauugguua
91


 


hsa-miR-208a
MIMAT0000241
AUAAGACGagcaaaaagcuugu
92


 


hsa-miR-129-5p
MIMAT0000242
CUUUUUGCggucugggcuugc
93


 


hsa-miR-129*
MIMAT0004548
AAGCCCUUaccccaaaaaguau
94


 


hsa-miR-148a*
MIMAT0004549
AAAGUUCUgagacacuccgacu
95


 


hsa-miR-148a
MIMAT0000243
UCAGUGCAcuacagaacuuugu
96


 


hsa-miR-30c
MIMAT0000244
UGUAAACAuccuacacucucagc
97


 


hsa-miR-30c-2*
MIMAT0004550
CUGGGAGAaggcuguuuacucu
98


 


hsa-miR-30d
MIMAT0000245
UGUAAACAuccccgacuggaag
99


 


hsa-miR-30d*
MIMAT0004551
CUUUCAGUcagauguuugcugc
100


 


hsa-miR-139-5p
MIMAT0000250
UCUACAGUgcacgugucuccag
101


 


hsa-miR-139-3p
MIMAT0004552
GGAGACGCggcccuguuggagu
102


 


hsa-miR-147
MIMAT0000251
GUGUGUGGaaaugcuucugc
103


 


hsa-miR-7
MIMAT0000252
UGGAAGACuagugauuuuguugu
104


 


hsa-miR-7-1*
MIMAT0004553
CAACAAAUcacagucugccaua
105


 


hsa-miR-7-2*
MIMAT0004554
CAACAAAUcccagucuaccuaa
106


 


hsa-miR-10a
MIMAT0000253
UACCCUGUagauccgaauuugug
107


 


hsa-miR-10a*
MIMAT0004555
CAAAUUCGuaucuaggggaaua
108


 


hsa-miR-10b
MIMAT0000254
UACCCUGUagaaccgaauuugug
109


 


hsa-miR-10b*
MIMAT0004556
ACAGAUUCgauucuaggggaau
110


 


hsa-miR-34a
MIMAT0000255
UGGCAGUGucuuagcugguugu
111


 


hsa-miR-34a*
MIMAT0004557
CAAUCAGCaaguauacugcccu
112


 


hsa-miR-181a
MIMAT0000256
AACAUUCAacgcugucggugagu
113


 


hsa-miR-181a-2*
MIMAT0004558
ACCACUGAccguugacuguacc
114


 


hsa-miR-181b
MIMAT0000257
AACAUUCAuugcugucggugggu
115


 


hsa-miR-181c
MIMAT0000258
AACAUUCAaccugucggugagu
116


 


hsa-miR-181c*
MIMAT0004559
AACCAUCGaccguugaguggac
117


 


hsa-miR-182
MIMAT0000259
UUUGGCAAugguagaacucacacu
118


 


hsa-miR-182*
MIMAT0000260
UGGUUCUAgacuugccaacua
119


 


hsa-miR-183
MIMAT0000261
UAUGGCACugguagaauucacu
120


 


hsa-miR-183*
MIMAT0004560
GUGAAUUAccgaagggccauaa
121


 


hsa-miR-187*
MIMAT0004561
GGCUACAAcacaggacccgggc
122


 


hsa-miR-187
MIMAT0000262
UCGUGUCUuguguugcagccgg
123


 


hsa-miR-196a*
MIMAT0004562
CGGCAACAagaaacugccugag
124


 


hsa-miR-199b-5p
MIMAT0000263
CCCAGUGUuuagacuaucuguuc
125


 


hsa-miR-199b-3p
MIMAT0004563
ACAGUAGUcugcacauugguua
91


 


hsa-miR-203
MIMAT0000264
GUGAAAUGuuuaggaccacuag
126


 


hsa-miR-204
MIMAT0000265
UUCCCUUUgucauccuaugccu
127


 


hsa-miR-205
MIMAT0000266
UCCUUCAUuccaccggagucug
128


 


hsa-miR-205*
MIMAT0009197
GAUUUCAGuggagugaaguuc
129


 


hsa-miR-210
MIMAT0000267
CUGUGCGUgugacagcggcuga
130


 


hsa-miR-211
MIMAT0000268
UUCCCUUUgucauccuucgccu
131


 


hsa-miR-212
MIMAT0000269
UAACAGUCuccagucacggcc
132


 


hsa-miR-181a*
MIMAT0000270
ACCAUCGAccguugauuguacc
133


 


hsa-miR-214*
MIMAT0004564
UGCCUGUCuacacuugcugugc
134


 


hsa-miR-214
MIMAT0000271
ACAGCAGGcacagacaggcagu
135


 


hsa-miR-215
MIMAT0000272
AUGACCUAugaauugacagac
136


 


hsa-miR-216a
MIMAT0000273
UAAUCUCAgcuggcaacuguga
137


 


hsa-miR-217
MIMAT0000274
UACUGCAUcaggaacugauugga
138


 


hsa-miR-218
MIMAT0000275
UUGUGCUUgaucuaaccaugu
139


 


hsa-miR-218-1*
MIMAT0004565
AUGGUUCCgucaagcaccaugg
140


 


hsa-miR-218-2*
MIMAT0004566
CAUGGUUCugucaagcaccgcg
141


 


hsa-miR-219-5p
MIMAT0000276
UGAUUGUCcaaacgcaauucu
142


 


hsa-miR-219-1-3p
MIMAT0004567
AGAGUUGAgucuggacgucccg
143


 


hsa-miR-220a
MIMAT0000277
CCACACCGuaucugacacuuu
144


 


hsa-miR-221*
MIMAT0004568
ACCUGGCAuacaauguagauuu
145


 


hsa-miR-221
MIMAT0000278
AGCUACAUugucugcuggguuuc
146


 


hsa-miR-222*
MIMAT0004569
CUCAGUAGccaguguagauccu
147


 


hsa-miR-222
MIMAT0000279
AGCUACAUcuggcuacugggu
148


 


hsa-miR-223*
MIMAT0004570
CGUGUAUUugacaagcugaguu
149


 


hsa-miR-223
MIMAT0000280
UGUCAGUUugucaaauacccca
150


 


hsa-miR-224
MIMAT0000281
CAAGUCACuagugguuccguu
151


 


hsa-miR-224*
MIMAT0009198
AAAAUGGUgcccuagugacuaca
152


 


hsa-miR-200b*
MIMAT0004571
CAUCUUACugggcagcauugga
153


 


hsa-miR-200b
MIMAT0000318
UAAUACUGccugguaaugauga
154


 


hsa-let-7g
MIMAT0000414
UGAGGUAGuaguuuguacaguu
155


 


hsa-let-7g*
MIMAT0004584
CUGUACAGgccacugccuugc
156


 


hsa-let-7i
MIMAT0000415
UGAGGUAGuaguuugugcuguu
157


 


hsa-let-7i*
MIMAT0004585
CUGCGCAAgcuacugccuugcu
158


 


hsa-miR-1
MIMAT0000416
UGGAAUGUaaagaaguauguau
159


 


hsa-miR-15b
MIMAT0000417
UAGCAGCAcaucaugguuuaca
160


 


hsa-miR-15b*
MIMAT0004586
CGAAUCAUuauuugcugcucua
161


 


hsa-miR-23b*
MIMAT0004587
UGGGUUCCuggcaugcugauuu
162


 


hsa-miR-23b
MIMAT0000418
AUCACAUUgccagggauuacc
163


 


hsa-miR-27b*
MIMAT0004588
AGAGCUUAgcugauuggugaac
164


 


hsa-miR-27b
MIMAT0000419
UUCACAGUggcuaaguucugc
165


 


hsa-miR-30b
MIMAT0000420
UGUAAACAuccuacacucagcu
166


 


hsa-miR-30b*
MIMAT0004589
CUGGGAGGuggauguuuacuuc
167


 


hsa-miR-122
MIMAT0000421
UGGAGUGUgacaaugguguuug
168


 


hsa-miR-122*
MIMAT0004590
AACGCCAUuaucacacuaaaua
169


 


hsa-miR-124*
MIMAT0004591
CGUGUUCAcagcggaccuugau
170


 


hsa-miR-124
MIMAT0000422
UAAGGCACgcggugaaugcc
171


 


hsa-miR-125b
MIMAT0000423
UCCCUGAGacccuaacuuguga
172


 


hsa-miR-125b-1*
MIMAT0004592
ACGGGUUAggcucuugggagcu
173


 


hsa-miR-128
MIMAT0000424
UCACAGUGaaccggucucuuu
174


 


hsa-miR-130a*
MIMAT0004593
UUCACAUUgugcuacugucugc
175


 


hsa-miR-130a
MIMAT0000425
CAGUGCAAuguuaaaagggcau
176


 


hsa-miR-132*
MIMAT0004594
ACCGUGGCuuucgauuguuacu
177


 


hsa-miR-132
MIMAT0000426
UAACAGUCuacagccauggucg
178


 


hsa-miR-133a
MIMAT0000427
UUUGGUCCccuucaaccagcug
179


 


hsa-miR-135a
MIMAT0000428
UAUGGCUUuuuauuccuauguga
180


 


hsa-miR-135a*
MIMAT0004595
UAUAGGGAuuggagccguggcg
181


 


hsa-miR-137
MIMAT0000429
UUAUUGCUuaagaauacgcguag
182


 


hsa-miR-138
MIMAT0000430
AGCUGGUGuugugaaucaggccg
183


 


hsa-miR-138-2*
MIMAT0004596
GCUAUUUCacgacaccaggguu
184


 


hsa-miR-140-5p
MIMAT0000431
CAGUGGUUuuacccuaugguag
185


 


hsa-miR-140-3p
MIMAT0004597
UACCACAGgguagaaccacgg
186


 


hsa-miR-141*
MIMAT0004598
CAUCUUCCaguacaguguugga
187


 


hsa-miR-141
MIMAT0000432
UAACACUGucugguaaagaugg
188


 


hsa-miR-142-5p
MIMAT0000433
CAUAAAGUagaaagcacuacu
189


 


hsa-miR-142-3p
MIMAT0000434
UGUAGUGUuuccuacuuuaugga
190


 


hsa-miR-143*
MIMAT0004599
GGUGCAGUgcugcaucucuggu
191


 


hsa-miR-143
MIMAT0000435
UGAGAUGAagcacuguagcuc
192


 


hsa-miR-144*
MIMAT0004600
GGAUAUCAucauauacuguaag
193


 


hsa-miR-144
MIMAT0000436
UACAGUAUagaugauguacu
194


 


hsa-miR-145
MIMAT0000437
GUCCAGUUuucccaggaaucccu
195


 


hsa-miR-145*
MIMAT0004601
GGAUUCCUggaaauacuguucu
196


 


hsa-miR-152
MIMAT0000438
UCAGUGCAugacagaacuugg
197


 


hsa-miR-153
MIMAT0000439
UUGCAUAGucacaaaagugauc
198


 


hsa-miR-191
MIMAT0000440
CAACGGAAucccaaaagcagcug
199


 


hsa-miR-191*
MIMAT0001618
GCUGCGCUuggauuucgucccc
200


 


hsa-miR-9
MIMAT0000441
UCUUUGGUuaucuagcuguauga
201


 


hsa-miR-9*
MIMAT0000442
AUAAAGCUagauaaccgaaagu
202


 


hsa-miR-125a-5p
MIMAT0000443
UCCCUGAGacccuuuaaccuguga
203


 


hsa-miR-125a-3p
MIMAT0004602
ACAGGUGAgguucuugggagcc
204


 


hsa-miR-125b-2*
MIMAT0004603
UCACAAGUcaggcucuugggac
205


 


hsa-miR-126*
MIMAT0000444
CAUUAUUAcuuuugguacgcg
206


 


hsa-miR-126
MIMAT0000445
UCGUACCGugaguaauaaugcg
207


 


hsa-miR-127-5p
MIMAT0004604
CUGAAGCUcagagggcucugau
208


 


hsa-miR-127-3p
MIMAT0000446
UCGGAUCCgucugagcuuggcu
209


 


hsa-miR-129-3p
MIMAT0004605
AAGCCCUUaccccaaaaagcau
210


 


hsa-miR-134
MIMAT0000447
UGUGACUGguugaccagagggg
211


 


hsa-miR-136
MIMAT0000448
ACUCCAUUuguuuugaugaugga
212


 


hsa-miR-136*
MIMAT0004606
CAUCAUCGucucaaaugagucu
213


 


hsa-miR-138-1*
MIMAT0004607
GCUACUUCacaacaccagggcc
214


 


hsa-miR-146a
MIMAT0000449
UGAGAACUgaauuccauggguu
215


 


hsa-miR-146a*
MIMAT0004608
CCUCUGAAauucaguucuucag
216


 


hsa-miR-149
MIMAT0000450
UCUGGCUCcgugucuucacuccc
217


 


hsa-miR-149*
MIMAT0004609
AGGGAGGGacgggggcugugc
218


 


hsa-miR-150
MIMAT0000451
UCUCCCAAcccuuguaccagug
219


 


hsa-miR-150*
MIMAT0004610
CUGGUACAggccugggggacag
220


 


hsa-miR-154
MIMAT0000452
UAGGUUAUccguguugccuucg
221


 


hsa-miR-154*
MIMAT0000453
AAUCAUACacgguugaccuauu
222


 


hsa-miR-184
MIMAT0000454
UGGACGGAgaacugauaagggu
223


 


hsa-miR-185
MIMAT0000455
UGGAGAGAaaggcaguuccuga
224


 


hsa-miR-185*
MIMAT0004611
AGGGGCUGgcuuuccucugguc
225


 


hsa-miR-186
MIMAT0000456
CAAAGAAUucuccuuuugggcu
226


 


hsa-miR-186*
MIMAT0004612
GCCCAAAGgugaauuuuuuggg
227


 


hsa-miR-188-5p
MIMAT0000457
CAUCCCUUgcaugguggaggg
228


 


hsa-miR-188-3p
MIMAT0004613
CUCCCACAugcaggguuugca
229


 


hsa-miR-190
MIMAT0000458
UGAUAUGUuugauauauuaggu
230


 


hsa-miR-193a-5p
MIMAT0004614
UGGGUCUUugcgggcgagauga
231


 


hsa-miR-193a-3p
MIMAT0000459
AACUGGCCuacaaagucccagu
232


 


hsa-miR-194
MIMAT0000460
UGUAACAGcaacuccaugugga
233


 


hsa-miR-195
MIMAT0000461
UAGCAGCAcagaaauauuggc
234


 


hsa-miR-195*
MIMAT0004615
CCAAUAUUggcugugcugcucc
235


 


hsa-miR-206
MIMAT0000462
UGGAAUGUaaggaagugugugg
236


 


hsa-miR-320a
MIMAT0000510
AAAAGCUGgguugagagggcga
237


 


hsa-miR-200c*
MIMAT0004657
CGUCUUACccagcaguguuugg
238


 


hsa-miR-200c
MIMAT0000617
UAAUACUGccggguaaugaugga
239


 


hsa-miR-155
MIMAT0000646
UUAAUGCUaaucgugauaggggu
240


 


hsa-miR-155*
MIMAT0004658
CUCCUACAuauuagcauuaaca
241


 


hsa-miR-194*
MIMAT0004671
CCAGUGGGgcugcuguuaucug
242


 


hsa-miR-106b
MIMAT0000680
UAAAGUGCugacagugcagau
243


 


hsa-miR-106b*
MIMAT0004672
CCGCACUGuggguacuugcugc
244


 


hsa-miR-29c*
MIMAT0004673
UGACCGAUuucuccugguguuc
245


 


hsa-miR-29c
MIMAT0000681
UAGCACCAuuugaaaucgguua
246


 


hsa-miR-30c-1*
MIMAT0004674
CUGGGAGAggguuguuuacucc
247


 


hsa-miR-200a*
MIMAT0001620
CAUCUUACcggacagugcugga
248


 


hsa-miR-200a
MIMAT0000682
UAACACUGucugguaacgaugu
249


 


hsa-miR-302a*
MIMAT0000683
ACUUAAACguggauguacuugcu
250


 


hsa-miR-302a
MIMAT0000684
UAAGUGCUuccauguuuugguga
251


 


hsa-miR-219-2-3p
MIMAT0004675
AGAAUUGUggcuggacaucugu
252


 


hsa-miR-34b*
MIMAT0000685
UAGGCAGUgucauuagcugauug
253


 


hsa-miR-34b
MIMAT0004676
CAAUCACUaacuccacugccau
254


 


hsa-miR-34c-5p
MIMAT0000686
AGGCAGUGuaguuagcugauugc
255


 


hsa-miR-34c-3p
MIMAT0004677
AAUCACUAaccacacggccagg
256


 


hsa-miR-299-5p
MIMAT0002890
UGGUUUACcgucccacauacau
257


 


hsa-miR-299-3p
MIMAT0000687
UAUGUGGGaugguaaaccgcuu
258


 


hsa-miR-301a
MIMAT0000688
CAGUGCAAuaguauugucaaagc
259


 


hsa-miR-99b
MIMAT0000689
CACCCGUAgaaccgaccuugcg
260


 


hsa-miR-99b*
MIMAT0004678
CAAGCUCGugucuguggguccg
261


 


hsa-miR-296-5p
MIMAT0000690
AGGGCCCCcccucaauccugu
262


 


hsa-miR-296-3p
MIMAT0004679
GAGGGUUGgguggaggcucucc
263


 


hsa-miR-130b*
MIMAT0004680
ACUCUUUCccuguugcacuac
264


 


hsa-miR-130b
MIMAT0000691
CAGUGCAAugaugaaagggcau
265


 


hsa-miR-30e
MIMAT0000692
UGUAAACAuccuugacuggaag
266


 


hsa-miR-30e*
MIMAT0000693
CUUUCAGUcggauguuuacagc
267


 


hsa-miR-26a-2*
MIMAT0004681
CCUAUUCUugauuacuuguuuc
268


 


hsa-miR-361-5p
MIMAT0000703
UUAUCAGAaucuccagggguac
269


 


hsa-miR-361-3p
MIMAT0004682
UCCCCCAGgugugauucugauuu
270


 


hsa-miR-362-5p
MIMAT0000705
AAUCCUUGgaaccuaggugugagu
271


 


hsa-miR-362-3p
MIMAT0004683
AACACACCuauucaaggauuca
272


 


hsa-miR-363*
MIMAT0003385
CGGGUGGAucacgaugcaauuu
273


 


hsa-miR-363
MIMAT0000707
AAUUGCACgguauccaucugua
274


 


hsa-miR-365
MIMAT0000710
UAAUGCCCcuaaaaauccuuau
275


 


hsa-miR-365*
MIMAT0009199
AGGGACUUucaggggcagcugu
276


 


hsa-miR-302b*
MIMAT0000714
ACUUUAACauggaagugcuuuc
277


 


hsa-miR-302b
MIMAT0000715
UAAGUGCUuccauguuuuaguag
278


 


hsa-miR-302c*
MIMAT0000716
UUUAACAUggggguaccugcug
279


 


hsa-miR-302c
MIMAT0000717
UAAGUGCUuccauguuucagugg
280


 


hsa-miR-302d*
MIMAT0004685
ACUUUAACauggaggcacuugc
281


 


hsa-miR-302d
MIMAT0000718
UAAGUGCUuccauguuugagugu
282


 


hsa-miR-367*
MIMAT0004686
ACUGUUGCuaauaugcaacucu
283


 


hsa-miR-367
MIMAT0000719
AAUUGCACuuuagcaaugguga
284


 


hsa-miR-376c
MIMAT0000720
AACAUAGAggaaauuccacgu
285


 


hsa-miR-369-5p
MIMAT0001621
AGAUCGACcguguuauauucgc
286


 


hsa-miR-369-3p
MIMAT0000721
AAUAAUACaugguugaucuuu
287


 


hsa-miR-370
MIMAT0000722
GCCUGCUGggguggaaccuggu
288


 


hsa-miR-371-5p
MIMAT0004687
ACUCAAACugugggggcacu
289


 


hsa-miR-371-3p
MIMAT0000723
AAGUGCCGccaucuuuugagugu
290


 


hsa-miR-372
MIMAT0000724
AAAGUGCUgcgacauuugagcgu
291


 


hsa-miR-373*
MIMAT0000725
ACUCAAAAugggggcgcuuucc
292


 


hsa-miR-373
MIMAT0000726
GAAGUGCUucgauuuuggggugu
293


 


hsa-miR-374a
MIMAT0000727
UUAUAAUAcaaccugauaagug
294


 


hsa-miR-374a*
MIMAT0004688
CUUAUCAGauuguauuguaauu
295


 


hsa-miR-375
MIMAT0000728
UUUGUUCGuucggcucgcguga
296


 


