# Patent

## Title
Modified dicer polypeptide and methods of use thereof

## Abstract

A modified Dicer polypeptide is provided, which modified Dicer polypeptide exhibits enhanced catalytic activity. Also provided is a method for producing small regulatory RNAs from a dsRNA, involving contacting a dsRNA with a subject modified Dicer. Small regulatory RNAs produced by a subject method find use in a variety of applications, including research and therapeutic applications.

## Background
CROSS-REFERENCE
This application claims the benefit of U.S. Provisional Patent Application No. 61/070,286, filed Mar. 21, 2008, which application is incorporated herein by reference in its entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with government support under Grant No. R01-GM073794 awarded by the National Institutes of Health. The government has certain rights in the invention.


BACKGROUND
Dicer is a large multi-domain enzyme responsible for cytoplasmic production of both microRNAs (miRNAs) and short interfering RNAs (siRNAs) during sequence-directed gene regulation by RNAi. As a member of the Ribonuclease III family of proteins, Dicer recognizes the 5′ and 3′ helical ends of double-stranded RNA substrates and cleaves a specific distance away to produce 21-27 nucleotide products. Dicer helps these mi- and siRNAs to load onto Argonaute proteins together with other protein components of the RNA-induced silencing complex (RISC). Once bound to target mRNAs, miRNAs typically regulate protein expression by controlling the level of translation, whereas siRNAs direct cleavage and subsequent degradation of complementary mRNAs.
Production of siRNAs is useful in various research and therapeutic applications. There is a need in the art for an enzyme that efficiently generates siRNAs from a double-stranded RNA substrate.
LITERATURE


    U.S. Patent Publication No. 2007/0031417; U.S. Patent Publication No. 2003/0224432; WO 03/093430; MacRae and Doudna (2007) Curr. Opin. Struct. Biol. 17:138;


SUMMARY OF THE INVENTION
A modified Dicer polypeptide is provided, which modified Dicer polypeptide exhibits enhanced catalytic activity. Also provided is a method for producing a small regulator RNA from a dsRNA, involving contacting a dsRNA with a subject modified Dicer. Small regulatory RNAs produced by a subject method find use in a variety of applications, including research and therapeutic applications.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-C depict domain structures and expression of human Dicer proteins.
FIGS. 2A-D depict single-turnover activity of Dicer proteins. FIG. 2A provides nucleotide sequences of 37ab double-stranded RNA substrate (upper sequence: SEQ ID NO:5; lower sequence: SEQ ID NO:6); and a nucleotide sequence of pre-hlet-7 (SEQ ID NO:7).
FIGS. 3A-C depict binding affinity of Dicer proteins to duplex RNAs.
FIGS. 4A-C depict kinetic analysis of wild-type and ΔDEAD hDcr proteins.
FIG. 5 depicts the results of a 1-anilino-naphthalene-8-sulfonate (ANS) binding assay.
FIGS. 6A and 6B depict interaction of human Dicer with TRBP mediated by DEAD domain.
FIG. 7 depicts the amino acid sequence of a wild-type human Dicer polypeptide.
FIG. 8 depicts an amino acid sequence of a DExD/H-box domain.
FIG. 9 depicts the amino acid sequence of an exemplary modified Dicer polypeptide, which lacks a DExD/H-box domain.
FIG. 10 depicts the amino acid sequence of an exemplary modified Dicer polypeptide, which has a single amino acid substitution in the DExD/H-box domain.
FIGS. 11A-I depict an amino acid sequence alignment of Dicer polypeptides from various mammalian species. Sequence 1: SEQ ID NO:1; Sequence 2: SEQ ID NO:21; Sequence 3: SEQ ID NO:22; Sequence 4: SEQ ID NO:23; Sequence 5: SEQ ID NO:24.



DEFINITIONS
The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
A “protein coding sequence” or a sequence that “encodes” a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ terminus and a translation stop codon at the 3′ terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic nucleic acids. A transcription termination sequence will usually be located 3′ to the coding sequence.
A “small interfering” or “short interfering RNA” or siRNA is a RNA duplex of nucleotides that is targeted to a gene interest (a “target gene”). An “RNA duplex” refers to the structure formed by the complementary pairing between two regions of a RNA molecule or between two separate RNA molecules. siRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene. In some embodiments, the length of the duplex of siRNAs is less than 30 nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length. In some embodiments, the length of the duplex is 19-25 nucleotides in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpin structure can also contain 3′ or 5′ overhang portions. In some embodiments, the overhang is a 3′ or a 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.
MicroRNAs (miRNAs) are encoded by genes, which encode transcripts containing short double-stranded RNA hairpins. mRNAs are transcribed as longer precursors, termed pre-miRNAs, which can be 50 to 80 nucleotides in length, and which are sometimes found in clusters and frequently found in introns. Upon transcription, miRNAs undergo nuclear cleavage by an RNase III endonuclease, producing the 60-70-nt stem-loop precursor miRNA (pre-miRNA) with a 5′ phosphate and a 2-nt 3 overhang. The pre-miRNAs are cleaved by Dicer about two helical turns away from the ends of the pre-miRNA stem loop, producing double-stranded RNA with strands that are approximately the same length (21 to 24 nucleotides), and possess the characteristic 5′-phosphate and 3′-hydroxyl termini. One of the strands of this short-lived intermediate accumulates as the mature miRNA and is subsequently incorporated into a ribonucleoprotein complex, the miRNP. mRNAs interact with target RNAs at specific sites to induce cleavage of the message or inhibit translation.