hsa-miR-376a*
MIMAT0003386
GUAGAUUCuccuucuaugagua
297


 


hsa-miR-376a
MIMAT0000729
AUCAUAGAggaaaauccacgu
298


 


hsa-miR-377*
MIMAT0004689
AGAGGUUGcccuuggugaauuc
299


 


hsa-miR-377
MIMAT0000730
AUCACACAaaggcaacuuuugu
300


 


hsa-miR-378*
MIMAT0000731
CUCCUGACuccagguccugugu
301


 


hsa-miR-378
MIMAT0000732
ACUGGACUuggagucagaagg
302


 


hsa-miR-379
MIMAT0000733
UGGUAGACuauggaacguagg
303


 


hsa-miR-379*
MIMAT0004690
UAUGUAACaugguccacuaacu
304


 


hsa-miR-380*
MIMAT0000734
UGGUUGACcauagaacaugcgc
305


 


hsa-miR-380
MIMAT0000735
UAUGUAAUaugguccacaucuu
306


 


hsa-miR-381
MIMAT0000736
UAUACAAGggcaagcucucugu
307


 


hsa-miR-382
MIMAT0000737
GAAGUUGUucgugguggauucg
308


 


hsa-miR-383
MIMAT0000738
AGAUCAGAaggugauuguggcu
309


 


hsa-miR-340
MIMAT0004692
UUAUAAAGcaaugagacugauu
310


 


hsa-miR-340*
MIMAT0000750
UCCGUCUCaguuacuuuauagc
311


 


hsa-miR-330-5p
MIMAT0004693
UCUCUGGGccugugucuuaggc
312


 


hsa-miR-330-3p
MIMAT0000751
GCAAAGCAcacggccugcagaga
313


 


hsa-miR-328
MIMAT0000752
CUGGCCCUcucugcccuuccgu
314


 


hsa-miR-342-5p
MIMAT0004694
AGGGGUGCuaucugugauuga
315


 


hsa-miR-342-3p
MIMAT0000753
UCUCACACagaaaucgcacccgu
316


 


hsa-miR-337-5p
MIMAT0004695
GAACGGCUucauacaggaguu
317


 


hsa-miR-337-3p
MIMAT0000754
CUCCUAUAugaugccuuucuuc
318


 


hsa-miR-323-5p
MIMAT0004696
AGGUGGUCcguggcgcguucgc
319


 


hsa-miR-323-3p
MIMAT0000755
CACAUUACacggucgaccucu
320


 


hsa-miR-326
MIMAT0000756
CCUCUGGGcccuuccuccag
321


 


hsa-miR-151-5p
MIMAT0004697
UCGAGGAGcucacagucuagu
322


 


hsa-miR-151-3p
MIMAT0000757
CUAGACUGaagcuccuugagg
323


 


hsa-miR-135b
MIMAT0000758
UAUGGCUUuucauuccuauguga
324


 


hsa-miR-135b*
MIMAT0004698
AUGUAGGGcuaaaagccauggg
325


 


hsa-miR-148b*
MIMAT0004699
AAGUUCUGuuauacacucaggc
326


 


hsa-miR-148b
MIMAT0000759
UCAGUGCAucacagaacuuugu
327


 


hsa-miR-331-5p
MIMAT0004700
CUAGGUAUggucccagggaucc
328


 


hsa-miR-331-3p
MIMAT0000760
GCCCCUGGgccuauccuagaa
329


 


hsa-miR-324-5p
MIMAT0000761
CGCAUCCCcuagggcauuggugu
330


 


hsa-miR-324-3p
MIMAT0000762
ACUGCCCCaggugcugcugg
331


 


hsa-miR-338-5p
MIMAT0004701
AACAAUAUccuggugcugagug
332


 


hsa-miR-338-3p
MIMAT0000763
UCCAGCAUcagugauuuuguug
333


 


hsa-miR-339-5p
MIMAT0000764
UCCCUGUCcuccaggagcucacg
334


 


hsa-miR-339-3p
MIMAT0004702
UGAGCGCCucgacgacagagccg
335


 


hsa-miR-335
MIMAT0000765
UCAAGAGCaauaacgaaaaaugu
336


 


hsa-miR-335*
MIMAT0004703
UUUUUCAUuauugcuccugacc
337


 


hsa-miR-133b
MIMAT0000770
UUUGGUCCccuucaaccagcua
338


 


hsa-miR-325
MIMAT0000771
CCUAGUAGguguccaguaagugu
339


 


hsa-miR-345
MIMAT0000772
GCUGACUCcuaguccagggcuc
340


 


hsa-miR-346
MIMAT0000773
UGUCUGCCcgcaugccugccucu
341


 


hsa-miR-384
MIMAT0001075
AUUCCUAGaaauuguucaua
342


 


hsa-miR-196b
MIMAT0001080
UAGGUAGUuuccuguuguuggg
343


 


hsa-miR-196b*
MIMAT0009201
UCGACAGCacgacacugccuuc
344


 


hsa-miR-422a
MIMAT0001339
ACUGGACUuagggucagaaggc
345


 


hsa-miR-423-5p
MIMAT0004748
UGAGGGGCagagagcgagacuuu
346


 


hsa-miR-423-3p
MIMAT0001340
AGCUCGGUcugaggccccucagu
347


 


hsa-miR-424
MIMAT0001341
CAGCAGCAauucauguuuugaa
348


 


hsa-miR-424*
MIMAT0004749
CAAAACGUgaggcgcugcuau
349


 


hsa-miR-425
MIMAT0003393
AAUGACACgaucacucccguuga
350


 


hsa-miR-425*
MIMAT0001343
AUCGGGAAugucguguccgccc
351


 


hsa-miR-18b
MIMAT0001412
UAAGGUGCaucuagugcaguuag
352


 


hsa-miR-18b*
MIMAT0004751
UGCCCUAAaugccccuucuggc
353


 


hsa-miR-20b
MIMAT0001413
CAAAGUGCucauagugcagguag
354


 


hsa-miR-20b*
MIMAT0004752
ACUGUAGUaugggcacuuccag
355


 


hsa-miR-448
MIMAT0001532
UUGCAUAUguaggaugucccau
356


 


hsa-miR-429
MIMAT0001536
UAAUACUGucugguaaaaccgu
357


 


hsa-miR-449a
MIMAT0001541
UGGCAGUGuauuguuagcuggu
358


 


hsa-miR-450a
MIMAT0001545
UUUUGCGAuguguuccuaauau
359


 


hsa-miR-431
MIMAT0001625
UGUCUUGCaggccgucaugca
360


 


hsa-miR-431*
MIMAT0004757
CAGGUCGUcuugcagggcuucu
361


 


hsa-miR-433
MIMAT0001627
AUCAUGAUgggcuccucggugu
362


 


hsa-miR-329
MIMAT0001629
AACACACCugguuaaccucuuu
363


 


hsa-miR-451
MIMAT0001631
AAACCGUUaccauuacugaguu
364


 


hsa-miR-452
MIMAT0001635
AACUGUUUgcagaggaaacuga
365


 


hsa-miR-452*
MIMAT0001636
CUCAUCUGcaaagaaguaagug
366


 


hsa-miR-409-5p
MIMAT0001638
AGGUUACCcgagcaacuuugcau
367


 


hsa-miR-409-3p
MIMAT0001639
GAAUGUUGcucggugaaccccu
368


 


hsa-miR-412
MIMAT0002170
ACUUCACCugguccacuagccgu
369


 


hsa-miR-410
MIMAT0002171
AAUAUAACacagauggccugu
370


 


hsa-miR-376b
MIMAT0002172
AUCAUAGAggaaaauccauguu
371


 


hsa-miR-483-5p
MIMAT0004761
AAGACGGGaggaaagaagggag
372


 


hsa-miR-483-3p
MIMAT0002173
UCACUCCUcuccucccgucuu
373


 


hsa-miR-484
MIMAT0002174
UCAGGCUCaguccccucccgau
374


 


hsa-miR-485-5p
MIMAT0002175
AGAGGCUGgccgugaugaauuc
375


 


hsa-miR-485-3p
MIMAT0002176
GUCAUACAcggcucuccucucu
376


 


hsa-miR-486-5p
MIMAT0002177
UCCUGUACugagcugccccgag
377


 


hsa-miR-486-3p
MIMAT0004762
CGGGGCAGcucaguacaggau
378


 


hsa-miR-487a
MIMAT0002178
AAUCAUACagggacauccaguu
379


 


hsa-miR-488*
MIMAT0002804
CCCAGAUAauggcacucucaa
380


 


hsa-miR-488
MIMAT0004763
UUGAAAGGcuauuucuugguc
381


 


hsa-miR-489
MIMAT0002805
GUGACAUCacauauacggcagc
382


 


hsa-miR-490-5p
MIMAT0004764
CCAUGGAUcuccaggugggu
383


 


hsa-miR-490-3p
MIMAT0002806
CAACCUGGaggacuccaugcug
384


 


hsa-miR-491-5p
MIMAT0002807
AGUGGGGAacccuuccaugagg
385


 


hsa-miR-491-3p
MIMAT0004765
CUUAUGCAagauucccuucuac
386


 


hsa-miR-511
MIMAT0002808
GUGUCUUUugcucugcaguca
387


 


hsa-miR-146b-5p
MIMAT0002809
UGAGAACUgaauuccauaggcu
388


 


hsa-miR-146b-3p
MIMAT0004766
UGCCCUGUggacucaguucugg
389


 


hsa-miR-202*
MIMAT0002810
UUCCUAUGcauauacuucuuug
390


 


hsa-miR-202
MIMAT0002811
AGAGGUAUagggcaugggaa
391


 


hsa-miR-492
MIMAT0002812
AGGACCUGcgggacaagauucuu
392


 


hsa-miR-493*
MIMAT0002813
UUGUACAUgguaggcuuucauu
393


 


hsa-miR-493
MIMAT0003161
UGAAGGUCuacugugugccagg
394


 


hsa-miR-432
MIMAT0002814
UCUUGGAGuaggucauugggugg
395


 


hsa-miR-432*
MIMAT0002815
CUGGAUGGcuccuccaugucu
396


 


hsa-miR-494
MIMAT0002816
UGAAACAUacacgggaaaccuc
397


 


hsa-miR-495
MIMAT0002817
AAACAAACauggugcacuucuu
398


 


hsa-miR-496
MIMAT0002818
UGAGUAUUacauggccaaucuc
399


 


hsa-miR-193b*
MIMAT0004767
CGGGGUUUugagggcgagauga
400


 


hsa-miR-193b
MIMAT0002819
AACUGGCCcucaaagucccgcu
401


 


hsa-miR-497
MIMAT0002820
CAGCAGCAcacugugguuugu
402


 


hsa-miR-497*
MIMAT0004768
CAAACCACacugugguguuaga
403


 


hsa-miR-181d
MIMAT0002821
AACAUUCAuuguugucggugggu
404


 


hsa-miR-512-5p
MIMAT0002822
CACUCAGCcuugagggcacuuuc
405


 


hsa-miR-512-3p
MIMAT0002823
AAGUGCUGucauagcugagguc
406


 


hsa-miR-498
MIMAT0002824
UUUCAAGCcagggggcguuuuuc
407


 


hsa-miR-520e
MIMAT0002825
AAAGUGCUuccuuuuugaggg
408


 


hsa-miR-515-5p
MIMAT0002826
UUCUCCAAaagaaagcacuuucug
409


 


hsa-miR-515-3p
MIMAT0002827
GAGUGCCUucuuuuggagcguu
410


 


hsa-miR-519e*
MIMAT0002828
UUCUCCAAaagggagcacuuuc
411


 


hsa-miR-519e
MIMAT0002829
AAGUGCCUccuuuuagaguguu
412


 


hsa-miR-520f
MIMAT0002830
AAGUGCUUccuuuuagaggguu
413


 


hsa-miR-519c-5p
MIMAT0002831
CUCUAGAGggaagcgcuuucug
414


 


hsa-miR-519c-3p
MIMAT0002832
AAAGUGCAucuuuuuagaggau
415


 


hsa-miR-520a-5p
MIMAT0002833
CUCCAGAGggaaguacuuucu
416


 


hsa-miR-520a-3p
MIMAT0002834
AAAGUGCUucccuuuggacugu
417


 


hsa-miR-526b
MIMAT0002835
CUCUUGAGggaagcacuuucugu
418


 


hsa-miR-526b*
MIMAT0002836
GAAAGUGCuuccuuuuagaggc
419


 


hsa-miR-519b-5p
MIMAT0005454
CUCUAGAGggaagcgcuuucug
414


 


hsa-miR-519b-3p
MIMAT0002837
AAAGUGCAuccuuuuagagguu
420


 


hsa-miR-525-5p
MIMAT0002838
CUCCAGAGggaugcacuuucu
421


 


hsa-miR-525-3p
MIMAT0002839
GAAGGCGCuucccuuuagagcg
422


 


hsa-miR-523*
MIMAT0005449
CUCUAGAGggaagcgcuuucug
414


 


hsa-miR-523
MIMAT0002840
GAACGCGCuucccuauagagggu
423


 


hsa-miR-518f*
MIMAT0002841
CUCUAGAGggaagcacuuucuc
424


 


hsa-miR-518f
MIMAT0002842
GAAAGCGCuucucuuuagagg
425


 


hsa-miR-520b
MIMAT0002843
AAAGUGCUuccuuuuagaggg
426


 


hsa-miR-518b
MIMAT0002844
CAAAGCGCuccccuuuagaggu
427


 


hsa-miR-526a
MIMAT0002845
CUCUAGAGggaagcacuuucug
428


 


hsa-miR-520c-5p
MIMAT0005455
CUCUAGAGggaagcacuuucug
428


 


hsa-miR-520c-3p
MIMAT0002846
AAAGUGCUuccuuuuagagggu
429


 


hsa-miR-518c*
MIMAT0002847
UCUCUGGAgggaagcacuuucug
430


 


hsa-miR-518c
MIMAT0002848
CAAAGCGCuucucuuuagagugu
431


 


hsa-miR-524-5p
MIMAT0002849
CUACAAAGggaagcacuuucuc
432


 


hsa-miR-524-3p
MIMAT0002850
GAAGGCGCuucccuuuggagu
433


 


hsa-miR-517*
MIMAT0002851
CCUCUAGAuggaagcacugucu
434


 


hsa-miR-517a
MIMAT0002852
AUCGUGCAucccuuuagagugu
435


 


hsa-miR-519d
MIMAT0002853
CAAAGUGCcucccuuuagagug
436


 


hsa-miR-521
MIMAT0002854
AACGCACUucccuuuagagugu
437


 


hsa-miR-520d-5p
MIMAT0002855
CUACAAAGggaagcccuuuc
438


 


hsa-miR-520d-3p
MIMAT0002856
AAAGUGCUucucuuuggugggu
439


 


hsa-miR-517b
MIMAT0002857
UCGUGCAUcccuuuagaguguu
440


 


hsa-miR-520g
MIMAT0002858
ACAAAGUGcuucccuuuagagugu
441


 


hsa-miR-516b
MIMAT0002859
AUCUGGAGguaagaagcacuuu
442


 


hsa-miR-516b*
MIMAT0002860
UGCUUCCUuucagagggu
443


 


hsa-miR-518e*
MIMAT0005450
CUCUAGAGggaagcgcuuucug
414


 


hsa-miR-518e
MIMAT0002861
AAAGCGCUucccuucagagug
444


 


hsa-miR-518a-5p
MIMAT0005457
CUGCAAAGggaagcccuuuc
445


 


hsa-miR-518a-3p
MIMAT0002863
GAAAGCGCuucccuuugcugga
446


 


hsa-miR-518d-5p
MIMAT0005456
CUCUAGAGggaagcacuuucug
428


 


hsa-miR-518d-3p
MIMAT0002864
CAAAGCGCuucccuuuggagc
447


 


hsa-miR-517c
MIMAT0002866
AUCGUGCAuccuuuuagagugu
448


 


hsa-miR-520h
MIMAT0002867
ACAAAGUGcuucccuuuagagu
449


 


hsa-miR-522*
MIMAT0005451
CUCUAGAGggaagcgcuuucug
414


 


hsa-miR-522
MIMAT0002868
AAAAUGGUucccuuuagagugu
450


 


hsa-miR-519a*
MIMAT0005452
CUCUAGAGggaagcgcuuucug
414


 


hsa-miR-519a
MIMAT0002869
AAAGUGCAuccuuuuagagugu
451


 


hsa-miR-527
MIMAT0002862
CUGCAAAGggaagcccuuuc
445


 


hsa-miR-516a-5p
MIMAT0004770
UUCUCGAGgaaagaagcacuuuc
452


 


hsa-miR-516a-3p
MIMAT0006778
UGCUUCCUuucagagggu
443


 


hsa-miR-499-5p
MIMAT0002870
UUAAGACUugcagugauguuu
453


 


hsa-miR-499-3p
MIMAT0004772
AACAUCACagcaagucugugcu
454


 


hsa-miR-500
MIMAT0004773
UAAUCCUUgcuaccugggugaga
455


 


hsa-miR-500*
MIMAT0002871
AUGCACCUgggcaaggauucug
456


 


hsa-miR-501-5p
MIMAT0002872
AAUCCUUUgucccugggugaga
457


 


hsa-miR-501-3p
MIMAT0004774
AAUGCACCcgggcaaggauucu
458


 


hsa-miR-502-5p
MIMAT0002873
AUCCUUGCuaucugggugcua
459


 


hsa-miR-502-3p
MIMAT0004775
AAUGCACCugggcaaggauuca
460


 


hsa-miR-503
MIMAT0002874
UAGCAGCGggaacaguucugcag
461


 


hsa-miR-504
MIMAT0002875
AGACCCUGgucugcacucuauc
462


 


hsa-miR-505*
MIMAT0004776
GGGAGCCAggaaguauugaugu
463


 


hsa-miR-505
MIMAT0002876
CGUCAACAcuugcugguuuccu
464


 


hsa-miR-513a-5p
MIMAT0002877
UUCACAGGgaggugucau
465


 


hsa-miR-513a-3p
MIMAT0004777
UAAAUUUCaccuuucugagaagg
466


 


hsa-miR-506
MIMAT0002878
UAAGGCACccuucugaguaga
467


 


hsa-miR-507
MIMAT0002879
UUUUGCACcuuuuggagugaa
468


 


hsa-miR-508-5p
MIMAT0004778
UACUCCAGagggcgucacucaug
469


 


hsa-miR-508-3p
MIMAT0002880
UGAUUGUAgccuuuuggaguaga
470


 


hsa-miR-509-5p
MIMAT0004779
UACUGCAGacaguggcaauca
471


 


hsa-miR-509-3p
MIMAT0002881
UGAUUGGUacgucuguggguag
472


 


hsa-miR-510
MIMAT0002882
UACUCAGGagaguggcaaucac
473


 


hsa-miR-514
MIMAT0002883
AUUGACACuucugugaguaga
474


 


hsa-miR-532-5p
MIMAT0002888
CAUGCCUUgaguguaggaccgu
475


 


hsa-miR-532-3p
MIMAT0004780
CCUCCCACacccaaggcuugca
476


 


hsa-miR-455-5p
MIMAT0003150
UAUGUGCCuuuggacuacaucg
477


 


hsa-miR-455-3p
MIMAT0004784
GCAGUCCAugggcauauacac
478


 


hsa-miR-539
MIMAT0003163
GGAGAAAUuauccuuggugugu
479


 


hsa-miR-544
MIMAT0003164
AUUCUGCAuuuuuagcaaguuc
480


 


hsa-miR-545*
MIMAT0004785
UCAGUAAAuguuuauuagauga
481


 


hsa-miR-545
MIMAT0003165
UCAGCAAAcauuuauugugugc
482


 


hsa-miR-487b
MIMAT0003180
AAUCGUACagggucauccacuu
483


 


hsa-miR-551a
MIMAT0003214
GCGACCCAcucuugguuucca
484


 


hsa-miR-552
MIMAT0003215
AACAGGUGacugguuagacaa
485


 


hsa-miR-553
MIMAT0003216
AAAACGGUgagauuuuguuuu
486


 


hsa-miR-554
MIMAT0003217
GCUAGUCCugacucagccagu
487


 


hsa-miR-92b*
MIMAT0004792
AGGGACGGgacgcggugcagug
488


 


hsa-miR-92b
MIMAT0003218
UAUUGCACucgucccggccucc
489


 


hsa-miR-555
MIMAT0003219
AGGGUAAGcugaaccucugau
490


 


hsa-miR-556-5p
MIMAT0003220
GAUGAGCUcauuguaauaugag
491


 


hsa-miR-556-3p
MIMAT0004793
AUAUUACCauuagcucaucuuu
492


 


hsa-miR-557
MIMAT0003221
GUUUGCACgggugggccuugucu
493


 


hsa-miR-558
MIMAT0003222
UGAGCUGCuguaccaaaau
494


 


hsa-miR-559
MIMAT0003223
UAAAGUAAauaugcaccaaaa
495


 


hsa-miR-561
MIMAT0003225
CAAAGUUUaagauccuugaagu
496


 


hsa-miR-562
MIMAT0003226
AAAGUAGCuguaccauuugc
497


 


hsa-miR-563
MIMAT0003227
AGGUUGACauacguuuccc
498


 


hsa-miR-564
MIMAT0003228
AGGCACGGugucagcaggc
499


 


hsa-miR-566
MIMAT0003230
GGGCGCCUgugaucccaac
500


 


hsa-miR-567
MIMAT0003231
AGUAUGUUcuuccaggacagaac
501


 


hsa-miR-568
MIMAT0003232
AUGUAUAAauguauacacac
502


 


hsa-miR-552b*
MIMAT0004794
GAAAUCAAgcgugggugagacc
503


 


hsa-miR-551b
MIMAT0003233
GCGACCCAuacuugguuucag
504


 


hsa-miR-569
MIMAT0003234
AGUUAAUGaauccuggaaagu
505


 


hsa-miR-570
MIMAT0003235
CGAAAACAgcaauuaccuuugc
506


 


hsa-miR-571
MIMAT0003236
UGAGUUGGccaucugagugag
507


 


hsa-miR-572
MIMAT0003237
GUCCGCUCggcgguggccca
508


 


hsa-miR-573
MIMAT0003238
CUGAAGUGauguguaacugaucag
509


 


hsa-miR-574-5p
MIMAT0004795
UGAGUGUGugugugugagugugu
510


 


hsa-miR-574-3p
MIMAT0003239
CACGCUCAugcacacacccaca
511


 


hsa-miR-575
MIMAT0003240
GAGCCAGUuggacaggagc
512


 


hsa-miR-576-5p
MIMAT0003241
AUUCUAAUuucuccacgucuuu
513


 


hsa-miR-576-3p
MIMAT0004796
AAGAUGUGgaaaaauuggaauc
514


 


hsa-miR-577
MIMAT0003242
UAGAUAAAauauugguaccug
515


 


hsa-miR-578
MIMAT0003243
CUUCUUGUgcucuaggauugu
516


 


hsa-miR-579
MIMAT0003244
UUCAUUUGguauaaaccgcgauu
517


 


hsa-miR-580
MIMAT0003245
UUGAGAAUgaugaaucauuagg
518


 


hsa-miR-581
MIMAT0003246
UCUUGUGUucucuagaucagu
519


 


hsa-miR-582-5p
MIMAT0003247
UUACAGUUguucaaccaguuacu
520


 


hsa-miR-582-3p
MIMAT0004797
UAACUGGUugaacaacugaacc
521


 


hsa-miR-583
MIMAT0003248
CAAAGAGGaaggucccauuac
522


 


hsa-miR-584
MIMAT0003249
UUAUGGUUugccugggacugag
523


 


hsa-miR-585
MIMAT0003250
UGGGCGUAucuguaugcua
524


 


hsa-miR-548a-3p
MIMAT0003251
CAAAACUGgcaauuacuuuugc
525


 


hsa-miR-586
MIMAT0003252
UAUGCAUUguauuuuuaggucc
526


 


hsa-miR-587
MIMAT0003253
UUUCCAUAggugaugagucac
527


 


hsa-miR-548b-5p
MIMAT0004798
AAAAGUAAuugugguuuuggcc
528


 


hsa-miR-548b-3p
MIMAT0003254
CAAGAACCucaguugcuuuugu
529


 


hsa-miR-588
MIMAT0003255
UUGGCCACaauggguuagaac
530


 


hsa-miR-589
MIMAT0004799
UGAGAACCacgucugcucugag
531


 


hsa-miR-589*
MIMAT0003256
UCAGAACAaaugccgguucccaga
532


 


hsa-miR-550
MIMAT0004800
AGUGCCUGagggaguaagagccc
533


 


hsa-miR-550*
MIMAT0003257
UGUCUUACucccucaggcacau
534


 


hsa-miR-590-5p
MIMAT0003258
GAGCUUAUucauaaaagugcag
535


 


hsa-miR-590-3p
MIMAT0004801
UAAUUUUAuguauaagcuagu
536


 


hsa-miR-591
MIMAT0003259
AGACCAUGgguucucauugu
537


 


hsa-miR-592
MIMAT0003260
UUGUGUCAauaugcgaugaugu
538


 


hsa-miR-593*
MIMAT0003261
AGGCACCAgccaggcauugcucagc
539


 


hsa-miR-593
MIMAT0004802
UGUCUCUGcugggguuucu
540


 


hsa-miR-595
MIMAT0003263
GAAGUGUGccguggugugucu
541


 


hsa-miR-596
MIMAT0003264
AAGCCUGCccggcuccucggg
542


 


hsa-miR-597
MIMAT0003265
UGUGUCACucgaugaccacugu
543


 


hsa-miR-598
MIMAT0003266
UACGUCAUcguugucaucguca
544


 


hsa-miR-599
MIMAT0003267
GUUGUGUCaguuuaucaaac
545


 


hsa-miR-548a-5p
MIMAT0004803
AAAAGUAAuugcgaguuuuacc
546


 


hsa-miR-600
MIMAT0003268
ACUUACAGacaagagccuugcuc
547


 


hsa-miR-601
MIMAT0003269
UGGUCUAGgauuguuggaggag
548


 


hsa-miR-602
MIMAT0003270
GACACGGGcgacagcugcggccc
549


 


hsa-miR-603
MIMAT0003271
CACACACUgcaauuacuuuugc
550


 


hsa-miR-604
MIMAT0003272
AGGCUGCGgaauucaggac
551


 


hsa-miR-605
MIMAT0003273
UAAAUCCCauggugccuucuccu
552


 


hsa-miR-606
MIMAT0003274
AAACUACUgaaaaucaaagau
553


 


hsa-miR-607
MIMAT0003275
GUUCAAAUccagaucuauaac
554


 


hsa-miR-608
MIMAT0003276
AGGGGUGGuguugggacagcuccgu
555


 


hsa-miR-609
MIMAT0003277
AGGGUGUUucucucaucucu
556


 


hsa-miR-610
MIMAT0003278
UGAGCUAAaugugugcuggga
557


 


hsa-miR-611
MIMAT0003279
GCGAGGACcccucggggucugac
558


 


hsa-miR-612
MIMAT0003280
GCUGGGCAgggcuucugagcuccuu
559


 


hsa-miR-613
MIMAT0003281
AGGAAUGUuccuucuuugcc
560


 


hsa-miR-614
MIMAT0003282
GAACGCCUguucuugccaggugg
561


 


hsa-miR-615-5p
MIMAT0004804
GGGGGUCCccggugcucggauc
562


 


hsa-miR-615-3p
MIMAT0003283
UCCGAGCCugggucucccucuu
563


 


hsa-miR-616*
MIMAT0003284
ACUCAAAAcccuucagugacuu
564


 


hsa-miR-616
MIMAT0004805
AGUCAUUGgaggguuugagcag
565


 


hsa-miR-548c-5p
MIMAT0004806
AAAAGUAAuugcgguuuuugcc
566


 


hsa-miR-548c-3p
MIMAT0003285
CAAAAAUCucaauuacuuuugc
567


 


hsa-miR-617
MIMAT0003286
AGACUUCCcauuugaagguggc
568


 


hsa-miR-618
MIMAT0003287
AAACUCUAcuuguccuucugagu
569


 


hsa-miR-619
MIMAT0003288
GACCUGGAcauguuugugcccagu
570


 


hsa-miR-620
MIMAT0003289
AUGGAGAUagauauagaaau
571


 


hsa-miR-621
MIMAT0003290
GGCUAGCAacagcgcuuaccu
572


 


hsa-miR-622
MIMAT0003291
ACAGUCUGcugagguuggagc
573


 


hsa-miR-623
MIMAT0003292
AUCCCUUGcaggggcuguugggu
574


 


hsa-miR-624*
MIMAT0003293
UAGUACCAguaccuuguguuca
575


 


hsa-miR-624
MIMAT0004807
CACAAGGUauugguauuaccu
576


 


hsa-miR-625
MIMAT0003294
AGGGGGAAaguucuauagucc
577


 


hsa-miR-625*
MIMAT0004808
GACUAUAGaacuuucccccuca
578


 


hsa-miR-626
MIMAT0003295
AGCUGUCUgaaaaugucuu
579


 


hsa-miR-627
MIMAT0003296
GUGAGUCUcuaagaaaagagga
580


 


hsa-miR-628-5p
MIMAT0004809
AUGCUGACauauuuacuagagg
581


 


hsa-miR-628-3p
MIMAT0003297
UCUAGUAAgaguggcagucga
582


 


hsa-miR-629
MIMAT0004810
UGGGUUUAcguugggagaacu
583


 


hsa-miR-629*
MIMAT0003298
GUUCUCCCaacguaagcccagc
584


 


hsa-miR-630
MIMAT0003299
AGUAUUCUguaccagggaaggu
585


 


hsa-miR-631
MIMAT0003300
AGACCUGGcccagaccucagc
586


 


hsa-miR-33b
MIMAT0003301
GUGCAUUGcuguugcauugc
587


 


hsa-miR-33b*
MIMAT0004811
CAGUGCCUcggcagugcagccc
588


 


hsa-miR-632
MIMAT0003302
GUGUCUGCuuccuguggga
589


 


hsa-miR-633
MIMAT0003303
CUAAUAGUaucuaccacaauaaa
590


 


hsa-miR-634
MIMAT0003304
AACCAGCAccccaacuuuggac
591


 


hsa-miR-635
MIMAT0003305
ACUUGGGCacugaaacaaugucc
592


 


hsa-miR-636
MIMAT0003306
UGUGCUUGcucgucccgcccgca
593


 


hsa-miR-637
MIMAT0003307
ACUGGGGGcuuucgggcucugcgu
594


 


hsa-miR-638
MIMAT0003308
AGGGAUCGcgggcggguggcggccu
595


 


hsa-miR-639
MIMAT0003309
AUCGCUGCgguugcgagcgcugu
596


 


hsa-miR-640
MIMAT0003310
AUGAUCCAggaaccugccucu
597


 


hsa-miR-641
MIMAT0003311
AAAGACAUaggauagagucaccuc
598


 


hsa-miR-642
MIMAT0003312
GUCCCUCUccaaaugugucuug
599


 


hsa-miR-643
MIMAT0003313
ACUUGUAUgcuagcucagguag
600


 


hsa-miR-644
MIMAT0003314
AGUGUGGCuuucuuagagc
601


 


hsa-miR-645
MIMAT0003315
UCUAGGCUgguacugcuga
602


 


hsa-miR-646
MIMAT0003316
AAGCAGCUgccucugaggc
603


 


hsa-miR-647
MIMAT0003317
GUGGCUGCacucacuuccuuc
604


 


hsa-miR-648
MIMAT0003318
AAGUGUGCagggcacuggu
605


 


hsa-miR-649
MIMAT0003319
AAACCUGUguuguucaagaguc
606


 


hsa-miR-650
MIMAT0003320
AGGAGGCAgcgcucucaggac
607


 


hsa-miR-651
MIMAT0003321
UUUAGGAUaagcuugacuuuug
608


 


hsa-miR-652
MIMAT0003322
AAUGGCGCcacuaggguugug
609


 


hsa-miR-548d-5p
MIMAT0004812
AAAAGUAAuugugguuuuugcc
610


 


hsa-miR-548d-3p
MIMAT0003323
CAAAAACCacaguuucuuuugc
611


 


hsa-miR-661
MIMAT0003324
UGCCUGGGucucuggccugcgcgu
612


 


hsa-miR-662
MIMAT0003325
UCCCACGUuguggcccagcag
613


 


hsa-miR-663
MIMAT0003326
AGGCGGGGcgccgcgggaccgc
614


 


hsa-miR-449b
MIMAT0003327
AGGCAGUGuauuguuagcuggc
615


 


hsa-miR-449b*
MIMAT0009203
CAGCCACAacuacccugccacu
616


 


hsa-miR-653
MIMAT0003328
GUGUUGAAacaaucucuacug
617


 


hsa-miR-411
MIMAT0003329
UAGUAGACcguauagcguacg
618


 


hsa-miR-411*
MIMAT0004813
UAUGUAACacgguccacuaacc
619


 


hsa-miR-654-5p
MIMAT0003330
UGGUGGGCcgcagaacaugugc
620


 


hsa-miR-654-3p
MIMAT0004814
UAUGUCUGcugaccaucaccuu
621


 


hsa-miR-655
MIMAT0003331
AUAAUACAugguuaaccucuuu
622


 


hsa-miR-656
MIMAT0003332
AAUAUUAUacagucaaccucu
623


 


hsa-miR-549
MIMAT0003333
UGACAACUauggaugagcucu
624


 


hsa-miR-657
MIMAT0003335
GGCAGGUUcucacccucucuagg
625


 


hsa-miR-658
MIMAT0003336
GGCGGAGGgaaguagguccguuggu
626


 


hsa-miR-659
MIMAT0003337
CUUGGUUCagggagggucccca
627


 


hsa-miR-660
MIMAT0003338
UACCCAUUgcauaucggaguug
628


 


hsa-miR-421
MIMAT0003339
AUCAACAGacauuaauugggcgc
629


 


hsa-miR-542-5p
MIMAT0003340
UCGGGGAUcaucaugucacgaga
630


 


hsa-miR-542-3p
MIMAT0003389
UGUGACAGauugauaacugaaa
631


 


hsa-miR-758
MIMAT0003879
UUUGUGACcugguccacuaacc
632


 


hsa-miR-1264
MIMAT0005791
CAAGUCUUauuugagcaccuguu
633


 


hsa-miR-671-5p
MIMAT0003880
AGGAAGCCcuggaggggcuggag
634


 


hsa-miR-671-3p
MIMAT0004819
UCCGGUUCucagggcuccacc
635


 


hsa-miR-668
MIMAT0003881
UGUCACUCggcucggcccacuac
636


 


hsa-miR-767-5p
MIMAT0003882
UGCACCAUgguugucugagcaug
637


 


hsa-miR-767-3p
MIMAT0003883
UCUGCUCAuaccccaugguuucu
638


 


hsa-miR-1224-5p
MIMAT0005458
GUGAGGACucgggaggugg
639


 


hsa-miR-1224-3p
MIMAT0005459
CCCCACCUccucucuccucag
640


 


hsa-miR-320b
MIMAT0005792
AAAAGCUGgguugagagggcaa
641


 


hsa-miR-320c
MIMAT0005793
AAAAGCUGgguugagagggu
642


 


hsa-miR-1296
MIMAT0005794
UUAGGGCCcuggcuccaucucc
643


 


hsa-miR-1468
MIMAT0006789
CUCCGUUUgccuguuucgcug
644


 


hsa-miR-1323
MIMAT0005795
UCAAAACUgaggggcauuuucu
645


 


hsa-miR-1271
MIMAT0005796
CUUGGCACcuagcaagcacuca
646


 


hsa-miR-1301
MIMAT0005797
UUGCAGCUgccugggagugacuuc
647


 


hsa-miR-454*
MIMAT0003884
ACCCUAUCaauauugucucugc
648


 


hsa-miR-454
MIMAT0003885
UAGUGCAAuauugcuuauagggu
649


 


hsa-miR-1185
MIMAT0005798
AGAGGAUAcccuuuguauguu
650


 


hsa-miR-449c
MIMAT0010251
UAGGCAGUguauugcuagcggcugu
651


 


hsa-miR-449c*
MIMAT0013771
UUGCUAGUugcacuccucucugu
652


 


hsa-miR-1283
MIMAT0005799
UCUACAAAggaaagcgcuuucu
653


 


hsa-miR-769-5p
MIMAT0003886
UGAGACCUcuggguucugagcu
654


 


hsa-miR-769-3p
MIMAT0003887
CUGGGAUCuccggggucuugguu
655


 


hsa-miR-766
MIMAT0003888
ACUCCAGCcccacagccucagc
656


 


hsa-miR-762
MIMAT0010313
GGGGCUGGggccggggccgagc
657


 


hsa-miR-802
MIMAT0004185
CAGUAACAaagauucauccuugu
658


 


hsa-miR-670
MIMAT0010357
GUCCCUGAguguauguggug
659


 


hsa-miR-1298
MIMAT0005800
UUCAUUCGgcuguccagaugua
660


 


hsa-miR-2113
MIMAT0009206
AUUUGUGCuuggcucugucac
661


 


hsa-miR-761
MIMAT0010364
GCAGCAGGgugaaacugacaca
662


 


hsa-miR-764
MIMAT0010367
GCAGGUGCucacuuguccuccu
663


 


hsa-miR-759
MIMAT0010497
GCAGAGUGcaaacaauuuugac
664


 


hsa-miR-765
MIMAT0003945
UGGAGGAGaaggaaggugaug
665


 


hsa-miR-770-5p
MIMAT0003948
UCCAGUACcacgugucagggcca
666


 


hsa-miR-675
MIMAT0004284
UGGUGCGGagagggcccacagug
667


 


hsa-miR-675*
MIMAT0006790
CUGUAUGCccucaccgcuca
668


 


hsa-miR-298
MIMAT0004901
AGCAGAAGcagggagguucuccca
669


 


hsa-miR-891a
MIMAT0004902
UGCAACGAaccugagccacuga
670


 


hsa-miR-300
MIMAT0004903
UAUACAAGggcagacucucucu
671


 


hsa-miR-886-5p
MIMAT0004905
CGGGUCGGaguuagcucaagcgg
672


 


hsa-miR-886-3p
MIMAT0004906
CGCGGGUGcuuacugacccuu
673


 


hsa-miR-892a
MIMAT0004907
CACUGUGUccuuucugcguag
674


 


hsa-miR-220b
MIMAT0004908
CCACCACCgugucugacacuu
675


 


hsa-miR-450b-5p
MIMAT0004909
UUUUGCAAuauguuccugaaua
676


 


hsa-miR-450b-3p
MIMAT0004910
UUGGGAUCauuuugcauccaua
677


 


hsa-miR-874
MIMAT0004911
CUGCCCUGgcccgagggaccga
678


 


hsa-miR-890
MIMAT0004912
UACUUGGAaaggcaucaguug
679


 


hsa-miR-891b
MIMAT0004913
UGCAACUUaccugagucauuga
680


 


hsa-miR-220c
MIMAT0004915
ACACAGGGcuguugugaagacu
681


 


hsa-miR-888
MIMAT0004916
UACUCAAAaagcugucaguca
682


 


hsa-miR-888*
MIMAT0004917
GACUGACAccucuuugggugaa
683


 


hsa-miR-892b
MIMAT0004918
CACUGGCUccuuucuggguaga
684


 


hsa-miR-541*
MIMAT0004919
AAAGGAUUcugcugucggucccacu
685


 


hsa-miR-541
MIMAT0004920
UGGUGGGCacagaaucuggacu
686


 


hsa-miR-889
MIMAT0004921
UUAAUAUCggacaaccauugu
687


 


hsa-miR-875-5p
MIMAT0004922
UAUACCUCaguuuuaucaggug
688


 


hsa-miR-875-3p
MIMAT0004923
CCUGGAAAcacugagguugug
689


 


hsa-miR-876-5p
MIMAT0004924
UGGAUUUCuuugugaaucacca
690


 


hsa-miR-876-3p
MIMAT0004925
UGGUGGUUuacaaaguaauuca
691


 


hsa-miR-708
MIMAT0004926
AAGGAGCUuacaaucuagcuggg
692


 


hsa-miR-708*
MIMAT0004927
CAACUAGAcugugagcuucuag
693


 


hsa-miR-147b
MIMAT0004928
GUGUGCGGaaaugcuucugcua
694


 


hsa-miR-190b
MIMAT0004929
UGAUAUGUuugauauuggguu
695


 


hsa-miR-744
MIMAT0004945
UGCGGGGCuagggcuaacagca
696


 


hsa-miR-744*
MIMAT0004946
CUGUUGCCacuaaccucaaccu
697


 


hsa-miR-885-5p
MIMAT0004947
UCCAUUACacuacccugccucu
698


 


hsa-miR-885-3p
MIMAT0004948
AGGCAGCGggguguaguggaua
699


 


hsa-miR-877
MIMAT0004949
GUAGAGGAgauggcgcaggg
700


 


hsa-miR-877*
MIMAT0004950
UCCUCUUCucccuccucccag
701


 


hsa-miR-887
MIMAT0004951
GUGAACGGgcgccaucccgagg
702


 