The term “naturally-occurring” as used herein as applied to a nucleic acid, a cell, or an organism, refers to a nucleic acid, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is naturally occurring.
As used herein the term “isolated” is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.
As used herein, the term “exogenous nucleic acid” refers to a nucleic acid that is not normally or naturally found in and/or produced by a given bacterium, organism, or cell in nature. As used herein, the term “endogenous nucleic acid” refers to a nucleic acid that is normally found in and/or produced by a given bacterium, organism, or cell in nature. An “endogenous nucleic acid” is also referred to as a “native nucleic acid” or a nucleic acid that is “native” to a given bacterium, organism, or cell.
The term “heterologous,” as used herein in the context of a genetically modified host cell, refers to a polypeptide wherein at least one of the following is true: (a) the polypeptide is foreign (“exogenous”) to (i.e., not naturally found in) the host cell; (b) the polypeptide is naturally found in (e.g., is “endogenous to”) a given host microorganism or host cell but is either produced in an unnatural (e.g., greater than expected or greater than naturally found) amount in the cell, or differs in nucleotide sequence from the endogenous nucleotide sequence such that the same encoded protein (having the same or substantially the same amino acid sequence) as found endogenously is produced in an unnatural (e.g., greater than expected or greater than naturally found) amount in the cell.
The term “heterologous,” as used herein in the context of a chimeric polypeptide, refers to two components that are defined by structures derived from different sources. For example, where “heterologous” is used in the context of a chimeric polypeptide (e.g., a chimeric modified Dicer enzyme), the chimeric polypeptide includes operably linked amino acid sequences that can be derived from different polypeptides (e.g., a first amino acid sequence from modified Dicer enzyme; and a second amino acid sequence other than a modified Dicer enzyme). Similarly, “heterologous” in the context of a polynucleotide encoding a chimeric polypeptide includes operably linked nucleotide sequences that can be derived from different coding regions (e.g., a first nucleotide sequence encoding a modified Dicer enzyme; and a second nucleotide sequence encoding a polypeptide other than a modified Dicer enzyme).
“Recombinant,” as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5′ or 3′ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see “DNA regulatory sequences”, below).
Thus, e.g., the term “recombinant” polynucleotide or “recombinant” nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
Similarly, the term “recombinant” polypeptide refers to a polypeptide which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino sequence through human intervention. Thus, e.g., a polypeptide that comprises a heterologous amino acid sequence is recombinant.
By “construct” or “vector” is meant a recombinant nucleic acid, generally recombinant DNA, which has been generated for the purpose of the expression and/or propagation of a specific nucleotide sequence(s), or is to be used in the construction of other recombinant nucleotide sequences.
The terms “DNA regulatory sequences,” “control elements,” and “regulatory elements,” used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate expression of a coding sequence and/or production of an encoded polypeptide in a host cell.
The term “transformation” is used interchangeably herein with “genetic modification” and refers to a permanent or transient genetic change induced in a cell following introduction of new nucleic acid (i.e., DNA exogenous to the cell). Genetic change (“modification”) can be accomplished either by incorporation of the new DNA into the genome of the host cell, or by transient or stable maintenance of the new DNA as an episomal element. Where the cell is a eukaryotic cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. In prokaryotic cells, permanent changes can be introduced into the chromosome or via extrachromosomal elements such as plasmids and expression vectors, which may contain one or more selectable markers to aid in their maintenance in the recombinant host cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e. in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.
“Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. As used herein, the terms “heterologous promoter” and “heterologous control regions” refer to promoters and other control regions that are not normally associated with a particular nucleic acid in nature. For example, a “transcriptional control region heterologous to a coding region” is a transcriptional control region that is not normally associated with the coding region in nature.
A “host cell,” as used herein, denotes an in vivo or in vitro eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which eukaryotic or prokaryotic cells can be, or have been, used as recipients for a nucleic acid (e.g., an expression vector that comprises a nucleotide sequence encoding one or more biosynthetic pathway gene products such as mevalonate pathway gene products), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector. For example, a subject prokaryotic host cell is a genetically modified prokaryotic host cell (e.g., a bacterium), by virtue of introduction into a suitable prokaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to (not normally found in nature in) the prokaryotic host cell, or a recombinant nucleic acid that is not normally found in the prokaryotic host cell; and a subject eukaryotic host cell is a genetically modified eukaryotic host cell, by virtue of introduction into a suitable eukaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.
The term “conservative amino acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm nih.gov/BLAST. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).
Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a modified Dicer polypeptide” includes a plurality of such polypeptide and reference to “the siRNA” includes reference to one or more siRNAs and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
DETAILED DESCRIPTION
A modified Dicer polypeptide is provided, which modified Dicer polypeptide exhibits enhanced catalytic activity. Also provided is a method for producing small regulator RNAs (e.g., siRNAs and miRNAs) from a dsRNA, involving contacting a dsRNA with a subject modified Dicer. siRNAs and miRNAs produced by a subject method find use in a variety of applications, including research and therapeutic applications.