hsa-miR-665
MIMAT0004952
ACCAGGAGgcugaggccccu
703


 


hsa-miR-873
MIMAT0004953
GCAGGAACuugugagucuccu
704


 


hsa-miR-543
MIMAT0004954
AAACAUUCgcggugcacuucuu
705


 


hsa-miR-374b
MIMAT0004955
AUAUAAUAcaaccugcuaagug
706


 


hsa-miR-374b*
MIMAT0004956
CUUAGCAGguuguauuaucauu
707


 


hsa-miR-760
MIMAT0004957
CGGCUCUGggucugugggga
708


 


hsa-miR-301b
MIMAT0004958
CAGUGCAAugauauugucaaagc
709


 


hsa-miR-216b
MIMAT0004959
AAAUCUCUgcaggcaaauguga
710


 


hsa-miR-208b
MIMAT0004960
AUAAGACGaacaaaagguuugu
711


 


hsa-miR-920
MIMAT0004970
GGGGAGCUguggaagcagua
712


 


hsa-miR-921
MIMAT0004971
CUAGUGAGggacagaaccaggauuc
713


 


hsa-miR-922
MIMAT0004972
GCAGCAGAgaauaggacuacguc
714


 


hsa-miR-924
MIMAT0004974
AGAGUCUUgugaugucuugc
715


 


hsa-miR-509-3-5p
MIMAT0004975
UACUGCAGacguggcaaucaug
716


 


hsa-miR-933
MIMAT0004976
UGUGCGCAgggagaccucuccc
717


 


hsa-miR-934
MIMAT0004977
UGUCUACUacuggagacacugg
718


 


hsa-miR-935
MIMAT0004978
CCAGUUACcgcuuccgcuaccgc
719


 


hsa-miR-936
MIMAT0004979
ACAGUAGAgggaggaaucgcag
720


 


hsa-miR-937
MIMAT0004980
AUCCGCGCucugacucucugcc
721


 


hsa-miR-938
MIMAT0004981
UGCCCUUAaaggugaacccagu
722


 


hsa-miR-939
MIMAT0004982
UGGGGAGCugaggcucugggggug
723


 


hsa-miR-940
MIMAT0004983
AAGGCAGGgcccccgcucccc
724


 


hsa-miR-941
MIMAT0004984
CACCCGGCugugugcacaugugc
725


 


hsa-miR-942
MIMAT0004985
UCUUCUCUguuuuggccaugug
726


 


hsa-miR-943
MIMAT0004986
CUGACUGUugccguccuccag
727


 


hsa-miR-944
MIMAT0004987
AAAUUAUUguacaucggaugag
728


 


hsa-miR-297
MIMAT0004450
AUGUAUGUgugcaugugcaug
729


 


hsa-miR-1178
MIMAT0005823
UUGCUCACuguucuucccuag
730


 


hsa-miR-1179
MIMAT0005824
AAGCAUUCuuucauugguugg
731


 


hsa-miR-1180
MIMAT0005825
UUUCCGGCucgcgugggugugu
732


 


hsa-miR-1181
MIMAT0005826
CCGUCGCCgccacccgagccg
733


 


hsa-miR-1182
MIMAT0005827
GAGGGUCUugggagggaugugac
734


 


hsa-miR-1183
MIMAT0005828
CACUGUAGgugauggugagagugggca
735


 


hsa-miR-1184
MIMAT0005829
CCUGCAGCgacuugauggcuucc
736


 


hsa-miR-1225-5p
MIMAT0005572
GUGGGUACggcccagugggggg
737


 


hsa-miR-1225-3p
MIMAT0005573
UGAGCCCCugugccgcccccag
738


 


hsa-miR-1226*
MIMAT0005576
GUGAGGGCaugcaggccuggaugggg
739


 


hsa-miR-1226
MIMAT0005577
UCACCAGCccuguguucccuag
740


 


hsa-miR-1227
MIMAT0005580
CGUGCCACccuuuuccccag
741


 


hsa-miR-1228*
MIMAT0005582
GUGGGCGGgggcaggugugug
742


 


hsa-miR-1228
MIMAT0005583
UCACACCUgccucgcccccc
743


 


hsa-miR-1229
MIMAT0005584
CUCUCACCacugcccucccacag
744


 


hsa-miR-1231
MIMAT0005586
GUGUCUGGgcggacagcugc
745


 


hsa-miR-1233
MIMAT0005588
UGAGCCCUguccucccgcag
746


 


hsa-miR-1234
MIMAT0005589
UCGGCCUGaccacccaccccac
747


 


hsa-miR-1236
MIMAT0005591
CCUCUUCCccuugucucuccag
748


 


hsa-miR-1237
MIMAT0005592
UCCUUCUGcuccgucccccag
749


 


hsa-miR-1238
MIMAT0005593
CUUCCUCGucugucugcccc
750


 


hsa-miR-1200
MIMAT0005863
CUCCUGAGccauucugagccuc
751


 


hsa-miR-1201
MIMAT0005864
AGCCUGAUuaaacacaugcucuga
752


 


hsa-miR-1202
MIMAT0005865
GUGCCAGCugcagugggggag
753


 


hsa-miR-1203
MIMAT0005866
CCCGGAGCcaggaugcagcuc
754


 


hsa-miR-663b
MIMAT0005867
GGUGGCCCggccgugccugagg
755


 


hsa-miR-1204
MIMAT0005868
UCGUGGCCuggucuccauuau
756


 


hsa-miR-1205
MIMAT0005869
UCUGCAGGguuugcuuugag
757


 


hsa-miR-1206
MIMAT0005870
UGUUCAUGuagauguuuaagc
758


 


hsa-miR-1207-5p
MIMAT0005871
UGGCAGGGaggcugggagggg
759


 


hsa-miR-1207-3p
MIMAT0005872
UCAGCUGGcccucauuuc
760


 


hsa-miR-1208
MIMAT0005873
UCACUGUUcagacaggcgga
761


 


hsa-miR-548e
MIMAT0005874
AAAAACUGagacuacuuuugca
762


 


hsa-miR-548j
MIMAT0005875
AAAAGUAAuugcggucuuuggu
763


 


hsa-miR-1285
MIMAT0005876
UCUGGGCAacaaagugagaccu
764


 


hsa-miR-1286
MIMAT0005877
UGCAGGACcaagaugagcccu
765


 


hsa-miR-1287
MIMAT0005878
UGCUGGAUcagugguucgaguc
766


 


hsa-miR-1289
MIMAT0005879
UGGAGUCCaggaaucugcauuuu
767


 


hsa-miR-1290
MIMAT0005880
UGGAUUUUuggaucaggga
768


 


hsa-miR-1291
MIMAT0005881
UGGCCCUGacugaagaccagcagu
769


 


hsa-miR-548k
MIMAT0005882
AAAAGUACuugcggauuuugcu
770


 


hsa-miR-1293
MIMAT0005883
UGGGUGGUcuggagauuugugc
771


 


hsa-miR-1294
MIMAT0005884
UGUGAGGUuggcauuguugucu
772


 


hsa-miR-1295
MIMAT0005885
UUAGGCCGcagaucuggguga
773


 


hsa-miR-1297
MIMAT0005886
UUCAAGUAauucaggug
774


 


hsa-miR-1299
MIMAT0005887
UUCUGGAAuucugugugaggga
775


 


hsa-miR-5481
MIMAT0005889
AAAAGUAUuugcggguuuuguc
776


 


hsa-miR-1302
MIMAT0005890
UUGGGACAuacuuaugcuaaa
777


 


hsa-miR-1303
MIMAT0005891
UUUAGAGAcggggucuugcucu
778


 


hsa-miR-1304
MIMAT0005892
UUUGAGGCuacagugagaugug
779


 


hsa-miR-1305
MIMAT0005893
UUUUCAACucuaaugggagaga
780


 


hsa-miR-1243
MIMAT0005894
AACUGGAUcaauuauaggagug
781


 


hsa-miR-548f
MIMAT0005895
AAAAACUGuaauuacuuuu
782


 


hsa-miR-1244
MIMAT0005896
AAGUAGUUgguuuguaugagaugguu
783


 


hsa-miR-1245
MIMAT0005897
AAGUGAUCuaaaggccuacau
784


 


hsa-miR-1246
MIMAT0005898
AAUGGAUUuuuggagcagg
785


 


hsa-miR-1247
MIMAT0005899
ACCCGUCCcguucguccccgga
786


 


hsa-miR-1248
MIMAT0005900
ACCUUCUUguauaagcacugugcuaaa
787


 


hsa-miR-1249
MIMAT0005901
ACGCCCUUcccccccuucuuca
788


 


hsa-miR-1250
MIMAT0005902
ACGGUGCUggauguggccuuu
789


 


hsa-miR-1251
MIMAT0005903
ACUCUAGCugccaaaggcgcu
790


 


hsa-miR-1253
MIMAT0005904
AGAGAAGAagaucagccugca
791


 


hsa-miR-1254
MIMAT0005905
AGCCUGGAagcuggagccugcagu
792


 


hsa-miR-1255a
MIMAT0005906
AGGAUGAGcaaagaaaguagauu
793


 


hsa-miR-1256
MIMAT0005907
AGGCAUUGacuucucacuagcu
794


 


hsa-miR-1257
MIMAT0005908
AGUGAAUGauggguucugacc
795


 


hsa-miR-1258
MIMAT0005909
AGUUAGGAuuaggucguggaa
796


 


hsa-miR-1259
MIMAT0005910
AUAUAUGAugacuuagcuuuu
797


 


hsa-miR-1260
MIMAT0005911
AUCCCACCucugccacca
798


 


hsa-miR-548g
MIMAT0005912
AAAACUGUaauuacuuuuguac
799


 


hsa-miR-1261
MIMAT0005913
AUGGAUAAggcuuuggcuu
800


 


hsa-miR-1262
MIMAT0005914
AUGGGUGAauuuguagaaggau
801


 


hsa-miR-1263
MIMAT0005915
AUGGUACCcuggcauacugagu
802


 


hsa-miR-548n
MIMAT0005916
CAAAAGUAauuguggauuuugu
803


 


hsa-miR-548m
MIMAT0005917
CAAAGGUAuuugugguuuuug
804


 


hsa-miR-1265
MIMAT0005918
CAGGAUGUggucaaguguuguu
805


 


hsa-miR-548o
MIMAT0005919
CCAAAACUgcaguuacuuuugc
806


 


hsa-miR-1266
MIMAT0005920
CCUCAGGGcuguagaacagggcu
807


 


hsa-miR-1267
MIMAT0005921
CCUGUUGAaguguaaucccca
808


 


hsa-miR-1268
MIMAT0005922
CGGGCGUGgugguggggg
809


 


hsa-miR-1269
MIMAT0005923
CUGGACUGagccgugcuacugg
810


 


hsa-miR-1270
MIMAT0005924
CUGGAGAUauggaagagcugugu
811


 


hsa-miR-1272
MIMAT0005925
GAUGAUGAuggcagcaaauucugaaa
812


 


hsa-miR-1273
MIMAT0005926
GGGCGACAaagcaagacucuuucuu
813


 


hsa-miR-1274a
MIMAT0005927
GUCCCUGUucaggcgcca
814


 


hsa-miR-548h
MIMAT0005928
AAAAGUAAucgcgguuuuuguc
815


 


hsa-miR-1275
MIMAT0005929
GUGGGGGAgaggcuguc
816


 


hsa-miR-1276
MIMAT0005930
UAAAGAGCccuguggagaca
817


 


hsa-miR-302e
MIMAT0005931
UAAGUGCUuccaugcuu
818


 


hsa-miR-302f
MIMAT0005932
UAAUUGCUuccauguuu
819


 


hsa-miR-1277
MIMAT0005933
UACGUAGAuauauauguauuuu
820


 


hsa-miR-548p
MIMAT0005934
UAGCAAAAacugcaguuacuuu
821


 


hsa-miR-548i
MIMAT0005935
AAAAGUAAuugcggauuuugcc
822


 


hsa-miR-1278
MIMAT0005936
UAGUACUGugcauaucaucuau
823


 


hsa-miR-1279
MIMAT0005937
UCAUAUUGcuucuuucu
824


 


hsa-miR-1274b
MIMAT0005938
UCCCUGUUcgggcgcca
825


 


hsa-miR-1281
MIMAT0005939
UCGCCUCCuccucuccc
826


 


hsa-miR-1282
MIMAT0005940
UCGUUUGCcuuuuucugcuu
827


 


hsa-miR-1284
MIMAT0005941
UCUAUACAgacccuggcuuuuc
828


 


hsa-miR-1288
MIMAT0005942
UGGACUGCccugaucuggaga
829


 


hsa-miR-1292
MIMAT0005943
UGGGAACGgguuccggcagacgcug
830


 


hsa-miR-1252
MIMAT0005944
AGAAGGAAauugaauucauuua
831


 


hsa-miR-1255b
MIMAT0005945
CGGAUGAGcaaagaaagugguu
832


 


hsa-miR-1280
MIMAT0005946
UCCCACCGcugccaccc
833


 


hsa-miR-1308
MIMAT0005947
GCAUGGGUgguucagugg
834


 


hsa-miR-664*
MIMAT0005948
ACUGGCUAgggaaaaugauuggau
835


 


hsa-miR-664
MIMAT0005949
UAUUCAUUuauccccagccuaca
836


 


hsa-miR-1306
MIMAT0005950
ACGUUGGCucugguggug
837


 


hsa-miR-1307
MIMAT0005951
ACUCGGCGuggcgucggucgug
838


 


hsa-miR-513b
MIMAT0005788
UUCACAAGgaggugucauuuau
839


 


hsa-miR-513c
MIMAT0005789
UUCUCAAGgaggugucguuuau
840


 


hsa-miR-1321
MIMAT0005952
CAGGGAGGugaaugugau
841


 


hsa-miR-1322
MIMAT0005953
GAUGAUGCugcugaugcug
842


 


hsa-miR-720
MIMAT0005954
UCUCGCUGgggccucca
843


 


hsa-miR-1197
MIMAT0005955
UAGGACACauggucuacuucu
844


 


hsa-miR-1324
MIMAT0005956
CCAGACAGaauucuaugcacuuuc
845


 


hsa-miR-1469
MIMAT0007347
CUCGGCGCggggcgcgggcucc
846


 


hsa-miR-1470
MIMAT0007348
GCCCUCCGcccgugcaccccg
847


 


hsa-miR-1471
MIMAT0007349
GCCCGCGUguggagccaggugu
848


 


hsa-miR-1537
MIMAT0007399
AAAACCGUcuaguuacaguugu
849


 


hsa-miR-1538
MIMAT0007400
CGGCCCGGgcugcugcuguuccu
850


 


hsa-miR-1539
MIMAT0007401
UCCUGCGCgucccagaugccc
851


 


hsa-miR-103-as
MIMAT0007402
UCAUAGCCcuguacaaugcugcu
852


 


hsa-miR-320d
MIMAT0006764
AAAAGCUGgguugagagga
853


 


hsa-miR-1825
MIMAT0006765
UCCAGUGCccuccucucc
854


 


hsa-miR-1826
MIMAT0006766
AUUGAUCAucgacacuucgaacgcaau
855


 


hsa-miR-1827
MIMAT0006767
UGAGGCAGuagauugaau
856


 


hsa-miR-1908
MIMAT0007881
CGGCGGGGacggcgauugguc
857


 


hsa-miR-1909*
MIMAT0007882
UGAGUGCCggugccugcccug
858


 


hsa-miR-1909
MIMAT0007883
CGCAGGGGccgggugcucaccg
859


 


hsa-miR-1910
MIMAT0007884
CCAGUCCUgugccugccgccu
860


 


hsa-miR-1911
MIMAT0007885
UGAGUACCgccaugucuguuggg
861


 


hsa-miR-1911*
MIMAT0007886
CACCAGGCauuguggucucc
862


 


hsa-miR-1912
MIMAT0007887
UACCCAGAgcaugcagugugaa
863


 


hsa-miR-1913
MIMAT0007888
UCUGCCCCcuccgcugcugcca
864


 


hsa-miR-1914
MIMAT0007889
CCCUGUGCccggcccacuucug
865


 


hsa-miR-1914*
MIMAT0007890
GGAGGGGUcccgcacugggagg
866


 


hsa-miR-1915*
MIMAT0007891
ACCUUGCCuugcugcccgggcc
867


 


hsa-miR-1915
MIMAT0007892
CCCCAGGGcgacgcggcggg
868


 


hsa-miR-1972
MIMAT0009447
UCAGGCCAggcacaguggcuca
869


 


hsa-miR-1973
MIMAT0009448
ACCGUGCAaagguagcaua
870


 


hsa-miR-1975
MIMAT0009450
CCCCCACAaccgcgcuugacuagcu
871


 


hsa-miR-1976
MIMAT0009451
CCUCCUGCccuccuugcugu
872


 


hsa-miR-1979
MIMAT0009454
CUCCCACUgcuucacuugacua
873


 


hsa-miR-2052
MIMAT0009977
UGUUUUGAuaacaguaaugu
874


 


hsa-miR-2053
MIMAT0009978
GUGUUAAUuaaaccucuauuuac
875


 


hsa-miR-2054
MIMAT0009979
CUGUAAUAuaaauuuaauuuauu
876


 


hsa-miR-2110
MIMAT0010133
UUGGGGAAacggccgcugagug
877


 


hsa-miR-2114
MIMAT0011156
UAGUCCCUuccuugaagcgguc
878


 


hsa-miR-2114*
MIMAT0011157
CGAGCCUCaagcaagggacuu
879


 


hsa-miR-2115
MIMAT0011158
AGCUUCCAugacuccugaugga
880


 


hsa-miR-2115*
MIMAT0011159
CAUCAGAAuucauggaggcuag
881


 


hsa-miR-2116
MIMAT0011160
GGUUCUUAgcauaggaggucu
882


 


hsa-miR-2116*
MIMAT0011161
CCUCCCAUgccaagaacuccc
883


 


hsa-miR-2117
MIMAT0011162
UGUUCUCUuugccaaggacag
884


 


hsa-miR-548q
MIMAT0011163
GCUGGUGCaaaaguaauggcgg
885


 


hsa-miR-2276
MIMAT0011775
UCUGCAAGugucagaggcgagg
886


 


hsa-miR-2277
MIMAT0011777
UGACAGCGcccugccuggcuc
887


 


hsa-miR-2278
MIMAT0011778
GAGAGCAGuguguguugccugg
888


 


hsa-miR-711
MIMAT0012734
GGGACCCAgggagagacguaag
889


 


hsa-miR-718
MIMAT0012735
CUUCCGCCccgccgggcgucg
890


 


hsa-miR-2861
MIMAT0013802
GGGGCCUGgcggugggcgg
891


 


hsa-miR-2909
MIMAT0013863
GUUAGGGCcaacaucucuugg
892


 


hsa-miR-3115
MIMAT0014977
AUAUGGGUuuacuaguuggu
893


 


hsa-miR-3116
MIMAT0014978
UGCCUGGAacauaguagggacu
894


 


hsa-miR-3117
MIMAT0014979
AUAGGACUcauauagugccag
895


 


hsa-miR-3118
MIMAT0014980
UGUGACUGcauuaugaaaauucu
896


 


hsa-miR-3119
MIMAT0014981
UGGCUUUUaacuuugauggc
897


 


hsa-miR-3120
MIMAT0014982
CACAGCAAguguagacaggca
898


 


hsa-miR-3121
MIMAT0014983
UAAAUAGAguaggcaaaggaca
899


 


hsa-miR-3122
MIMAT0014984
GUUGGGACaagaggacggucuu
900


 


hsa-miR-3123
MIMAT0014985
CAGAGAAUuguuuaauc
901


 


hsa-miR-3124
MIMAT0014986
UUCGCGGGcgaaggcaaaguc
902


 


hsa-miR-548s
MIMAT0014987
AUGGCCAAaacugcaguuauuuu
903


 


hsa-miR-3125
MIMAT0014988
UAGAGGAAgcuguggagaga
904


 


hsa-miR-3126-5p
MIMAT0014989
UGAGGGACagaugccagaagca
905


 


hsa-miR-3126-3p
MIMAT0015377
CAUCUGGCauccgucacacaga
906


 


hsa-miR-3127
MIMAT0014990
AUCAGGGCuuguggaaugggaag
907


 


hsa-miR-3128
MIMAT0014991
UCUGGCAAguaaaaaacucucau
908


 


hsa-miR-3129
MIMAT0014992
GCAGUAGUguagagauugguuu
909


 


hsa-miR-3130-5p
MIMAT0014995
UACCCAGUcuccggugcagcc
910


 


hsa-miR-3130-3p
MIMAT0014994
GCUGCACCggagacuggguaa
911


 


hsa-miR-3131
MIMAT0014996
UCGAGGACugguggaagggccuu
912


 


hsa-miR-3132
MIMAT0014997
UGGGUAGAgaaggagcucagagga
913


 


hsa-miR-3133
MIMAT0014998
UAAAGAACucuuaaaacccaau
914


 


hsa-miR-378b
MIMAT0014999
ACUGGACUuggaggcagaa
915


 


hsa-miR-3134
MIMAT0015000
UGAUGGAUaaaagacuacauauu
916


 


hsa-miR-3135
MIMAT0015001
UGCCUAGGcugagacugcagug
917


 


hsa-miR-466
MIMAT0015002
AUACACAUacacgcaacacacau
918


 


hsa-miR-3136
MIMAT0015003
CUGACUGAauagguagggucauu
919


 


hsa-miR-544b
MIMAT0015004
ACCUGAGGuugugcauuucuaa
920


 


hsa-miR-3137
MIMAT0015005
UCUGUAGCcugggagcaauggggu
921


 


hsa-miR-3138
MIMAT0015006
UGUGGACAgugagguagagggagu
922


 


hsa-miR-3139
MIMAT0015007
UAGGAGCUcaacagaugccuguu
923


 


hsa-miR-3140
MIMAT0015008
AGCUUUUGggaauucagguagu
924


 


hsa-miR-548t
MIMAT0015009
CAAAAGUGaucgugguuuuug
925


 


hsa-miR-3141
MIMAT0015010
GAGGGCGGguggaggagga
926


 


hsa-miR-3142
MIMAT0015011
AAGGCCUUucugaaccuucaga
927


 


hsa-miR-3143
MIMAT0015012
AUAACAUUguaaagcgcuucuuucg
928


 


hsa-miR-548u
MIMAT0015013
CAAAGACUgcaauuacuuuugcg
929


 


hsa-miR-3144-5p
MIMAT0015014
AGGGGACCaaagagauauauag
930


 


hsa-miR-3144-3p
MIMAT0015015
AUAUACCUguucggucucuuua
931


 


hsa-miR-3145
MIMAT0015016
AGAUAUUUugaguguuuggaauug
932


 


hsa-miR-1273c
MIMAT0015017
GGCGACAAaacgagacccuguc
933


 


hsa-miR-3146
MIMAT0015018
CAUGCUAGgauagaaagaaugg
934


 


hsa-miR-3147
MIMAT0015019
GGUUGGGCagugaggaggguguga
935


 


hsa-miR-548v
MIMAT0015020
AGCUACAGuuacuuuugcacca
936


 


hsa-miR-3148
MIMAT0015021
UGGAAAAAacuggugugugcuu
937


 


hsa-miR-3149
MIMAT0015022
UUUGUAUGgauauguguguguau
938


 


hsa-miR-3150
MIMAT0015023
CUGGGGAGauccucgagguugg
939


 


hsa-miR-3151
MIMAT0015024
GGUGGGGCaaugggaucaggu
940


 


hsa-miR-3152
MIMAT0015025
UGUGUUAGaauaggggcaauaa
941


 


hsa-miR-3153
MIMAT0015026
GGGGAAAGcgaguagggacauuu
942


 


hsa-miR-3074
MIMAT0015027
GAUAUCAGcucaguaggcaccg
943


 


hsa-miR-3154
MIMAT0015028
CAGAAGGGgaguugggagcaga
944


 


hsa-miR-3155
MIMAT0015029
CCAGGCUCugcagugggaacu
945


 


hsa-miR-3156
MIMAT0015030
AAAGAUCUggaagugggagaca
946


 


hsa-miR-3157
MIMAT0015031
UUCAGCCAggcuagugcagucu
947


 


hsa-miR-3158
MIMAT0015032
AAGGGCUUccucucugcaggac
948


 


hsa-miR-3159
MIMAT0015033
UAGGAUUAcaagugucggccac
949


 


hsa-miR-3160
MIMAT0015034
AGAGCUGAgacuagaaagccca
950


 


hsa-miR-3161
MIMAT0015035
CUGAUAAGaacagaggcccagau
951


 


hsa-miR-3162
MIMAT0015036
UUAGGGAGuagaaggguggggag
952


 


hsa-miR-3163
MIMAT0015037
UAUAAAAUgagggcaguaagac
953


 


hsa-miR-3164
MIMAT0015038
UGUGACUUuaagggaaauggcg
954


 


hsa-miR-3165
MIMAT0015039
AGGUGGAUgcaaugugaccuca
955


 


hsa-miR-3166
MIMAT0015040
CGCAGACAaugccuacuggccua
956


 


hsa-miR-1260b
MIMAT0015041
AUCCCACCacugccaccau
957


 


hsa-miR-3167
MIMAT0015042
AGGAUUUCagaaauacuggugu
958


 


hsa-miR-3168
MIMAT0015043G
AGUUCUAcagucagac
959


 


hsa-miR-3169
MIMAT0015044
UAGGACUGugcuuggcacauag
960


 


hsa-miR-3170
MIMAT0015045
CUGGGGUUcugagacagacagu
961


 


hsa-miR-3171
MIMAT0015046
AGAUGUAUggaaucuguauauauc
962


 


hsa-miR-3172
MIMAT0015047
UGGGGUUUugcaguccuua
963


 


hsa-miR-3173
MIMAT0015048
AAAGGAGGaaauaggcaggcca
964


 


hsa-miR-1193
MIMAT0015049
GGGAUGGUagaccggugacgugc
965


 


hsa-miR-323b-5p
MIMAT0001630
AGGUUGUCcguggugaguucgca
966


 


hsa-miR-323b-3p
MIMAT0015050
CCCAAUACacggucgaccucuu
967


 


hsa-miR-3174
MIMAT0015051
UAGUGAGUuagagaugcagagcc
968


 


hsa-miR-3175
MIMAT0015052
CGGGGAGAgaacgcagugacgu
969


 


hsa-miR-3176
MIMAT0015053
ACUGGCCUgggacuaccgg
970


 


hsa-miR-3177
MIMAT0015054
UGCACGGCacuggggacacgu
971


 


hsa-miR-3178
MIMAT0015055
GGGGCGCGgccggaucg
972


 


hsa-miR-3179
MIMAT0015056
AGAAGGGGugaaauuuaaacgu
973


 


hsa-miR-3180-5p
MIMAT0015057
CUUCCAGAcgcuccgccccacgucg
974


 


hsa-miR-3180-3p
MIMAT0015058
UGGGGCGGagcuuccggaggcc
975


 


hsa-miR-548w
MIMAT0015060
AAAAGUAAcugcgguuuuugccu
976


 


hsa-miR-3181
MIMAT0015061
AUCGGGCCcucggcgccgg
977


 


hsa-miR-3182
MIMAT0015062
GCUUCUGUaguguaguc
978


 


hsa-miR-3183
MIMAT0015063
GCCUCUCUcggagucgcucgga
979


 


hsa-miR-3184
MIMAT0015064
UGAGGGGCcucagaccgagcuuuu
980


 


hsa-miR-3185
MIMAT0015065
AGAAGAAGgcggucggucugcgg
981


 


hsa-miR-3065-5p
MIMAT0015066
UCAACAAAaucacugaugcugga
982


 


hsa-miR-3065-3p
MIMAT0015378
UCAGCACCaggauauuguuggag
983


 


hsa-miR-3186-5p
MIMAT0015067
CAGGCGUCugucuacguggcuu
984


 


hsa-miR-3186-3p
MIMAT0015068
UCACGCGGagagauggcuuug
985


 


hsa-miR-3187
MIMAT0015069
UUGGCCAUggggcugcgcgg
986


 


hsa-miR-3188
MIMAT0015070
AGAGGCUUugugcggauacgggg
987


 


hsa-miR-3189
MIMAT0015071
CCCUUGGGucugaugggguag
988


 


hsa-miR-320e
MIMAT0015072
AAAGCUGGguugagaagg
989


 


hsa-miR-3190-5p
MIMAT0015073
UGUGGAAGguagacggccagaga
990


 


hsa-miR-3190-3p
MIMAT0015074
UGGAAGGUagacggccagagag
991


 


hsa-miR-3191
MIMAT0015075
UGGGGACGuagcuggccagacag
992


 


hsa-miR-3192
MIMAT0015076
UCUGGGAGguuguagcaguggaa
993


 


hsa-miR-3193
MIMAT0015077
UCCUGCGUaggaucugaggagu
994


 


hsa-miR-3194
MIMAT0015078
GGCCAGCCaccaggagggcug
995


 


hsa-miR-3195
MIMAT0015079
CGCGCCGGgcccggguu
996


 


hsa-miR-3196
MIMAT0015080
CGGGGCGGcaggggccuc
997


 


hsa-miR-548x
MIMAT0015081
UAAAAACUgcaauuacuuuca
998


 


hsa-miR-3197
MIMAT0015082
GGAGGCGCaggcucggaaaggcg
999


 


hsa-miR-3198
MIMAT0015083
GUGGAGUCcuggggaauggaga
1000


 


hsa-miR-3199
MIMAT0015084
AGGGACUGccuuaggagaaaguu
1001


 


hsa-miR-3200
MIMAT0015085
CACCUUGCgcuacucaggucug
1002


 


hsa-miR-3201
MIMAT0015086
GGGAUAUGaagaaaaau
1003


 


hsa-miR-514b-5p
MIMAT0015087
UUCUCAAGagggaggcaaucau
1004


 


hsa-miR-514b-3p
MIMAT0015088
AUUGACACcucugugagugga
1005


 


hsa-miR-3202
MIMAT0015089
UGGAAGGGagaagagcuuuaau
1006


 


hsa-miR-1273d
MIMAT0015090
GAACCCAUgagguugaggcugcagu
1007


 


hsa-miR-4295
MIMAT0016844
CAGUGCAAuguuuuccuu
1008


 


hsa-miR-4296
MIMAT0016845
AUGUGGGCucaggcuca
1009


 


hsa-miR-4297
MIMAT0016846
UGCCUUCCugucugug
1010


 


hsa-miR-378c
MIMAT0016847
ACUGGACUuggagucagaagagugg
1011


 


hsa-miR-4293
MIMAT0016848
CAGCCUGAcaggaacag
1012


 


hsa-miR-4294
MIMAT0016849
GGGAGUCUacagcaggg
1013


 


hsa-miR-4301
MIMAT0016850
UCCCACUAcuucacuuguga
1014


 


hsa-miR-4299
MIMAT0016851
GCUGGUGAcaugagaggc
1015


 


hsa-miR-4298
MIMAT0016852
CUGGGACAggaggaggaggcag
1016


 


hsa-miR-4300
MIMAT0016853
UGGGAGCUggacuacuuc
1017


 


hsa-miR-4304
MIMAT0016854
CCGGCAUGuccagggca
1018


 


hsa-miR-4302
MIMAT0016855
CCAGUGUGgcucagcgag
1019


 


hsa-miR-4303
MIMAT0016856
UUCUGAGCugaggacag
1020


 


hsa-miR-4305
MIMAT0016857
CCUAGACAccuccaguuc
1021


 


hsa-miR-4306
MIMAT0016858
UGGAGAGAaaggcagua
1022


 


hsa-miR-4309
MIMAT0016859
CUGGAGUCuaggauucca
1023


 


hsa-miR-4307
MIMAT0016860
AAUGUUUUuuccuguuucc
1024


 


hsa-miR-4308
MIMAT0016861
UCCCUGGAguuucuucuu
1025


 


hsa-miR-4310
MIMAT0016862
GCAGCAUUcauguccc
1026


 


hsa-miR-4311
MIMAT0016863
GAAAGAGAgcugagugug
1027


 


hsa-miR-4312
MIMAT0016864
GGCCUUGUuccugucccca
1028


 


hsa-miR-4313
MIMAT0016865
AGCCCCCUggccccaaaccc
1029


 


hsa-miR-4315
MIMAT0016866
CCGCUUUCugagcuggac
1030


 


hsa-miR-4316
MIMAT0016867
GGUGAGGCuagcuggug
1031


 


hsa-miR-4314
MIMAT0016868
CUCUGGGAaaugggacag
1032


 


hsa-miR-4318
MIMAT0016869
CACUGUGGguacaugcu
1033


 


hsa-miR-4319
MIMAT0016870
UCCCUGAGcaaagccac
1034


 


hsa-miR-4320
MIMAT0016871
GGGAUUCUguagcuuccu
1035


 


hsa-miR-4317
MIMAT0016872
ACAUUGCCagggaguuu
1036


 


hsa-miR-4322
MIMAT0016873
CUGUGGGCucagcgcgugggg
1037


 


hsa-miR-4321
MIMAT0016874
UUAGCGGUggaccgcccugcg
1038


 


hsa-miR-4323
MIMAT0016875
CAGCCCCAcagccucaga
1039


 


hsa-miR-4324
MIMAT0016876
CCCUGAGAcccuaaccuuaa
1040


 


hsa-miR-4256
MIMAT0016877
AUCUGACCugaugaaggu
1041


 


hsa-miR-4257
MIMAT0016878
CCAGAGGUggggacugag
1042


 


hsa-miR-4258
MIMAT0016879
CCCCGCCAccgccuugg
1043


 


hsa-miR-4259
MIMAT0016880
CAGUUGGGucuaggggucagga
1044


 


hsa-miR-4260
MIMAT0016881
CUUGGGGCauggaguccca
1045


 


hsa-miR-4253
MIMAT0016882
AGGGCAUGuccagggggu
1046


 


hsa-miR-4251
MIMAT0016883
CCUGAGAAaagggccaa
1047


 


hsa-miR-4254
MIMAT0016884
GCCUGGAGcuacuccaccaucuc
1048


 


hsa-miR-4255
MIMAT0016885
CAGUGUUCagagaugga
1049


 


hsa-miR-4252
MIMAT0016886
GGCCACUGagucagcacca
1050


 


hsa-miR-4325
MIMAT0016887
UUGCACUUgucucaguga
1051


 


hsa-miR-4326
MIMAT0016888
UGUUCCUCugucucccagac
1052


 


hsa-miR-4327
MIMAT0016889
GGCUUGCAugggggacugg
1053


 


hsa-miR-4261
MIMAT0016890
AGGAAACAgggaccca
1054


 


hsa-miR-4265
MIMAT0016891
CUGUGGGCucagcucuggg
1055


 


hsa-miR-4266
MIMAT0016892
CUAGGAGGccuuggcc
1056


 


hsa-miR-4267
MIMAT0016893
UCCAGCUCgguggcac
1057


 


hsa-miR-4262
MIMAT0016894
GACAUUCAgacuaccug
1058


 


hsa-miR-2355
MIMAT0016895
AUCCCCAGauacaauggacaa
1059


 


hsa-miR-4268
MIMAT0016896
GGCUCCUCcucucaggaugug
1060


 


hsa-miR-4269
MIMAT0016897
GCAGGCACagacagcccuggc
1061


 


hsa-miR-4263
MIMAT0016898
AUUCUAAGugccuuggcc
1062


 


hsa-miR-4264
MIMAT0016899
ACUCAGUCauggucauu
1063


 


hsa-miR-4270
MIMAT0016900
UCAGGGAGucaggggagggc
1064


 


hsa-miR-4271
MIMAT0016901
GGGGGAAGaaaaggugggg
1065


 


hsa-miR-4272
MIMAT0016902
CAUUCAACuagugauugu
1066


 


hsa-miR-4273
MIMAT0016903
GUGUUCUCugauggacag
1067


 


hsa-miR-4276
MIMAT0016904
CUCAGUGAcucaugugc
1068


 


hsa-miR-4275
MIMAT0016905
CCAAUUACcacuucuuu
1069


 


hsa-miR-4274
MIMAT0016906
CAGCAGUCccucccccug
1070


 


hsa-miR-4281
MIMAT0016907
GGGUCCCGgggagggggg
1071


 


hsa-miR-4277
MIMAT0016908
GCAGUUCUgagcacaguacac
1072


 


hsa-miR-4279
MIMAT0016909
CUCUCCUCccggcuuc
1073


 


hsa-miR-4278
MIMAT0016910
CUAGGGGGuuugcccuug
1074


 


hsa-miR-4280
MIMAT0016911
GAGUGUAGuucugagcagagc
1075


 


hsa-miR-4282
MIMAT0016912
UAAAAUUUgcauccagga
1076


 


hsa-miR-4285
MIMAT0016913
GCGGCGAGuccgacucau
1077


 


hsa-miR-4283
MIMAT0016914
UGGGGCUCagcgaguuu
1078


 


hsa-miR-4284
MIMAT0016915
GGGCUCACaucaccccau
1079


 


hsa-miR-4286
MIMAT0016916
ACCCCACUccugguacc
1080


 


hsa-miR-4287
MIMAT0016917
UCUCCCUUgagggcacuuu
1081


 


hsa-miR-4288
MIMAT0016918
UUGUCUGCugaguuucc
1082


 


hsa-miR-4292
MIMAT0016919
CCCCUGGGccggccuugg
1083


 


hsa-miR-4289
MIMAT0016920
GCAUUGUGcagggcuauca
1084


 


hsa-miR-4290
MIMAT0016921
UGCCCUCCuuucuucccuc
1085


 


hsa-miR-4291
MIMAT0016922
UUCAGCAGgaacagcu
1086


 


hsa-miR-4329
MIMAT0016923
CCUGAGACccuaguuccac
1087


 


hsa-miR-4330
MIMAT0016924
CCUCAGAUcagagccuugc
1088


 


hsa-miR-500b
MIMAT0016925
AAUCCUUGcuaccugggu
1089


 


hsa-miR-4328
MIMAT0016926
CCAGUUUUcccaggauu
1090









EXAMPLES
Example 1
Segmented miRNA Mimetics
MicroRNAs (miRNAs) are a class of  ̃22 nt noncoding RNAs that play important roles in regulating gene expression in plants and animals. MicroRNAs are usually produced by a process in which a RNA pol II transcript is cut by Drosha to produce a precursor hairpin, which is cut by Dicer in the cytoplasm to produce a two stranded duplex that is incorporated into Argonaute (Ago) proteins. After elimination of the passenger strand by cleavage or helicase activity, the guide strand can then bind to complementary target RNAs. Studies have found that the most prevalent aspect of miRNA target recognition is complementary binding to a target 3′ UTR by the miRNA seed region (positions 2 through 8 at the 5′ end of the guide strand), leading to downregulation at mRNA and protein levels.
Ago2 mediated cleavage of the passenger strand has been found to be important for assembly of siRNAs and some miRNAs and a nicked passenger strand was found to rescue the activity of an siRNA containing a phosphorothioate bond that prevented passenger strand cleavage by Ago. This concept has been applied to the design of siRNAs (Bramsen et al., 2007 supra), where passenger segmentation was found to maintain siRNA activity (while eliminating passenger strand activity), while guide segmentation was found to eliminate the desired siRNA activity.
Applicant has surprisingly found that, as opposed to siRNA, segmentation is well tolerated in the guide strand of microRNA. Applicant demonstrates herein that this segmentation provides for the alternative design of various miRNA mimetics that include nicks and gaps, as well as substitutions and insertions that can confer additional properties toward therapeutic use.
Materials and Methods
Oligonucleotides used to obtain data in this Example were synthesized at Sigma-Aldrich or Merck & Co. using standard methodologies. Annealing was accomplished by mixing single stranded RNA at 10 uM in 10 mM TrisHCl/50 mM NaCl and heating at 95° C. for 2 minutes before slowly cooling to 37° C. over the course of 1 hour.
The RNA oligonucleotides synthesized are shown in the following Table II.









TABLE II 





Name
Sequence
SEQ ID NO.