Modified Dicer Polypeptides
The present invention provides modified Dicer polypeptides. A subject modified Dicer polypeptide exhibits enhanced catalytic activity, i.e., exhibits enhanced double-stranded RNA (dsRNA) endonuclease activity, compared an unmodified Dicer polypeptide, e.g., compared to a naturally-occurring Dicer polypeptide.
In some embodiments, the kcat of a subject modified Dicer polypeptide is higher than the kcat of an unmodified Dicer polypeptide. kcat is the turnover number (i.e., the number of times an enzyme active site converts substrate to product per unit time), and is expressed as an inverse time unit. In some embodiments, the Km of a subject modified Dicer polypeptide is lower than the Km of an unmodified Dicer polypeptide. Km is the Michaelis-Menten constant, i.e., the substrate concentration needed to achieve a half-maximum enzyme velocity. In some embodiments, the efficiency of a subject modified Dicer polypeptide is greater than the efficiency of an unmodified Dicer polypeptide, where efficiency is expressed as kcat divided by Km, or kcat×Km−1.
In some embodiments, the kcat of a subject modified Dicer polypeptide is higher than the kcat of an unmodified Dicer, e.g., the kcat of a subject modified Dicer polypeptide is at least about 25%, at least about 50%, at least about 100% (or 2-fold), at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold, or more than 100-fold, higher than the kcat of an unmodified Dicer.
In some embodiments, the kcat of a subject modified Dicer polypeptide is higher than the kcat of an unmodified Dicer, e.g., the kcat of a subject modified Dicer polypeptide is from about 25% higher to about 50% higher, from about 50% higher to about 2-fold higher to about 5-fold higher, from about 5-fold higher to about 10-fold higher, from about 10-fold higher to about 15-fold higher, from about 15-fold higher to about 20-fold higher, from about 20-fold higher to about 25-fold higher, from about 25-fold higher to about 30-fold higher, from about 30-fold higher to about 40-fold higher, from about 40-fold higher to about 50-fold higher, from about 50-fold higher to about 60-fold higher, from about 60-fold higher to about 70-fold higher, from about 70-fold higher to about 80-fold higher, from about 80-fold higher to about 90-fold higher, from about 90-fold higher to about 100-fold higher, or more than 100-fold higher, than the kcat of an unmodified Dicer.
For example, in some embodiments, the kcat of a subject modified Dicer polypeptide is higher than the kcat of a Dicer polypeptide having the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1, e.g., the kcat of a subject modified Dicer polypeptide is from about 25% higher to about 50% higher, from about 50% higher to about 2-fold higher to about 5-fold higher, from about 5-fold higher to about 10-fold higher, from about 10-fold higher to about 15-fold higher, from about 15-fold higher to about 20-fold higher, from about 20-fold higher to about 25-fold higher, from about 25-fold higher to about 30-fold higher, from about 30-fold higher to about 40-fold higher, from about 40-fold higher to about 50-fold higher, from about 50-fold higher to about 60-fold higher, from about 60-fold higher to about 70-fold higher, from about 70-fold higher to about 80-fold higher, from about 80-fold higher to about 90-fold higher, from about 90-fold higher to about 100-fold higher, or more than 100-fold higher, than the kcat of a Dicer polypeptide having the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1.
To measure kcat, any suitable double-stranded RNA substrate can be used. As one non-limiting example, a double-stranded RNA of the following sequence can be used:









(SEQ ID NO: 5)



5′-UGAGGUAGUAGGUUGUAUAGUUUGAAAGUUCACGAUU-3′


 



(SEQ ID NO: 6)



3′-UUACUCCAUCAUCCAACAUAUCAAACUUUCAAGUGCU-5′






where the two sequences form a double-stranded RNA. This double-stranded RNA (dsRNA) is referred to in the Examples as “37ab.”
Thus, for example, in some embodiments, the kcat of a subject modified Dicer polypeptide is higher than the kcat of a Dicer polypeptide having the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1, e.g., the kcat of a subject modified Dicer polypeptide is from about 25% higher to about 50% higher, from about 50% higher to about 2-fold higher to about 5-fold higher, from about 5-fold higher to about 10-fold higher, from about 10-fold higher to about 15-fold higher, from about 15-fold higher to about 20-fold higher, from about 20-fold higher to about 25-fold higher, from about 25-fold higher to about 30-fold higher, from about 30-fold higher to about 40-fold higher, from about 40-fold higher to about 50-fold higher, from about 50-fold higher to about 60-fold higher, from about 60-fold higher to about 70-fold higher, from about 70-fold higher to about 80-fold higher, from about 80-fold higher to about 90-fold higher, from about 90-fold higher to about 100-fold higher, or more than 100-fold higher, than the kcat of a Dicer polypeptide having the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1, when the 37ab dsRNA is used as a substrate.