G
UUAAGGCACGCGGUGAAUGCCA
1091


 


P
GCAUUCACCGCGUGCCUUAAAU
1092


 


G10
UUAAGGCACG
1093


 


G.10
CGGUGAAUGCCA
1094


 


G.9
GGUGAAUGCCA
1095


 


P10
GCAUUCACCG
1096


 


P.10
CGUGCCUUAAAU
1097


 


G10i
UUAAGGCACG-iB
1098


 


G10Cy3
UUAAGGCACG-Cy3
1099


 


58-mer
GCGUUCACCGCGGACCUUGAUUUAAAUGU
1100



CCAUACAAUUAAGGCACGCGGUGAAUGCC



 


48-mer
GCGUUCACCGCGGACCUUGAUUUAAAUGU
1101



CCAUACAAUUAAGGCACGCGGUGAAUGCC



 


10-mer
GGUGAAUGCC
1102









Sequences in the table above are shown in 5′ to 3′ orientation. “iB” denotes an inverted abasic, while “Cy3” denotes a Cy3 fluorescent dye molecule.
HCT-116 cells were cultured in McCoy's 5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, plated in 96-well culture plates at a density of 25,000 cells/well 24 hours prior to transfection, and then transfected using Opti-MEM I Reduced Serum Media (Gibco) and Lipofectamine 2000 (Invitrogen) with a final concentration of our miRNAs ranging from 30 nM down to 0.01 nM along a 12-point titration curve. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline and processed using the TaqMan® Gene Expression Cells-to-CTTM Kit (Applied Biosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCR using CD164-specific probes (Applied Biosystems) on an ABI Prism 7900HT Sequence Detector. Reverse transcription conditions were as follows: 60 minutes at 37° C. followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used for data normalization.
miRNAs were co-transfected with siCHECK2 vectors (Genscript) containing cloned target inserts, as shown in FIG. 14, consisting of a tandem repeat of a seed match to miR-124 (2×7a), a seed match containing additional 3′ complementarity to positions 13-17 of miR-124 (2×7a3p), or a full-length match to miR-124 (2×FL). A seed match with a two-base mutation (2×7aMutant) was used as a control. Twenty-four hours after transfection, transfection medium was replaced with fresh growth medium. Forty-eight hours after transfection, cells were lysed and both firefly and Renilla luciferase activity were measured using the Dual-GloTM Luciferase Assay System (Promega) on a Wallac EnVision 2103 Multilabel Reader (PerkinElmer).
HCT-116 cells were transfected with 10 nM miRNA duplex as described previously (Jackson et al 2006). RNA was extracted using RNeasy (Qiagen), amplified using the Ovation protocol (Nugen), and profiled on custom Affymetrix arrays (Rosetta Custom Human 1.0, Affymetrix). Array signals were analyzed with Affymetrix GeneChip Operating Software and Affymetrix Power Tools. UTR hexamer analysis was carried out as described previously (Jackson et al 2006).
Results
Structural variants of a miR-124 duplex were tested, wherein a nick was introduced 10 nucleotides from the 5′ end of either the guide (or miRNA) or passenger (or miRNA*) strand (FIG. 12A). Duplexes were transfected into cells and changes in the mRNA levels of miR-124 target CD164 were measured. Division of the passenger strand (G/P10.10) had little effect on miR-124 activity (FIG. 12B), leading to a slight increase in EC50 (0.12 nM to 0.29 nM). Division of the guide strand (G10.10/P) still allowed for miR-124 activity (EC50 of 0.22 nM), indicating that a continuous guide strand is not required for miRNA RISC activity. The addition of a one base gap between the guide halves, or capping of the junction with a Cy3 dye or an inverted abasic residue, still gave miR-124 activity, indicating that this activity was not a result of ligation of the guide halves. Toleration of guide strand segmentation is not a property only of the miR-124 sequence, as division of the guide strand in a miR-34 duplex still allowed for miR-34a activity (FIG. 9).
Microarrays were used to profile cells transfected with a miR-124 duplex containing the divided guide strand (FIG. 13A) to further confirm the targeting activity of a segmented microRNA duplex, in this instance at a genome-wide level. Analysis of the 3′ UTR sequences of the downregulated genes shows that the most significantly enriched hexamer is GCCTTA, which corresponds to the seed sequence of miR-124. Profiling of the effects of the segmented miR-124 duplex G10.10/P showed correlation (r=0.90) with profiling of the effects of the fully intact duplex (G/P), consistent with the preservation of the bulk of miR-124 targeting (FIG. 13B).
Previous work analyzing microarray profiles has shown that although the preponderance of miRNA targeting is due to seed sequence activity, a much smaller degree of downregulation can be attributed to other contributing factors, among them the supplementary binding of positions 13-16 of the miRNA. Microarray profiling of miR-124 targets containing supplementary 3′ binding was analyzed, and a shift following guide strand segmentation was detected that was indicative of a loss of supplementary 3′ binding activity in the divided miRNA (FIG. 16).
The effects of segmented miRNAs on luciferase reporter vectors were tested, using miRNAs whose 3′ UTRs had been engineered (FIG. 14A) to contain miRNA-complementary sites that constituted a full-length match (2×FL), a seed sequence match (2×7a), or a seed sequence plus supplementary 3′ match (2×7a3p). For an intact duplex (G/P), the repressive activity of miR-124 on luciferase activity was highest for 2×FL and followed the order 2×FL-2×7a3p>2×7a. (FIG. 14B) Similar behavior was seen with a segmented passenger strand (G/P10.10, FIG. 14C). However, when the guide strand was divided (G10.10/P, FIG. 14D), the activities of the 2×FL and 2×7a3p reporters became equivalent to that of 2×7a, showing that the discontinuity at position 10 of the guide strand prevents productive 3′ binding, while permitting seed-based activity from the 5′ half.
Activity of a segmented guide strand was tested in the context of a hairpin sequence that was designed to emulate the natural miR-124 hairpin. Appreciable activity was observed following guide strand division (FIG. 15B), indicating that processing of a hairpin into an Ago-recognizable duplex can occur in spite of a break in the guide strand.
Example 2
Segmented miRNA Mimetics Targeted to CD164
MicroRNAs can down-regulate gene expression by inhibiting translation of their target transcripts and/or mediating the degradation of these transcripts. This Example demonstrates that certain of the segmented miRNA mimetic constructs according to the present disclosure designed based on the corresponding naturally-occurring miRNAs are capable of doing the same. This example also indicates that segmented miRNA mimetics can comprise one or more locked nucleic acids (named “(L)”, underlined nucleotides are locked nucleic acid residues in Table III, IV, V and VI). Nicks are marked within the sequence as “(nick).” Gaps are marked within the sequence as “()” with each box indicating a one nucleotide gap.
Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table III, passenger strand shown on top and guide strand on bottom) and a non-targeting control “Universal Control (UC)” duplex were transfected into HCT116 DICERex5, a human colorectal cancer cell line with hypomorphic DICER function (Cummins, J. M., et al., PNAS 103:3687-3692, 2006). The transfections were carried out using Lipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions. RNA was isolated at 24 hours post-transfection using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The transcript abundance of the target gene, CD164, was measured by Taqman Real-time PCR and Biomek NX (Biomek FX Dual-96).
Passenger strand sequences in Table III are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.









TABLE III 









SEQ ID NOs.










Name
Sequence
P
G





miR-
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1091


124/124(p)/124
ACCGUAAGUGGCGCACGGAAUU (guide)




(g)





 


(124p)10(L).10
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1107/
1091


(L)/(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)
1108



 


(124p)10.10/
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1096/
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)
1097



 


(124p)12(L).8(L)/
(passenger) GCAUUCACCGCG(nick)UGCCUUAAAU
 1105/
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)
1106



 


(124p)10(L).8(L)/
(passenger) GCAUUCACCG(  )UGCCUUAAAU
 1107/
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)
1109



 


(124p)/(124g)10
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1103/


(L).10(L)
ACCGUAAGUGG(nick)CGCACGGAAUU (guide)

1104


 


(124p)/(124g)10.10
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1093/



ACCGUAAGUGG(nick)CGCACGGAAUU (guide)

1094


 


(124p)/(124g)10
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1103/


(L).9(L)
ACCGUAAGUGG( )GCACGGAAUU (guide)

1110


 


(124p)/(124g)10
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1103/


(L).8(L)
ACCGUAAGUG(  )GCACGGAAUU (guide)

1112


 


(124p)/(124g)10
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1103


(L).7(L)
ACCGUAAGU(   )GCACGGAAUU (guide)




 


(124p)/(124g)10
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1093


(L).7
ACCGUAAGU(   )GCACGGAAUU (guide)




 


(124p)/(124g)11
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1111/


(L).9(L)
ACCGUAAGUGG(nick)CGCACGGAAUU (guide)

1110


 


(124p)/(124g)11
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1111/


(L).8(L)
ACCGUAAGUG( )CGCACGGAAUU (guide)

1112


 


(124p)/(124g)11
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1111


(L).7(L)
ACCGUAAGU(  )CGCACGGAAUU (guide)




 


(124p)/(124g)12
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1113/


(L).8(L)
ACCGUAAGUG(nick)GCGCACGGAAUU (guide)

1112


 


(124p)/(124g)12
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1113


(L).7(L)
ACCGUAAGU( )GCGCACGGAAUU (guide)




 


(124p)/(124g)13
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1114


(L).7(L)
ACCGUAAGU(nick)GGCGCACGGAAUU (guide)









Results of the activities are shown in FIG. 7. In brief, most of the constructs tested demonstrated a capacity of knocking down CD164, a known target to the naturally-occurring endogenous miR-124. Constructs comprising nicks in one or both strands of the segmented miRNA mimetic demonstrated at least 25% of knockdown (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) achieved by the non-segmented miR-24 positive control.
Example 3
Segmented miRNA Mimetics Targeted to VAMP3
Segmented miRNA mimetics can be designed to include a discontinuity comprising a nick or gap in one or both strands of any miRNA sequence of the invention in which target specific silencing activity is maintained. In the following example, nicks and gaps were introduced into miR-124 miRNA mimetics and downstream target silencing was confirmed.
Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table IV) and a non-targeting control “Universal Control (UC)” duplex were transfected into HCT116 DICERex5, a human colorectal cancer cell line with hypomorphic DICER function (Cummins, J. M., et al., PNAS 103:3687-3692, 2006). The transfections were carried out using Lipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions. RNA was isolated at 24 hours post-transfection using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The transcript abundance of the target gene, VAMP3, was measured by Taqman Real-time PCR and Biomek NX (Biomek FX Dual-96).
Passenger strand sequences in Table IV are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.









TABLE IV 









SEQ ID




NO(s).










Name
Sequence
P
G





miR-124/
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1091


(124(p)/124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)10(L).10
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1107/
1091


(L)/(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)
1108



 


(124p)/(124g)10
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1103/


(L).10(L)
ACCGUAAGUGGC(nick)GCACGGAAUU (guide)

1104


 


(124p)/(124g)11
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1111/


(L).9(L)
ACCGUAAGUGG(nick)CGCACGGAAUU (guide)

1110


 


(124p)10(L).10
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1107/
 1103/


(L)/(124g)10
ACCGUAAGUGGC(nick)GCACGGAAUU (guide)
1108
1104


(L).10(L)





 


(124p)10(L).10
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1107/
 1093/


(L)/(124g)10.10
ACCGUAAGUGGC(nick)GCACGGAAUU (guide)
1108
1094


 


(124p)10.10/
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1096/
 1103/


(124g)10(L).10
ACCGUAAGUGGC(nick)GCACGGAAUU (guide)
1097
1104


(L)





 


(124p)10.10/
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1096/
 1093/


(124g)10.10
ACCGUAAGUGGC(nick)GCACGGAAUU (guide)
1097
1094


 


(124p)10(L).10
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1107/
 1111/


(L)/(124g)11
ACCGUAAGUGG(nick)CGCACGGAAUU (guide)
1108
1110


(L).9(L)





 


(124p)10.10/
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1096/
 1111/


(124g)11(L).9(L)
ACCGUAAGUGG(nick)CGCACGGAAUU (guide)
1097
1110


 


(124p)10(L).10
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1107/
 1113/


(L)/(124g)12
ACCGUAAGUG(nick)GCGCACGGAAUU (guide)
1108
1112


(L).8(L)





 


(124p)10.10/
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1096/
 1113/


(124g)12(L).8(L)
ACCGUAAGUG(nick)GCGCACGGAAUU (guide)
1097
1112


 


(124p)10(L).10
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1107/
1114


(L)/(124g)13
ACCGUAAGU(nick)GGCGCACGGAAUU (guide)
1108



(L).7(L)





 


(124p)10.10/
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1096/
1114


(124g)13(L).7(L)
ACCGUAAGU(nick)GGCGCACGGAAUU (guide)
1097



 


(124p)12(L).8
(passenger) GCAUUCACCGCG(nick)UGCCUUAAAU
 1105/
 1103/


(L)/(124g)10
ACCGUAAGUGGC(nick)GCACGGAAUU (guide)
1106
1104


(L).10(L)





 


(124p)12(L).8
(passenger) GCAUUCACCGCG(nick)UGCCUUAAAU
 1105/
 1103/


(L)/(124g)10.10
ACCGUAAGUGGC(nick)GCACGGAAUU (guide)
1106
1104


 


(124p)12(L).8
(passenger) GCAUUCACCGCG(nick)UGCCUUAAAU
 1105/
 1111/


(L)/(124g)11
ACCGUAAGUGG(nick)CGCACGGAAUU (guide)
1106
1110


(L).9(L)





 


(124p)12(L).8
(passenger) GCAUUCACCGCG(nick)UGCCUUAAAU
 1105/
 1113/


(L)/(124g)12
ACCGUAAGUG(nick)GCGCACGGAAUU (guide)
1106
1112


(L).8(L)





 


(124p)12(L).8 
(passenger) GCAUUCACCGCG(nick)UGCCUUAAAU
 1105/
1114


(L)/(124g)13
ACCGUAAGU(nick)GGCGCACGGAAUU (guide)
1106



(L).7(L)









Results of this example are shown in FIG. 8. A number of the segmented miRNA mimetic constructs of this example, each strand comprising two distinct contiguous stretches of nucleotides, achieved at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the knockdown effect as compared to the non-segmented duplex miR-124 mimetic.
Example 4
Segmented miRNA Mimetics of miR-124 Versus miR-34
Segmented miRNA mimetics can be designed to include a discontinuity comprising a nick or gap in any miRNA sequence of the invention in which the target specificity is maintained. In the following example, nicks and gaps were introduced into miR-124 and miR-34 miRNA mimetics and downstream target specificity determined.
Synthetic duplex mimetic of miR-124 and segmented miR-124 constructs (sequences shown in Table V) and a non-targeting control “Universal Control (UC)” duplex were transfected into HCT116 DICERex5, a human colorectal cancer cell line with hypomorphic DICER function (Cummins, J. M., et al., PNAS 103:3687-3692, 2006). The transfections were carried out using Lipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions. RNA was isolated at 24 hours post-transfection using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The transcript abundance of the target gene, CD 164, was measured by Taqman Real-time PCR and Biomek NX (Biomek FX Dual-96). The knockdown effect achieved by segmented miRNA-124 was also compared with the knockdown, or the lack thereof, achieved by segmented miRNA-34 and a duplex miR-34 mimetic, which are known to not target CD 164.
Synthetic duplex mimetic of miR-34 and segmented miR-34 constructs (sequences shown in Table V) and a non-targeting control “Universal Control (UC)” duplex were transfected into HCT116 DICERex5, a human colorectal cancer cell line with hypomorphic DICER function (Cummins, J. M., et al., PNAS 103:3687-3692, 2006). The transfections were carried out using Lipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions. RNA was isolated at 24 hours post-transfection using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The transcript abundance of the target gene, TK1, was measured by Taqman Real-time PCR and Biomek NX (Biomek FX Dual-96). The knockdown effect achieved by segmented miRNA-34 was also compared with the knockdown, or the lack thereof, achieved by segmented miRNA-124 and a duplex miR-124 mimetic, which are known to not target TK1.
The nucleotides that were changed to effectuate the mismatches are indicated in lower case letters in the sequences. Passenger strand sequences in Table V are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.








TABLE V 








SEQ ID











NO(s).










Name
Sequence
P
G





miR-124/(124p)/
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


miR-124 (blunt
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1115


end)
CGUAAGUGGCGCACGGAAUU (guide)




 


miR-124 (shorter
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1116


guide)
UAAGUGGCGCACGGAAUU (guide)




 


(124p)/(124g)10
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1103/


(L).10(L)
ACCGUAAGUGGC(nick)GCACGGAAUU (guide)

1104


 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1093/


10.10
ACCGUAAGUGGC(nick)GCACGGAAUU (guide)

1094


 


(124p)10.10/
(passenger) GCAUUCACCG(nick)CGUGCCUUAAAU
 1096/
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)
1097



 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1093/


10.10
ACCGUAAGUGGC(nick)GCACGGAAUU (guide)

1094


 


(124p)/(124g)11.9
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1117/



ACCGUAAGUGG(nick)CGCACGGAAUU (guide)

1118


 


(124p)/(124g)
(passenger) GCAUcuACCG(nick)CGUGCCUUAAAU
 1119/
 1093/


10.10m (mismatch)
ACCGUAgaUGGC(nick)GCACGGAAUU (guide)
1097
1120


 


(124p)/(124g)10
(passenger) GCAUUCACCG(nick)CGUcgCUUAAAU
 1096/
 1122/


m3.10 (mismatch)
ACCGUAAGUGGC(nick)GCAgcGAAUU (guide)
1121
1094


 


(124p)/(124g)10
(passenger) GCAUUCACCG(nick)CGUGCCUauAAU
 1096/
 1124/


m5.10 (mismatch)
ACCGUAAGUGG(nick)CGCACGGAuaU (guide)
1123
1118


 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1117/


10.9
ACCGUAAGUG( )CGCACGGAAUU (guide)

1125


 


(124p)/(124g)10
(passenger) GCAUUCACCGCGUcgCUUAAAU
1126
1127


m3.9 (mismatch)
ACCGUAAGU( )GCGCAgcGAAUU (guide)




 


(124p)/(124g)10
(passenger) GCAUUCACCG(nick)CGUGCCUauAAU
 1096/
 1128/


m5.9 (mismatch)
ACCGUAAGUG(nick)GCGCACGGAuaU (guide)

1112


 


miR-
(passenger) CACGAGCUAAGACACUGCUAAU
1093
1094


34a/(34p)/(34g)
UGGUGCUCGAUUCUGUGACGGU (guide)




 


(34p)/(34g)10.10
(passenger) CACGAGCUAAGACACUGCUAAU
1093
 1129/



UGGUGCUCGAUU(nick)CUGUGACGGU (guide)

1130


 


(34p)/(34g)11.9
(passenger) CACGAGCUAAGACACUGCUAAU
1093
 1131/



UGGUGCUCGAU(nick)UCUGUGACGGU (guide)

1132


 


(34p)/(34g)10.9
(passenger) CACGAGCUAAGACACUGCUAAU
1093
 1129/



UGGUGCUCGAU( )CUGUGACGGU (guide)

1132









The results are presented in FIGS. 9A and 9B. Certain segmented miRNA mimetic constructs of this example, achieved at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the knockdown effect as compared to their respective non-segmented duplex mimetics.
Example 5
Segmented miRNA Mimetics Comprising Abasic Insertions
Segmented miRNA mimetics can be designed to include a discontinuity comprising a nick or gap, in which one or more non-nucleotide moieties of the invention are inserted into the terminal portions of sequence adjacent to the nick or gap. In the following example, abasic moieties were used to cap the internal ends of nucleotide positions in the guide strand of a segmented miRNA mimetic. Likewise, insertions with other non-nucleotide moieties described herein or otherwise known in the art can be similarly performed by one of general skill following the methodologies herein.
Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table VI), comprising one or more inverted abasic modifications at the internal ends, and a non-targeting control “Universal Control (UC)” duplex were transfected into HCT116 DICERex5, a human colorectal cancer cell line with hypomorphic DICER function (Cummins, J. M., et al., PNAS 103:3687-3692, 2006). The transfections were carried out using Lipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions. RNA was isolated at 24 hours post-transfection using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The transcript abundance of the target genes, CD164 and VAMP3, was measured by Taqman Real-time PCR and Biomek NX (Biomek FX Dual-96).
The position of the inverted abasic group is indicated as “(i)” in both the names and the sequences of the following table. Passenger strand sequences in Table VI are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.








TABLE VI








SEQ ID











NO(s).










Name
Sequence
P
G





miR-124
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1091



ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1133


7(i).13(i)
ACCGUAAGUGGCGCA(i)(nick)(i)CGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1134


8(i).12(i)
ACCGUAAGUGGCGC(i)(nick)(i)ACGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1135


9(i).11(i)
ACCGUAAGUGGCG(i)(nick)(i)CACGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1136/


10(i).10(i)
ACCGUAAGUGGC(i)(nick)(i)GCACGGAAUU (guide)

1137


 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1138/


11(i).9(i)
ACCGUAAGUGG(i)(nick)(i)CGCACGGAAUU (guide)

1139


 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1140/


12(i).8(i)
ACCGUAAGUG(i)(nick)(i)GCGCACGGAAUU (guide)

1141


 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1142


13(i).7(i)
ACCGUAAGU(i)(nick)(i)GGCGCACGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1143


7(i).13
ACCGUAAGUGGCGCA(nick)(i)CGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1144


8(i).12
ACCGUAAGUGGCGC(nick)(i)ACGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1145


9(i).11
ACCGUAAGUGGCG(nick)(i)CACGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1136/


10(i).10
ACCGUAAGUGGC(nick)(i)GCACGGAAUU (guide)

1094


 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092 
 1138/


11(i).9
ACCGUAAGUGG(nick)(i)CGCACGGAAUU (guide)

1118


 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1140/


12(i).8
ACCGUAAGUG(nick)(i)GCGCACGGAAUU (guide)

1125


 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1142


13(i).7
ACCGUAAGU(nick)(i)GGCGCACGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1133


7.13(i)
ACCGUAAGUGGCGCA(i)(nick)CGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1134


8.12(i)
ACCGUAAGUGGCGC(i)(nick)ACGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1135


9.11(i)
ACCGUAAGUGGCG(i)(nick)CACGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1093/


10.10(i)
ACCGUAAGUGGC(i)(nick)GCACGGAAUU (guide)

1137


 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1117/


11.9(i)
ACCGUAAGUGG(i)(nick)CGCACGGAAUU (guide)

1139


 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
 1146/


12.8(i)
ACCGUAAGUG(i)(nick)GCGCACGGAAUU (guide)

1141


 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1147


13.7(i)
ACCGUAAGU(i)(nick)GGCGCACGGAAUU (guide)









The results of this example are indicated in FIGS. 10A and 10B. As can be seen from this example, modifying the internal ends with one or more deoxyabasic moieties or modifications can impart further improvement of activity to the segmented miRNA mimetics of the invention.
Example 6
Segmented miRNA Mimetics Comprising Abasic Substitutions
Segmented miRNA mimetics can be designed to include a discontinuity comprising one or more non-nucleotide substitutions of the invention that occupy deleted sequence portions. In the following example, abasic linkers were used to substitute deleted nucleotide positions in both the guide and passenger strands of a miRNA mimetic. Likewise, substitution with other non-nucleotide linking moieties described herein or otherwise known in the art can be similarly performed by one of general skill following the methodologies herein.
Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table VII), comprising one or more abasic substitutions and a non-targeting control “Universal Control (UC)” duplex were transfected into HCT116 DICERex5, a human colorectal cancer cell line with hypomorphic DICER function (Cummins, J. M., et al., PNAS 103:3687-3692 (2006). The transfections were carried out using Lipofectamine RNAiMax (Invitrogen) per the manufacturer's instructions. RNA was isolated at 24 hours post-transfection using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The transcript abundance of the target genes, CD164 and VAMP3, was measured by Taqman Real-time PCR and Biomek NX (Biomek FX Dual-96).
The positions of the abasic linker is indicated as “(ab)” in both the names and the sequences of the following table. Passenger strand sequences in Table VII are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.









TABLE VII 









SEQ ID




NO(s).










Name
Sequence
P
G





miR-124
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1091



ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)8(ab)211/
(passenger) GCAUUCAC(ab)(ab)CGUGCCUUAAAU
1148
1149


(124g)9(ab)212
ACCGUAAGUGG(ab)(ab)CACGGAAUU (guide)




 


(124p)7(ab)311/
(passenger) GCAUUCA(ab)(ab)(ab)CGUGCCUUAAAU
1150
1151


(124g)9(ab)313
ACCGUAAGUG(ab)(ab)(ab)CACGGAAUU (guide)




 


(124p)/6(ab)411/
(passenger) GCAUUC(ab)(ab)(ab)(ab)CGUGCCUUAAAU
1152
1153


(124g)9(ab)414
ACCGUAAGU(ab)(ab)(ab)(ab)CACGGAAUU (guide)




 


(124p)/5(ab)511/
(passenger) GCAUU(ab)(ab)(ab)(ab)(ab)CGUGCCUUAAAU
1154
1155


(124g)9(ab)515
ACCGUAAG(ab)(ab)(ab)(ab)(ab)CACGGAAUU (guide)




 


(124p)/7(ab)210/
(passenger) GCAUUCA(ab)(ab)GCGUGCCUUAAAU
1148
1156


(124g)10(ab)213
ACCGUAAGUG(ab)(ab)GCACGGAAUU (guide)




 


(124p)/6(ab)310/
(passenger) GCAUUC(ab)(ab)(ab)GCGUGCCUUAAAU
1157
1158


(124g)10(ab)314
ACCGUAAGU(ab)(ab)(ab)GCACGGAAUU (guide)




 


(124p)/5(ab)410/
(passenger) GCAUU(ab)(ab)(ab)(ab)GCGUGCCUUAAAU
1159
1160


(124g)10(ab)415
ACCGUAAG(ab)(ab)(ab)(ab)GCACGGAAUU (guide)




 


(124p)/4(ab)510/
(passenger) GCAU(ab)(ab)(ab)(ab)(ab)GCGUGCCUUAAAU
1161
1162


(124g)10(ab)516
ACCGUAA(ab)(ab)(ab)(ab)(ab)GCACGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1163


8(ab)211
ACCGUAAGUGGC(ab)(ab)ACGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1164


8(ab)312
ACCGUAAGUGG(ab)(ab)(ab)ACGGAAUU (guide)




 


(124p)/(124g)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1165


8(ab)413
ACCGUAAGUG(ab)(ab)(ab)(ab)ACGGAAUU (guide)




 


(124p)9(ab)11/
(passenger) GCAUUCACC(ab)CGUGCCUUAAAU
1166
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)8(ab)211/
(passenger) GCAUUCAC(ab)(ab)CGUGCCUUAAAU
1148
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)7(ab)311/
(passenger) GCAUUCA(ab)(ab)(ab)CGUGCCUUAAAU
1150
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)6(ab)411/
(passenger) GCAUUC(ab)(ab)(ab)(ab)CGUGCCUUAAAU
1152
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)5(ab)511/ 
(passenger) GCAUU(ab)(ab)(ab)(ab)(ab)CGUGCCUUAAAU
1154
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)4(ab)611/
(passenger) GCAU(ab)(ab)(ab)(ab)(ab)(ab)CGUGCCUUAAAU
1167
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)8(ab)10/
(passenger) GCAUUCAC(ab)GCGUGCCUUAAAU
1168
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)7(ab)210/
(passenger) GCAUUCA(ab)(ab)GCGUGCCUUAAAU
1169
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)6(ab)310/
(passenger) GCAUUC(ab)(ab)(ab)GCGUGCCUUAAAU
1170
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)5(ab)410/
(passenger) GCAUU(ab)(ab)(ab)(ab)GCGUGCCUUAAAU
1171
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)4(ab)510/
(passenger) GCAU(ab)(ab)(ab)(ab)(ab)GCGUGCCUUAAAU
1172
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)3(ab)610/
(passenger) GCA(ab)(ab)(ab)(ab)(ab)(ab)GCGUGCCUUAAAU
1173
1091


(124g)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


(124p)/(124g)10
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1156


(ab)213
ACCGUAAGUG(ab)(ab)GCACGGAAUU (guide)




 


(124p)/(124g)10
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1158


(ab)314
ACCGUAAGU(ab)(ab)(ab)GCACGGAAUU (guide)




 


(124p)/(124g)10
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1160


(ab)415
ACCGUAAG(ab)(ab)(ab)(ab)GCACGGAAUU (guide)




 


(124p)/(124g)10
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1162


(ab)516
ACCGUAA(ab)(ab)(ab)(ab)(ab)GCACGGAAUU (guide)




 


(124p)/(124g)11
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1174


(ab)214
ACCGUAAGU(ab)(ab)CGCACGGAAUU (guide)




 


(124p)/(124g)11
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1175


(ab)315
ACCGUAAG(ab)(ab)(ab)CGCACGGAAUU (guide)




 


(124p)/(124g)11
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1176


(ab)416
ACCGUAA(ab)(ab)(ab)(ab)CGCACGGAAUU (guide)




 


(124p)/(124g)11
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1177


(ab)517
ACCGUA(ab)(ab)(ab)(ab)(ab)CGCACGGAAUU (guide)









The results of this example are indicated in FIGS. 11A and 11B. As can be seen from this example, abasic substitutions can impart further advantageous properties to the segmented miRNA mimetics of the invention.
Example 7
Segmented miRNA Mimetics with Multiple Nucleotide Position Deletions and Substitutions
Segmented miRNA mimetics can be designed to include a discontinuity comprising one or more non-nucleotide substitutions of the invention that occupy deleted sequence portions of 1 or more nucleotide positions. In the following example, alkyl linkers were used to substitute deleted nucleotide positions in both the guide and passenger strands of a miRNA mimetic Likewise, substitution with other non-nucleotide linking moieties described herein or otherwise known in the art can be similarly performed by one of general skill following the methodologies herein.
Oligonucleotides comprising C3 and C6 linkers were synthesized using protocols well known in the art (solid phase synthesis) using commercially available phosphoramidites, then purified by reversed phase solid phase extraction (SPE). The C3 (C33H43N2O5P) and C6 (C36H49N2O5P) phosphoramidites were purchased from ChemGenes.
Briefly, the single strand oligonucleotides were synthesized using phosphoramidite chemistry on an automated solid-phase synthesizer, using procedures as are generally known in the art (see for example U.S. application Ser. No. 12/064,014). A synthesis column was packed with solid support derivatized with the first nucleoside residue (natural or chemically modified). Synthesis was initiated by detritylation of the acid labile 5′-O-dimethoxytrityl group to release the 5′-hydroxyl. A suitably protected phosphoramidite and a suitable activator in acetonitrile were delivered simultaneously to the synthesis column resulting in coupling of the amidite to the 5′-hydroxyl. The column was then washed with a solvent, such as acetonitrile. An oxidizing solution, such as an iodine solution was pumped through the column to oxidize the phosphite triester linkage P(III) to its phosphotriester P(V) analog. Unreacted 5′-hydroxyl groups were capped using reagents such as acetic anhydride in the presence of 2,6-lutidine and N-methylimidazole. The elongation cycle was resumed with the detritylation step for the next phosphoramidite incorporation. This process was repeated until the desired sequence was synthesized. The synthesis concluded with the final 5′-terminus protecting group (trityl or 5′-O-dimethoxytrityl).
Upon completion of the synthesis, the solid-support and associated oligonucleotide were dried under argon pressure or vacuum. Aqueous base was added and the mixture was heated to effect cleavage of the succinyl linkage, removal of the cyanoethyl phosphate protecting group, and deprotection of the exocyclic amine protection.
The following process was performed on single strands that do not contain ribonucleotides. After treating the solid support with the aqueous base, the mixture was filtered to separate the solid support from the deprotected crude synthesis material. The solid support was then rinsed with DMSO, which is combined with the filtrate. The resultant basic solution allows for retention of the 5′-O-dimethoxytrityl group to remain on the 5′ terminal position (trityl-on).
For single strands that contain ribonucleotides, the following process was performed. After treating the solid support with the aqueous base, the mixture was filtered to separate the solid support from the deprotected crude synthesis material. The solid support was then rinsed with dimethylsulfoxide (DMSO), which was combined with the filtrate. Fluoride reagent, such as triethylamine trihydrofluoride, was added to the mixture, and the solution was heated. The reaction was quenched with suitable buffer to provide a solution of crude single strand with the 5′-O-dimethoxytrityl group on the final 5′ terminal position.
The trityl-on solution of each crude single strand was purified using chromatographic purification, such as SPE RPC purification. The hydrophobic nature of the trityl group permits stronger retention of the desired full-length oligo than the non-tritylated truncated failure sequences. The failure sequences were selectively washed from the resin with a suitable solvent, such as low percent acetonitrile. Retained oligonucleotides were then detritylated on-column with trifluoroacetic acid to remove the acid-labile trityl group. Residual acid was washed from the column, a salt exchange was performed, and a final desalting of the material commenced. The full-length oligo was recovered in a purified form with an aqueous-organic solvent. The final product was then analyzed for purity (HPLC), identity (Maldi-TOF MS), and yield (UV A260). The oligos were dried via lyophilization or vacuum condensation.
Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table VIII), from which bases have been deleted, and a non-targeting control “Universal Control (UC3)” duplex were transfected into HCT-116 cells (wild-type) and cultured in McCoy's 5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These cells were plated in 96-well culture plates at a density of 6000 cells/well 24 hours prior to transfection. Transfection was carried out using Opti-MEM I Reduced Serum Media (Gibco) and Lipofectamine RNAiMax (Invitrogen) with a final concentration of our miRNAs at 10 nM. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline and processed using the TaqMan® Gene Expression Cells-to-CTTM Kit (Applied Biosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCR using gene-specific probes (Applied Biosystems) on an ABI Prism 7900HT Sequence Detector.
Reverse transcription conditions were as follows: 60 minutes at 37° C. followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used for data normalization. Knockdown of miR-124 targets was calculated as the two-fold change in target cDNA measured in experimentally-treated cells relative to the target cDNA measured in non-targeting control-treated cells.
The positions of deleted bases are indicated as “()” in both the names and the sequences of the following table. C3 and C6 linkers are identified as “(c3)” and “(c6)”, respectively. Passenger strand sequences in Table VIII are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.









TABLE VIII









SEQ ID




NO(s).










Name
Sequence
P
G





miR-124 (iP/G)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1091










ACCGUAAGUGGCGCACGGAAUU (guide)



 










iP9-10del/G11-
(passenger) GCAUUCAC( )( )CGUGCCUUAAAU
1097
1093









12del
ACCGUAAGUG( )( )GCACGGAAUU (guide)



 










iP8-10del/G11-
(passenger) GCAUUCA( )( )( )CGUGCCUUAAAU
1097
1093









13del
ACCGUAAGU( )( )( )GCACGGAAUU (guide)



 










iP7-10del/G11-
(passenger) GCAUUC( )( )( )( )CGUGCCUUAAAU
1097
1093









14del
ACCGUAAG( )( )( )( )GCACGGAAUU (guide)



 










iP7-11del/G10-
(passenger) GCAUUC( )( )( )( )( )GUGCCUUAAAU
1178
n/a









14del
ACCGUAAG( )( )( )( )( )CACGGAAUU (guide)



 










iP7-12del/G9-
(passenger) GCAUUC( )( )( )( )( )( )UGCCUUAAAU
1109
n/a


14del
ACCGUAAG( )( )( )( )( )( )ACGGAAUU (guide)




 


iP9-
(passenger) GCAUUCAC( )( )(c3)CGUGCCUUAAAU
1179
1180


10_1x_c3/G11-
ACCGUAAGUG( )( )(c3)GCACGGAAUU (guide)




12_1x_c3





 


iP8-
(passenger) GCAUUCA( )( )( )(c3)CGUGCCUUAAAU
1181
1182


10_1x_c3/G11-
ACCGUAAGU( )( )( )(c3)GCACGGAAUU (guide)




13_1x_c3





 


iP7-
(passenger) GCAUUC( )( )( )( )(c3)CGUGCCUUAAAU
1183
1184


10_1x_c3/G11-
ACCGUAAG( )( )( )( )(c3)GCACGGAAUU (guide)




14_1x_c3





 


iP7-
(passenger) GCAUUC( )( )( )( )( )(c3)GUGCCUUAAAU
1185
1186


11_1x_c3/G10-
ACCGUAAG( )( )( )( )( )(c3)CACGGAAUU (guide)




14_1x_c3





 


iP7-
(passenger) GCAUUC( )( )( )( )( )( )(c3)UGCCUUAAAU
1187
1188


12_1x_c3/G9-
ACCGUAAG( )( )( )( )( )( )(c3)ACGGAAUU (guide)




14_1x_c3





 


iP9-
(passenger) GCAUUCAC( )( )(c3)CGUGCCUUAAAU
1179
1189


10_1x_c3/G11-
ACCGUAAGUG( )( )(c6)GCACGGAAUU (guide)




12_1x_c6





 


iP8-
(passenger) GCAUUCA( )( )( )(c3)CGUGCCUUAAAU
1181
1190


10_1x_c3/G11-
ACCGUAAGU( )( )( )(c6)GCACGGAAUU (guide)




13_1x_c6





 


iP7-
(passenger) GCAUUC( )( )( )( )(c3)CGUGCCUUAAAU
1183
1191


10_1x_c3/G11-
ACCGUAAG( )( )( )( )(c6)GCACGGAAUU (guide)




14_1x_c6





 


iP7-
(passenger) GCAUUC( )( )( )( )( )(c3)GUGCCUUAAAU
1185
1192


11_1x_c3/G10-
ACCGUAAG( )( )( )( )( )(c6)CACGGAAUU (guide)




14_1x_c6





 


iP7-
(passenger) GCAUUC( )( )( )( )( )( )(c3)UGCCUUAAAU
1187
1193


12_1x_c3/G9-
ACCGUAAG( )( )( )( )( )( )(c6)ACGGAAUU (guide)




14_1x_c6









The results of this example are shown in FIGS. 17A and 17B. A number of the segmented miRNA mimetic constructs of this example achieved a significant knockdown effect as compared to the non-segmented duplex miR-124.
Example 8
Segmented miRNA Mimetics Comprising Small Substitutions
Segmented miRNA mimetics can be designed to include a discontinuity comprising non-nucleotide substitutions of the invention that occupy deleted nucleotide positions. In the following example, C3 alkyl linkers were used to substitute deleted nucleotide positions in both the guide and passenger strand of a miRNA mimetic Likewise, substitution with other non-nucleotide linking moieties described herein or otherwise known in the art can be similarly performed by one of general skill following the methodologies herein.
Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table IX), comprising C3 substitutions, and a non-targeting control “Universal Control (UC3)” duplex were transfected into HCT-116 cells (wild-type) and cultured in McCoy's 5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These cells were plated in 96-well culture plates at a density of 6000 cells/well 24 hours prior to transfection. Transfection was carried out using Opti-MEM I Reduced Serum Media (Gibco) and Lipofectamine RNAiMax (Invitrogen) with a final concentration of our miRNAs at 10 nM. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline and processed using the TaqMan® Gene Expression Cells-to-CTTM Kit (Applied Biosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCR using gene-specific probes (Applied Biosystems) on an ABI Prism 7900HT Sequence Detector.
Reverse transcription conditions were as follows: 60 minutes at 37° C. followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used for data normalization. Knockdown of miR-124 targets was calculated as the two-fold change in target cDNA measured in experimentally-treated cells relative to the target cDNA measured in non-targeting control-treated cells.
The positions of c3 substitutions are shown in both the names and the sequences of the following table. C3 linkers are identified as “(c3)”. Passenger strand sequences in Table IX are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.









TABLE IX 









SEQ ID NO(s).