In some embodiments, the kcat of a subject modified Dicer is from about 0.5×10−1 s−1 to about 1×10−5 s−1, e.g., from about 0.5×10−1 s−1 to about 1×10−1 s−1, from about 1×10−1 s−1 to about 5×10−1 s−1, from about 5×10−1 s−1 to about 1×10−2 s−1, from about 1×10−2 s−1 to about 5×10−2 s−1, from about 5×10−2 s−1 to about 1×10−3 s−1, from about 1×10−3 s−1 to about 5×104 s−1, or from about 5×104 s−1 to about 10−5 s−1.
In some embodiments, the Km of a subject modified Dicer polypeptide for a dsRNA substrate is lower than the Km of an unmodified Dicer polypeptide for the same dsRNA substrate, e.g., in some embodiments, the Km of a subject modified Dicer polypeptide for a dsRNA substrate is at least about 10% lower, at least about 15% lower, at least about 20% lower, at least about 25% lower, at least about 30% lower, at least about 35% lower, at least about 40% lower, at least about 45% lower, at least about 50% lower, at least about 60% lower, at least about 70% lower, at least about 80% lower, or at least about 90% lower, than the Km of an unmodified Dicer polypeptide. In some embodiments, the Km of a subject modified Dicer polypeptide for a dsRNA substrate is lower than the Km of a Dicer polypeptide having the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1, for the same dsRNA substrate, e.g., the Km of a subject modified Dicer polypeptide for a dsRNA substrate is at least about 10% lower, at least about 15% lower, at least about 20% lower, at least about 25% lower, at least about 30% lower, at least about 35% lower, at least about 40% lower, at least about 45% lower, at least about 50% lower, at least about 60% lower, at least about 70% lower, at least about 80% lower, or at least about 90% lower, than the Km of a Dicer polypeptide having the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1, for the same dsRNA substrate.
In some embodiments, the enzyme efficiency (the ratio of kcat to Km) of a subject modified Dicer polypeptide for processing a dsRNA substrate is greater than the efficiency of an unmodified Dicer polypeptide, e.g., the efficiency of a subject modified Dicer polypeptide is at least about 5-fold, at least about 7-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 100-fold, at least about 110-fold, at least about 120-fold, at least about 130-fold, at least about 140-fold, at least about 150-fold, at least about 160-fold, at least about 170-fold, at least about 180-fold, at least about 190-fold, at least about 200-fold, or more than 200-fold, greater than the efficiency of an unmodified Dicer polypeptide in processing the same substrate.
In some embodiments, the enzyme efficiency of a subject modified Dicer polypeptide for processing a dsRNA substrate is greater than the efficiency of a Dicer polypeptide having the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1, e.g., the enzyme efficiency of a subject modified Dicer polypeptide for processing a dsRNA substrate is at least about 5-fold, at least about 7-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 100-fold, at least about 110-fold, at least about 120-fold, at least about 130-fold, at least about 140-fold, at least about 150-fold, at least about 160-fold, at least about 170-fold, at least about 180-fold, at least about 190-fold, at least about 200-fold, or more than 200-fold, greater than the efficiency of a Dicer polypeptide having the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1 for processing the same dsRNA substrate.
In some embodiments, the enzyme efficiency of a subject modified Dicer polypeptide for processing the 37ab dsRNA substrate shown above is greater than the efficiency of a Dicer polypeptide having the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1, e.g., the enzyme efficiency of a subject modified Dicer polypeptide for processing a dsRNA substrate is at least about 5-fold, at least about 7-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 100-fold, at least about 110-fold, at least about 120-fold, at least about 130-fold, at least about 140-fold, at least about 150-fold, at least about 160-fold, at least about 170-fold, at least about 180-fold, at least about 190-fold, at least about 200-fold, or more than 200-fold, greater than the efficiency of a Dicer polypeptide having the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1 for processing the 37ab dsRNA substrate.
In some embodiments, a subject modified Dicer polypeptide lacks all or a portion of a DExD/H-box helicase domain The DExD/H-box helicase domain is an N-terminal domain found in many Dicer proteins, and is typically about 600 amino acids in length. In some embodiments, a subject modified Dicer polypeptide lacks from about 200 amino acids to about 250 amino acids, from about 250 amino acids to about 300 amino acids, from about 300 amino acids to about 350 amino acids, from about 350 amino acids to about 400 amino acids, from about 400 amino acids to about 450 amino acids, from about 450 amino acids to about 500 amino acids, from about 500 amino acids to about 550 amino acids, or from about 550 amino acids to about 600 amino acids of a DExD/H-box helicase domain. An exemplary DExD/H-box amino acid sequence is depicted in FIG. 8 (SEQ ID NO:2).
In some embodiments, a subject modified Dicer polypeptide lacks all or a portion of a DExD/H-box helicase domain, and comprises, in addition to two ribonuclease domains, a double-stranded RNA binding domain (dsRBD), a domain of unknown function (“DUF283”) domain, and a PAZ domain.
In some embodiments, a subject modified Dicer polypeptide has a length of from about 1100 amino acids to about 1150 amino acids, from about 1150 amino acids to about 1200 amino acids, from about 1200 amino acids to about 1250 amino acids, from about 1250 amino acids to about 1300 amino acids, from about 1300 amino acids to about 1325 amino acids, from about 1325 amino acids to about 1350 amino acids, from about 1350 amino acids to about 1375 amino acids, from about 1375 amino acids to about 1400 amino acids, from about 1400 amino acids to about 1425 amino acids, from about 1425 amino acids to about 1450 amino acids, from about 1450 amino acids to about 1475 amino acids, from about 1475 amino acids to about 1500 amino acids, from about 1500 amino acids to about 1525 amino acids, from about 1525 amino acids to about 1550 amino acids, from about 1550 amino acids to about 1575 amino acids, from about 1575 amino acids to about 1600 amino acids, from about 1600 amino acids to about 1625 amino acids, from about 1625 amino acids to about 1650 amino acids, from about 1650 amino acids to about 1675 amino acids, or from about 1675 amino acids to about 1700 amino acids.
The amino acid sequence of an exemplary modified Dicer polypeptide is depicted in FIG. 9. In some embodiments, a subject modified Dicer polypeptide: 1) lacks all or a portion of a DExD/H-box helicase domain; 2) has a length of from about 1100 amino acids to about 1150 amino acids, from about 1150 amino acids to about 1200 amino acids, from about 1200 amino acids to about 1250 amino acids, from about 1250 amino acids to about 1300 amino acids, from about 1300 amino acids to about 1320 amino acids; and 3) comprises an amino acid sequence having at least about 75%, at least about at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, amino acid sequence identity to a contiguous stretch of from about 1100 amino acids to about 1150 amino acids, from about 1150 amino acids to about 1200 amino acids, from about 1200 amino acids to about 1250 amino acids, from about 1250 amino acids to about 1300 amino acids, or from about 1300 amino acids to 1318 amino acids, of the amino acid sequence depicted in FIG. 9 (SEQ ID NO:3).
In some embodiments, a subject modified Dicer polypeptide comprises one or more amino acid substitutions, insertions, or deletions in the DExD/H-box domain (e.g., within amino acids 1 to about 604 of the amino acid sequence depicted in FIG. 7, and as set forth in SEQ ID NO:1), where the one or more amino acid substitutions, insertions, or deletions result in enhanced enzymatic activity (e.g., increased kcat and/or increased kcat×Km−1). In some embodiments, a subject modified Dicer polypeptide comprises one or more amino acid substitutions, insertions, or deletions in the DExD/H-box domain (e.g., within amino acids 63 to 71 of the amino acid sequence depicted in FIG. 7, and as set forth in SEQ ID NO:1), where the one or more amino acid substitutions, insertions, or deletions result in enhanced enzymatic activity (e.g., increased kcat and/or increased kcat×Km−1). In some embodiments, a subject modified Dicer polypeptide comprises one or more amino acid substitutions, insertions, or deletions in the DExD/H-box domain (e.g., within amino acids 175 to 178 of the amino acid sequence depicted in FIG. 7, and as set forth in SEQ ID NO:1), where the one or more amino acid substitutions, insertions, or deletions result in enhanced enzymatic activity (e.g., increased kcat and/or increased kcat×Km−1).
In some embodiments, a subject modified Dicer polypeptide comprises one or more amino acid substitutions in the DExD/H-box domain (e.g., within amino acids 1 to about 604 of the amino acid sequence depicted in FIG. 7, and as set forth in SEQ ID NO:1, where the one or more amino acid substitutions results in enhanced enzymatic activity (e.g., one or more of increased kcat, decreased Km, and increased kcat×Km−1).
As one non-limiting example, in some embodiments, a subject modified Dicer polypeptide comprises a K70A substitution in the DExD/H-box domain (e.g., within amino acids 1 to about 604 of the amino acid sequence depicted in FIG. 7, and as set forth in SEQ ID NO:1), or a K70A substitution at a corresponding amino acid position, compared to a Dicer polypeptide from a species other than human. For example, in some embodiments, a subject modified Dicer polypeptide: a) comprises a K70A substitution in the DExD/H-box domain, as shown in FIG. 10; b) shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity over a contiguous stretch of from about 1600 amino acids to about 1700 amino acids, from about 1700 amino acids to about 1800 amino acids, or from about 1800 amino acids to about 1921 amino acids, of the amino acid sequence depicted in FIG. 10 and set forth in SEQ ID NO:4; and c) enhanced enzymatic activity (e.g., one or more of increased kcat, decreased Km, and increased kcat×Km−1) compared to a Dicer polypeptide comprising the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1.
In some embodiments, a subject modified Dicer polypeptide comprises a K70A substitution in the DExD/H-box domain (e.g., within amino acids 1 to 604 of the amino acid sequence depicted in FIG. 7, and as set forth in SEQ ID NO:1), and shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity over a contiguous stretch of at least about 1100 amino acids, at least about 1200 amino acids, or at least about 1300 amino acids, of amino acids 605-1922 of the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1.