Name
Sequence
P
G





miR-124
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1091


(iP/G)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


iP22-22c3/G
(passenger) GCAUUCACCGCGUGCCUUAAA(c3)
1194
1091



ACCGUAAGUGGCGCACGGAAUU (guide)




 


iP21-21c3/G
(passenger) GCAUUCACCGCGUGCCUUAA(c3)U
1195
1091



ACCGUAAGUGGCGCACGGAAUU (guide)




 


iP20-
(passenger) GCAUUCACCGCGUGCCUUA(c3)AU
1196
1216


20c3/G1-1c3
ACCGUAAGUGGCGCACGGAAU(c3) (guide)




 


iP19-
(passenger) GCAUUCACCGCGUGCCUU(c3)AAU
1197
1217


19c3/G2-2c3
ACCGUAAGUGGCGCACGGAA(c3)U (guide)




 


iP18-
(passenger) GCAUUCACCGCGUGCCU(c3)AAAU 
1198
1218


18c3/G3-3c3
ACCGUAAGUGGCGCACGGA(c3)UU (guide)




 


iP17-
(passenger) GCAUUCACCGCGUGCC(c3)UAAAU
1199
1219


17c3/G4-4c3
ACCGUAAGUGGCGCACGG(c3)AUU (guide)




 


iP16-
(passenger) GCAUUCACCGCGUGC(c3)UUAAAU
1200
1220


16c3/G5-5c3
ACCGUAAGUGGCGCACG(c3)AAUU (guide)




 


iP15-
(passenger) GCAUUCACCGCGUG(c3)CUUAAAU
1201
1221


15c3/G6-6c3
ACCGUAAGUGGCGCAC(c3)GAAUU (guide)




 


iP14-
(passenger) GCAUUCACCGCGU(c3)CCUUAAAU
1202
1222


14c3/G7-7c3
ACCGUAAGUGGCGCA(c3)GGAAUU (guide)




 


iP13-
(passenger) GCAUUCACCGCG(c3)GCCUUAAAU
1203
1223


13c3/G8-8c3
ACCGUAAGUGGCGC(c3)CGGAAUU (guide)




 


iP12-
(passenger) GCAUUCACCGC(c3)UGCCUUAAAU
1204
1224


12c3/G9-9c3
ACCGUAAGUGGCG(c3)ACGGAAUU (guide)




 


iP11-11c3/
(passenger) GCAUUCACCG(c3)GUGCCUUAAAU
1205
1225


G10-10c3
ACCGUAAGUGGC(c3)CACGGAAUU (guide)




 


iP10-10c3/
(passenger) GCAUUCACC(c3)CGUGCCUUAAAU
1206
1226


G11-11c3
ACCGUAAGUGG(c3)GCACGGAAUU (guide)




 


iP9-9c3/G12-
(passenger) GCAUUCAC(c3)GCGUGCCUUAAAU
1207
1227


12c3
ACCGUAAGUG(c3)CGCACGGAAUU (guide)




 


iP8-8c3/G13-
(passenger) GCAUUCA(c3)CGCGUGCCUUAAAU
1208
1228


13c3
ACCGUAAGU(c3)GCGCACGGAAUU (guide)




 


iP7-7c3/G14-
(passenger) GCAUUC(c3)CCGCGUGCCUUAAAU
1209
1229


14c3
ACCGUAAG(c3)GGCGCACGGAAUU (guide)




 


iP6-6c3/G15-
(passenger) GCAUU(c3)ACCGCGUGCCUUAAAU
1210
1230


15c3
ACCGUAA(c3)UGGCGCACGGAAUU (guide)




 


iP5-5c3/G16-
(passenger) GCAU(c3)CACCGCGUGCCUUAAAU
1211
1231


16c3
ACCGUA(c3)GUGGCGCACGGAAUU (guide)




 


iP4-4c3/G17-
(passenger) GCA(c3)UCACCGCGUGCCUUAAAU
1212
1232


17c3
ACCGU(c3)AGUGGCGCACGGAAUU (guide)




 


iP3-3c3/G18-
(passenger) GC(c3)UUCACCGCGUGCCUUAAAU
1213
1233


18c3
ACCG(c3)AAGUGGCGCACGGAAUU (guide)




 


iP2-2c3/G19-
(passenger) G(c3)AUUCACCGCGUGCCUUAAAU
1214
1234


19c3
ACC(c3)UAAGUGGCGCACGGAAUU (guide)




 


iP1-1c3/G20-
(passenger) (c3)CAUUCACCGCGUGCCUUAAAU
1215
1235


20c3
AC(c3)GUAAGUGGCGCACGGAAUU (guide)




 


iP/G21-21c3
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1236



A(c3)CGUAAGUGGCGCACGGAAUU (guide)









The results of this example are shown in FIGS. 18A and 18B. A number of the segmented miRNA mimetics of this example showed improved knockdown in comparison to the non-segmented duplex miR-124.
Example 9
Segmented miRNA Mimetics Comprising Larger Substitutions
Segmented miRNA mimetics can be designed to include a discontinuity comprising non-nucleotide substitutions of the invention that occupy deleted nucleotide positions. In the following example, C6 alkyl linkers were used to substitute deleted nucleotide positions in both the guide and passenger strand of a miRNA mimetic Likewise, substitution with other larger non-nucleotide linking moieties described herein or otherwise known in the art can be similarly performed by one of general skill following the methodologies herein.
Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table X), comprising c6 substitutions, and a non-targeting control “Universal Control (UC3)” duplex were transfected into HCT-116 cells (wild-type) and cultured in McCoy's 5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These cells were plated in 96-well culture plates at a density of 6000 cells/well 24 hours prior to transfection. Transfection was carried out using Opti-MEM I Reduced Serum Media (Gibco) and Lipofectamine RNAiMax (Invitrogen) with a final concentration of our miRNAs at 10 nM. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline and processed using the TaqMan® Gene Expression Cells-to-CTTM Kit (Applied Biosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCR using gene-specific probes (Applied Biosystems) on an ABI Prism 7900HT Sequence Detector.
Reverse transcription conditions were as follows: 60 minutes at 37° C. followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used for data normalization. Knockdown of miR-124 targets was calculated as the two-fold change in target cDNA measured in experimentally-treated cells relative to the target cDNA measured in non-targeting control-treated cells.
The positions of c6 substitutions are shown in both the names and the sequences of the following table. C6 linkers are identified as “(c6)”. Passenger strand sequences in Table X are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.









TABLE X 









SEQ ID NO(s).










Name
Sequence
P
G





miR-124
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1091


(iP/G)
ACCGUAAGUGGCGCACGGAAUU (guide)




 


iP22-22c6/G
(passenger) GCAUUCACCGCGUGCCUUAAA(c6)
1237
1091



ACCGUAAGUGGCGCACGGAAUU (guide)




 


iP21-21c6/G
(passenger) GCAUUCACCGCGUGCCUUAA(c6)U
1238
1091



ACCGUAAGUGGCGCACGGAAUU (guide)




 


iP20-
(passenger) GCAUUCACCGCGUGCCUUA(c6)AU
1239
1259


20c6/G1-1c6
ACCGUAAGUGGCGCACGGAAU(c6) (guide)




 


iP19-
(passenger) GCAUUCACCGCGUGCCUU(c6)AAU
1240
1260


19c6/G2-2c6
ACCGUAAGUGGCGCACGGAA(c6)U (guide)




 


iP18-
(passenger) GCAUUCACCGCGUGCCU(c6)AAAU
1241
1261


18c6/G3-3c6
ACCGUAAGUGGCGCACGGA(c6)UU (guide)




 


iP17-
(passenger) GCAUUCACCGCGUGCC(c6)UAAAU
1242
1262


17c6/G4-4c6
ACCGUAAGUGGCGCACGG(c6)AUU (guide)




 


iP16-
(passenger) GCAUUCACCGCGUGC(c6)UUAAAU
1243
1263


16c6/G5-5c6
ACCGUAAGUGGCGCACG(c6)AAUU (guide)




 


iP15-
(passenger) GCAUUCACCGCGUG(c6)CUUAAAU
1244
1264


15c6/G6-6c6
ACCGUAAGUGGCGCAC(c6)GAAUU (guide)




 


iP14-
(passenger) GCAUUCACCGCGU(c6)CCUUAAAU
1245
1265


14c6/G7-7c6
ACCGUAAGUGGCGCA(c6)GGAAUU (guide)




 


iP13-
(passenger) GCAUUCACCGCG(c6)GCCUUAAAU
1246
1266


13c6/G8-8c6
ACCGUAAGUGGCGC(c6)CGGAAUU (guide)




 


iP12-
(passenger) GCAUUCACCGC(c6)UGCCUUAAAU
1247
1267


12c6/G9-9c6
ACCGUAAGUGGCG(c6)ACGGAAUU (guide)




 


iP11-
(passenger)GCAUUCACCG(c6)GUGCCUUAAAU
1248
1268


11c6/G10-10c6
ACCGUAAGUGGC(c6)CACGGAAUU (guide)




 


iP10-
(passenger) GCAUUCACC(c6)CGUGCCUUAAAU
1249
1269


10c6/G11-11c6
ACCGUAAGUGG(c6)GCACGGAAUU (guide)




 


iP9-9c6/G12-
(passenger) GCAUUCAC(c6)GCGUGCCUUAAAU
1250
1270


12c6
ACCGUAAGUG(c6)CGCACGGAAUU (guide)




 


iP8-8c6/G13-
(passenger) GCAUUCA(c6)CGCGUGCCUUAAAU
1251
1271


13c6
ACCGUAAGU(c6)GCGCACGGAAUU (guide)




 


iP7-7c6/G14-
(passenger) GCAUUC(c6)CCGCGUGCCUUAAAU
1252
1272


14c6
ACCGUAAG(c6)GGCGCACGGAAUU (guide)




 


iP6-6c6/G15-
(passenger) GCAUU(c6)ACCGCGUGCCUUAAAU
1253
1273


15c6
ACCGUAA(c6)UGGCGCACGGAAUU (guide)




 


iP5-5c6/G16-
(passenger) GCAU(c6)CACCGCGUGCCUUAAAU
1254
1274


16c6
ACCGUA(c6)GUGGCGCACGGAAUU (guide)




 


iP4-4c6/G17-
(passenger) GCA(c6)UCACCGCGUGCCUUAAAU
1255
1275


17c6
ACCGU(c6)AGUGGCGCACGGAAUU (guide)




 


iP3-3c6/G18-
(passenger) GC(c6)UUCACCGCGUGCCUUAAAU
1256
1276


18c6
ACCG(c6)AAGUGGCGCACGGAAUU (guide)




 


iP2-2c6/G19-
(passenger) G(c6)AUUCACCGCGUGCCUUAAAU
1257
1277


19c6
ACC(c6)UAAGUGGCGCACGGAAUU (guide)




 


iP1-1c6/G20-
(passenger) (c6)CAUUCACCGCGUGCCUUAAAU
1258
1278


20c6
AC(c6)GUAAGUGGCGCACGGAAUU (guide)




 


iP/G21-21c6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1279



A(c6)CGUAAGUGGCGCACGGAAUU (guide)









The results of this example are indicated in FIGS. 19A and 19B. A number of the segmented miRNA mimetics of this example showed increased knockdown in comparison to the non-segmented duplex miR-124.
Example 10
Segmented miRNA Mimetics Comprising Non-Nucleotide Insertions
Segmented miRNA mimetics can be designed to include a discontinuity comprising non-nucleotide insertions of the invention. In the following example, both small (C3) and larger (C6) alkyl moieties were used to connect segmented positions in both the guide and passenger strand of a miRNA mimetic. Likewise, insertions with other non-nucleotide linking moieties described herein or otherwise known in the art can be similarly performed by one of general skill following the methodologies herein.
Synthetic duplex mimetic of miR-124 and segmented miR-124 mimetic constructs (sequences shown in Table XI), comprising c3 and c6 insertions, and a non-targeting control “Universal Control (UC3)” duplex were transfected into HCT-116 cells (wild-type) and cultured in McCoy's 5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These cells were plated in 96-well culture plates at a density of 6000 cells/well 24 hours prior to transfection. Transfection was carried out using Opti-MEM I Reduced Serum Media (Gibco) and Lipofectamine RNAiMax (Invitrogen) with a final concentration of our miRNAs at 10 nM. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline and processed using the TaqMan® Gene Expression Cells-to-CTTM Kit (Applied Biosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCR using gene-specific probes (Applied Biosystems) on an ABI Prism 7900HT Sequence Detector.
Reverse transcription conditions were as follows: 60 minutes at 37° C. followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used for data normalization. Knockdown of miR-124 targets was calculated as the two-fold change in target cDNA measured in experimentally-treated cells relative to the target cDNA measured in non-targeting control-treated cells.
The positions of c3 and c6 insertions are shown in both the names and the sequences of the following table. C3 and C6 linkers are identified as “(c3)” and “(c6)”, respectively. Passenger strand sequences in Table XI are shown in 5′ to 3′ orientation and guide strand sequences are in 3′ to 5′ orientation.









TABLE XI 









SEQ ID NO(s).










Name
Sequence
P
G





miR-124 (iP/G)
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1091



ACCGUAAGUGGCGCACGGAAUU (guide)




 


iP/G1insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1280



ACCGUAAGUGGCGCACGGAAU(c6)U (guide)




 


iP/G2insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1281



ACCGUAAGUGGCGCACGGAA(c6)UU (guide)




 


iP/G3insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1282



ACCGUAAGUGGCGCACGGA(c6)AUU (guide)




 


iP/G4insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1283



ACCGUAAGUGGCGCACGG(c6)AAUU (guide)




 


iP/G5insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1284



ACCGUAAGUGGCGCACG(c6)GAAUU (guide)




 


iP/G6insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1285



ACCGUAAGUGGCGCAC(c6)GGAAUU (guide)




 


iP/G7insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1286



ACCGUAAGUGGCGCA(c6)CGGAAUU (guide)




 


iP/G8insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1287



ACCGUAAGUGGCGC(c6)ACGGAAUU (guide)




 


iP/G9insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1288



ACCGUAAGUGGCG(c6)CACGGAAUU (guide)




 


iP/G10insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1289



ACCGUAAGUGGC(c6)GCACGGAAUU (guide)




 


iP/G11insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1290



ACCGUAAGUGG(c6)CGCACGGAAUU (guide)




 


iP/G12insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1291



ACCGUAAGUG(c6)GCGCACGGAAUU (guide)




 


iP/G13insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1292



ACCGUAAGU(c6)GGCGCACGGAAUU (guide)




 


iP/G14insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1293



ACCGUAAG(c6)UGGCGCACGGAAUU (guide)




 


iP/G15insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1294



ACCGUAA(c6)GUGGCGCACGGAAUU (guide)




 


iP/G16insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1295



ACCGUA(c6)AGUGGCGCACGGAAUU (guide)




 


iP/G17insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1296



ACCGU(c6)AAGUGGCGCACGGAAUU (guide)




 


iP/G18insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1297



ACCG(c6)UAAGUGGCGCACGGAAUU (guide)




 


iP/G19insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1298



ACC(c6)GUAAGUGGCGCACGGAAUU (guide)




 


iP/G20insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1299



AC(c6)CGUAAGUGGCGCACGGAAUU (guide)




 


iP/G21insertc6
(passenger) GCAUUCACCGCGUGCCUUAAAU
1092
1300



A(c6)CCGUAAGUGGCGCACGGAAUU (guide)




 


iP10insertc6/G
(passenger) GCAUUCACCG(c6)CGUGCCUUAAAU
1301
1091



ACCGUAAGUGGCGCACGGAAUU (guide)




 


iP9insertc6/G
(passenger) GCAUUCACC(c6)GCGUGCCUUAAAU
1302
1091



ACCGUAAGUGGCGCACGGAAUU (guide)




 


iP8insertc6/G
(passenger) GCAUUCAC(c6)CGCGUGCCUUAAAU
1303
1091



ACCGUAAGUGGCGCACGGAAUU (guide)




 


iP7insertc6/G
(passenger) GCAUUCA(c6)CCGCGUGCCUUAAAU
1304
1091



ACCGUAAGUGGCGCACGGAAUU (guide)




 


iP6insertc6/G
(passenger) GCAUUC(c6)ACCGCGUGCCUUAAAU
1305
1091



ACCGUAAGUGGCGCACGGAAUU (guide)




 


iP10insertc6/
(passenger) GCAUUCACCG(c6)CGUGCCUUAAAU
1301
1289


G10insertc6
ACCGUAAGUGGC(c6)GCACGGAAUU (guide)




 


iP9insertc6/
(passenger) GCAUUCAC(c6)CGCGUGCCUUAAAU
1302
1290


G11insertc6
ACCGUAAGUG(c6)GCGCACGGAAUU (guide)




 


iP8insertc6/
(passenger) GCAUUCA(c6)CCGCGUGCCUUAAAU
1303
1291


G12insertc6
ACCGUAAGU(c6)GGCGCACGGAAUU (guide)




 


iP7insertc6/
(passenger) GCAUUC(c6)ACCGCGUGCCUUAAAU
1304
1292


G13insertc6
ACCGUAAG(c6)UGGCGCACGGAAUU (guide)




 


iP6insertc6/
(passenger) GCAUU(c6)CACCGCGUGCCUUAAAU
1305
1293


G14insertc6
ACCGUAA(c6)GUGGCGCACGGAAUU (guide)




 


iP10insertc3/
(passenger) GCAUUCACCG(c3)CGUGCCUUAAAU
1306
1307


G10insertc3
ACCGUAAGUGGC(c3)GCACGGAAUU (guide)









The results of this example are indicated in FIGS. 20A and 20B. A number of the segmented miRNA mimetics of this example showed increased knockdown in comparison to the non-segmented duplex miR-124.
All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

## Claims

1. A double-stranded miRNA mimetic molecule represented by Formula III:



wherein
(a) the molecule comprises a contiguous passenger strand and a guide strand where each line and its adjacent “N” represent a contiguous stretch of nucleotides, each of “X1,” “X2” and “X3” represent the number of nucleotides in each stretch, “G/N” represents a discontinuity in the guide strand, “Y1” represents a number of nucleotide positions in the discontinuity, and each group of dashed lines “ - - - ” and its adjacent “(W)” represents a terminal overhang that is optionally present, and each of “Z1” and “Z2” represents the number of overhanging nucleotides;
(b) X1 is an integer from 16 to 26, X2 is an integer from 2 to 20, X3 is an integer from 6 to 24, Y1 is an integer from 0 to 6, provided that the sum of X2, X3 and Y1 is an integer from 16 to 26; Zi and Z2 are independently integers from 0 to 4;
(c) NX3 comprises sequence having at least 6 contiguous nucleotides of the seed sequence of a miRNA sequence having any of SEQ ID NOs: 1-1090;
(d) NX2 and NX3 together comprise sequence having at least 50% homology to the miRNA sequence; and
(e) NX1 comprises sequence having at least 50% complementarity to the miRNA sequence.

2. The miRNA mimetic molecule of claim 1, wherein G/N comprises a nick in the guide strand.

3. The miRNA mimetic molecule of claim 1, wherein G/N comprises one or more gap(s) in the guide strand.

4. The miRNA mimetic molecule of claim 1, wherein G/N comprises one or more non-nucleotide substitutions in the guide strand.

5. The miRNA mimetic molecule of claim 1, wherein G/N comprises one or more non-nucleotide insertions in the guide strand.

6. The miRNA mimetic molecule of claim 1, wherein NX3 comprises a sequence having at least 7 contiguous nucleotides of the seed sequence of the miRNA.

7. The miRNA mimetic molecule of claim 1, wherein NX3 comprises a sequence having at least 8 contiguous nucleotides of the seed sequence of the miRNA.

8. The miRNA mimetic molecule of claim 1, wherein NX2 and NX3 together comprise a sequence having at least 60, 70, 80, or 90% homology to the miRNA sequence.

9. The miRNA mimetic molecule of claim 1, wherein NX1 comprises a sequence having at least 60, 70, 80, or 90% complementarity to the miRNA sequence.

10. A composition comprising the miRNA mimetic molecule of claim 1 and a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, buffer, stabilizer, or preservative.

11. The miRNA mimetic molecule of claim 1, wherein NX3 comprises sequence having at least 6 contiguous nucleotides of the seed sequence of a miRNA sequence of SEQ ID NO:168 or SEQ ID NO:169.

12. The miRNA mimetic molecule of claim 1, wherein NX3 comprises sequence having at least 6 contiguous nucleotides of the seed sequence of a miRNA sequence of SEQ ID NO:30 or SEQ ID NO: 31.

13. The miRNA molecule of claim 1, wherein NX3 comprises sequence having at least 6 contiguous nucleotides of the seed sequence of a miRNA sequence of SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:83.

14. The miRNA molecule of claim 1, wherein NX3 comprises sequence having at least 6 contiguous nucleotides of the seed sequence of a miRNA sequence of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.

15. The miRNA molecule of claim 1, wherein NX3 comprises sequence having at least 6 contiguous nucleotides of the seed sequence of a miRNA sequence of SEQ ID NO:109 or SEQ ID NO:110.

16. The miRNA molecule of claim 1, wherein NX3 comprises sequence having at least 6 contiguous nucleotides of the seed sequence of a miRNA sequence of SEQ ID NO:145 or SEQ ID NO:146.

17. The miRNA molecule of claim 1, wherein NX3 comprises sequence having at least 6 contiguous nucleotides of the seed sequence of a miRNA sequence of SEQ ID NO:57 or SEQ ID NO:58.

18. The miRNA molecule of claim 1, wherein NX3 comprises sequence having at least 6 contiguous nucleotides of the seed sequence of a miRNA sequence of SEQ ID NO:79 or SEQ ID NO:80.

19. The miRNA molecule of claim 1, wherein NX3 comprises sequence having at least 6 contiguous nucleotides of the seed sequence of a miRNA sequence of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:264 or SEQ ID NO:265.

20. The miRNA molecule of claim 1, wherein NX3 comprises sequence having at least 6 contiguous nucleotides of the seed sequence of a miRNA sequence of SEQ ID NO:118 or SEQ ID NO:119.

21. The miRNA mimetic molecule of claim 1, wherein the molecule comprises one or more 2′-deoxy modified nucleotides, 2′-O-methyl modified nucleotides, 2′-deoxy-2′-fluoro modified nucleotides or universal base modified nucleotides, or a combination thereof.

22. The miRNA mimetic molecule of claim 1, wherein the molecule comprises one or more phosphorothioate internucleotide linkages.

23. The miRNA mimetic molecule of claim 1, wherein the molecule comprises one or more of a ribonucleotide or a deoxyribonucleotide, or a combination thereof, that is chemically-modified at a nucleic acid sugar, base, or backbone.

24. The miRNA mimetic molecule of claim 1, wherein the molecule comprises one or more locked nucleic acid (LNA) nucleotides.

25. The miRNA mimetic molecule of claim 1, wherein the molecule comprises a ligand.

26. The miRNA mimetic molecule of claim 25, wherein the ligand is a galactose, a galactosamine, a folate, or a cholesterol.