As another example, a subject modified Dicer polypeptide comprises one or more amino acid substitutions, insertions, or deletions in the DExD/H-box domain (e.g., within amino acids 63 to 71 of the amino acid sequence depicted in FIG. 7, and as set forth in SEQ ID NO:1), where the one or more amino acid substitutions, insertions, or deletions result in enhanced enzymatic activity (e.g., increased kcat and/or increased kcat×Km−1). For example, in some embodiments, a subject modified Dicer polypeptide comprises one or more amino acid substitutions in the amino acid sequence CLNTGSGKT (SEQ ID NO:8) of the amino acid sequence depicted in FIG. 7, or a corresponding amino acid sequence of a Dicer polypeptide other than a human Dicer polypeptide. As shown in the amino acid sequence alignment presented in FIGS. 11A-I, the amino acid sequence CLNTGSGKT (SEQ ID NO:8) is conserved among Dicer polypeptides from various mammalian species.
For example, in some embodiments, a subject modified Dicer polypeptide comprises one or more non-conservative amino acid substitutions in the amino acid sequence CLNTGSGKT (SEQ ID NO:8) of the amino acid sequence depicted in FIG. 7, or a corresponding amino acid sequence of a Dicer polypeptide other than a human Dicer polypeptide. Exemplary, non-limiting examples of amino acid substitutions include, e.g., CLNDGSGKT (SEQ ID NO:9); CLNTPSGKT (SEQ ID NO:10); CLSTGSGKT (SEQ ID NO:11); and the like. For example, in some embodiments, a subject modified Dicer polypeptide: a) comprises a non-conservative amino acid substitution in the amino acid sequence CLNTGSGKT (SEQ ID NO:8; e.g., amino acids 63-71 of the amino acid sequence depicted in FIG. 7, or a corresponding amino acid sequence from a Dicer polypeptide other than a human Dicer polypeptide; b) shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity over a contiguous stretch of from about 1600 amino acids to about 1700 amino acids, from about 1700 amino acids to about 1800 amino acids, or from about 1800 amino acids to about 1921 amino acids, of the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1; and c) enhanced enzymatic activity (e.g., increased kcat and/or increased kcat×Km−1) compared to a Dicer polypeptide comprising the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1.
As another example, a subject modified Dicer polypeptide comprises one or more amino acid substitutions, insertions, or deletions in the DExD/H-box domain (e.g., within amino acids 175-178 of the amino acid sequence depicted in FIG. 7, and as set forth in SEQ ID NO:1), where the one or more amino acid substitutions, insertions, or deletions result in enhanced enzymatic activity (e.g., increased kcat and/or increased kcat×Km−1). For example, in some embodiments, a subject modified Dicer polypeptide comprises one or more amino acid substitutions in the amino acid sequence DECH (SEQ ID NO:25) of the amino acid sequence depicted in FIG. 7, or a corresponding amino acid sequence of a Dicer polypeptide other than a human Dicer polypeptide. As shown in the amino acid sequence alignment presented in FIGS. 11A-I, the amino acid sequence DECH (SEQ ID NO:25) is conserved among Dicer polypeptides from various mammalian species.
For example, in some embodiments, a subject modified Dicer polypeptide comprises one or more non-conservative amino acid substitutions in the amino acid sequence DECH (SEQ ID NO:25) of the amino acid sequence depicted in FIG. 7, or a corresponding amino acid sequence of a Dicer polypeptide other than a human Dicer polypeptide. For example, in some embodiments, a subject modified Dicer polypeptide: a) comprises a non-conservative amino acid substitution in the amino acid sequence DECH (SEQ ID NO:25; e.g., amino acids 175-178 of the amino acid sequence depicted in FIG. 7, or a corresponding amino acid sequence from a Dicer polypeptide other than a human Dicer polypeptide; b) shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity over a contiguous stretch of from about 1600 amino acids to about 1700 amino acids, from about 1700 amino acids to about 1800 amino acids, or from about 1800 amino acids to about 1921 amino acids, of the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1; and c) enhanced enzymatic activity (e.g., increased kcat and/or increased kcat×Km−1) compared to a Dicer polypeptide comprising the amino acid sequence depicted in FIG. 7 and set forth in SEQ ID NO:1.
In some embodiments, a modified Dicer polypeptide is a chimeric modified Dicer polypeptide, e.g., the modified Dicer polypeptide comprises a heterologous polypeptide. A heterologous polypeptide can be present at the carboxyl terminus, at the amino terminus, or at an internal site within the modified Dicer polypeptide. Suitable heterologous polypeptides include, e.g., epitope tags, including, but not limited to, hemagglutinin, FLAG, and the like; proteins that provide for a detectable signal, including, but not limited to, fluorescent proteins, enzymes (e.g., β-galactosidase, alkaline phosphatase, luciferase, horse radish peroxidase, etc.), and the like; polypeptides that facilitate purification or isolation of the fusion protein, e.g., metal ion binding polypeptides such as 6His tags, glutathione-S-transferase; etc.
For example, in some embodiments, a subject chimeric modified Dicer polypeptide: 1) lacks all or a portion of a DExD/H-box helicase domain; 2) comprises an amino acid sequence having at least about 75%, at least about at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, amino acid sequence identity to a contiguous stretch of from about 1100 amino acids to about 1150 amino acids, from about 1150 amino acids to about 1200 amino acids, from about 1200 amino acids to about 1250 amino acids, from about 1250 amino acids to about 1300 amino acids, or from about 1300 amino acids to 1318 amino acids, of the amino acid sequence depicted in FIG. 9; and 3) comprises a heterologous, non-Dicer, polypeptide fused in-frame to the modified Dicer.
Compositions
The present invention provides a composition comprising a subject modified Dicer polypeptide.
A subject composition can comprise, in addition to the modified Dicer polypeptide, one or more of: a salt, e.g., NaCl, MgCl, KCl, MgSO4, etc.; a buffering agent, e.g., a Tris buffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20, etc.; a protease inhibitor; and the like.
In some embodiments, a modified Dicer polypeptide present in a subject composition is pure, e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more than 99% pure, where “% purity” means that the modified Dicer polypeptide is the recited percent free from other proteins (e.g., proteins other than a subject modified Dicer polypeptide), other macromolecules, or contaminants that may be present during the production of the modified Dicer polypeptide.
Nucleic Acids
The present invention provides a nucleic acid comprising a nucleotide sequence encoding a subject modified Dicer polypeptide. A subject nucleic acid is useful for producing a subject modified Dicer polypeptide. A subject nucleic acid is recombinant. The present invention further provides a composition comprising a subject nucleic acid.
In some embodiments, a subject nucleic acid is an expression construct, e.g., an expression vector comprising a nucleotide sequence encoding a subject modified Dicer polypeptide, where the expression construct provides for production of the encoded modified Dicer polypeptide in an appropriate host cell. Suitable expression vectors include, but are not limited to, baculovirus vectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral vectors (e.g. viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, and the like), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as E. coli and yeast).
Suitable vectors for the production of a subject modified Dicer polypeptide in a prokaryotic cell include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as Escherichia coli. The following vectors are provided by way of example, for bacterial host cells: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, lambda-ZAP vectors (Stratagene); pTrc99a, pKK223-3, pDR540, and pRIT2T (Pharmacia). However, any other plasmid or other vector may be used so long as it is compatible with the host cell.
A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIPS, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into Saccharomyces cerevisiae (see, for example, Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incorporated by reference herein). These vectors can replicate in E. coli due the presence of the pBR322 on, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin can be used. In an illustrative embodiment, a subject modified Dicer polypeptide is produced recombinantly utilizing an expression vector generated by sub-cloning a nucleotide sequence encoding a subject modified Dicer polypeptide.
In some embodiments, the expression construct comprises a mammalian expression vector. Suitable mammalian expression vectors include those that contain both prokaryotic sequences, to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.
A subject modified Dicer polypeptide can be produced using an expression vector containing a nucleic acid encoding a Dicer polypeptide, operably linked to at least one transcriptional regulatory sequence. Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and are selected to direct expression of the encoded modified Dicer protein. Accordingly, the term transcriptional regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). For instance, any of a wide variety of expression control sequences, sequences that control the expression of a DNA sequence when operatively linked to it, may be used in these vectors to express DNA sequences encoding Dicer polypeptides to recombinantly produce Dicer. Such useful expression control sequences, include, for example, a viral LTR, such as the LTR of the Moloney murine leukemia virus, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAG or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage X, polyhedron promoter, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoS, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.
Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter (see, e.g., U.S. Patent Publication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93; Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter (Harborne et al. (1992) Mol. Micro. 6:2805-2813), and the like (see, e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al. (2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol. 10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, e.g., a dps promoter, an spy promoter, and the like; a promoter derived from the pathogenicity island SPI-2 (see, e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al. (2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g., Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl. Acids Res. 12:7035-7056); and the like.
Non-limiting examples of suitable eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Suitable promoters for expression in yeast include, but are not limited to, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, and TP1; and, e.g., AOX1 (e.g., for use in Pichia).
In some embodiments, the promoter is an inducible promoter. Suitable inducible promoters include, but are not limited to, the pL of bacteriophage Plac; Ptrp; Ptac (Ptrp-lac hybrid promoter); an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter, e.g., a lacZ promoter; a tetracycline-inducible promoter; an arabinose inducible promoter, e.g., PBAD (see, e.g., Guzman et al. (1995) J. Bacteriol. 177:4121-4130); a xylose-inducible promoter, e.g., Pxyl (see, e.g., Kim et al. (1996) Gene 181:71-76); a GAL1 promoter; a tryptophan promoter; a lac promoter; an alcohol-inducible promoter, e.g., a methanol-inducible promoter, an ethanol-inducible promoter; a raffinose-inducible promoter; a heat-inducible promoter, e.g., heat inducible lambda PL promoter, a promoter controlled by a heat-sensitive repressor (e.g., CI857-repressed lambda-based expression vectors; see, e.g., Hoffmann et al. (1999) FEMS Microbiol Lett. 177(2):327-34); and the like.
In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant, et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II. A constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. DM Glover, 1986, IRL Press, Wash., D.C.). Alternatively, vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
Compositions
The present invention provides a composition comprising a subject nucleic acid. A subject composition can comprise, in addition to a subject nucleic acid, one or more of: a salt, e.g., NaCl, MgCl, KCl, MgSO4, etc.; a buffering agent, e.g., a Tris buffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20, etc.; a nuclease inhibitor; glycerol; and the like.
Genetically Modified Host Cells
The present invention provides genetically modified host cells comprising a subject nucleic acid. Suitable host cells include, e.g., prokaryotic host cells (e.g., prokaryotic cells in vitro); and eukaryotic cells that are cultured as single-celled entities in vitro. Eukaryotic host cells include, e.g., insect cells; primary mammalian cells; immortalized mammalian cell lines; and the like. The present invention further provides composition comprising a subject genetically modified host cell.
Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus sp., Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al. (1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302. Examples of Salmonella strains which can be employed in the present invention include, but are not limited to, Salmonella typhi and S. typhimurium. Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Typically, the laboratory strain is one that is non-pathogenic. Non-limiting examples of other suitable bacteria include, but are not limited to, Bacillus subtilis, Pseudomonas pudita, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and the like. In some embodiments, the host cell is Escherichia coli. 
Suitable eukaryotic host cells include, but are not limited to, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas reinhardtii, and the like.
Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.
Suitable methods of genetic modification of a host cell include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e. in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995. To generate a subject genetically modified host cell, a subject nucleic acid is introduced stably or transiently into a host cell, using established techniques, including, but not limited to, electroporation, lithium acetate transformation, calcium phosphate precipitation, DEAE-dextran mediated transfection, liposome-mediated transfection, and the like. For stable transformation, a nucleic acid will generally further include a selectable marker, e.g., any of several well-known selectable markers such as neomycin resistance, ampicillin resistance, tetracycline resistance, chloramphenicol resistance, kanamycin resistance, and the like.
Compositions
The present invention provides a composition comprising a subject genetically modified host cell. A subject composition comprises a subject genetically modified host cell, and will in some embodiments comprise one or more further components, which components are selected based in part on the intended use of the genetically modified host cell, storage considerations, etc. Suitable components include, but are not limited to, salts; buffers; stabilizers; protease-inhibiting agents; nuclease-inhibiting agents; cell membrane- and/or cell wall-preserving compounds, e.g., glycerol, dimethylsulfoxide, etc.; nutritional media appropriate to the cell; and the like. In some embodiments, the cells are lyophilized
Production of a Subject Modified Dicer Polypeptide
A host cell is genetically modified with a subject nucleic acid, such that a subject modified Dicer polypeptide is produced in the genetically modified host cell, and the encoded modified Dicer polypeptide is produced by the cell. The genetically modified host cell is cultured in vitro under suitable conditions such that the modified Dicer polypeptide is produced. Where the nucleotide sequence encoding a subject modified Dicer polypeptide is operably linked to an inducible promoter, an inducer is added to the culture medium in which the genetically modified host cell is cultured.
The modified Dicer polypeptide can be recovered and isolated from the genetically modified host cell. In some embodiments, the modified Dicer polypeptide is purified, e.g., is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% pure. Any convenient protein purification procedures may be employed, where suitable protein purification methodologies are described in Guide to Protein Purification, (Deuthser ed.) (Academic Press, 1990). For example, a lysate may be prepared from a genetically modified host cell that expresses a subject modified Dicer polypeptide, and purified using any of a number of standard protein purification methods, e.g., high performance liquid chromatography, size exclusion chromatography, gel electrophoresis, affinity chromatography, and the like.
Utility
A subject modified Dicer polypeptide is useful for producing small regulatory RNAs, which in turn are useful in a number of applications, including basic research applications, drug screening/target validation, large scale functional library screening, and therapeutic applications. Thus, the present invention provides methods of producing a small regulatory RNA molecule from a substrate dsRNA molecule Small regulatory RNA molecules that can be produced using a subject method include siRNA and miRNA.
Methods of Producing a Small Regulatory RNA Molecule
The present invention provides methods of producing small regulatory RNA from a substrate dsRNA molecule, the methods generally involving contacting the substrate dsRNA molecule with a subject modified Dicer polypeptide, where the modified Dicer polypeptide efficiently produces a small regulatory RNA using the substrate dsRNA molecule. The methods described below are directed to producing siRNA; however, a subject method can be adapted for producing miRNA.
In some embodiments, a subject method provides for production of a plurality of small regulatory RNA molecules, e.g., a plurality of siRNA molecules or a plurality of miRNA molecules. By “plurality” is meant at least 2, at least about 5, or at least about 10, where the number of distinct siRNA or miRNA molecules produced from a given substrate dsRNA molecule in the subject methods can depend on the length of the substrate dsRNA molecule, but may be as high as about 25 or higher, e.g., about 100, or about 400 or higher.
The siRNA or miRNA product molecules can range in length from about 10 nucleotides (nt) to about 25 nt, e.g., from about 10 nt to about 15 nt, from about 15 nt to about 20 nt, or from about 20 nt to about 25 nt. In some embodiments, a subject modified Dicer polypeptide produces siRNA product molecules having a length of from about 19 nt to about 24 nt, from about 20 nt to about 24 nt, from about 21 nt to about 24 nt, or from about 21 nt to about 23 nt. In some embodiments, a subject modified Dicer polypeptide produces siRNA product molecules, where at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, of the siRNA molecules have a length of from 21 nt to 23 nt.
A subject modified Dicer polypeptide is contacted with a substrate dsRNA molecule. The length of the parent dsRNA molecule can vary, but generally the length is at least about 300 bp, at least about 500 bp, or at least about 1000 bp, where the length may be as long as about 2000 bp or longer, but often does not exceed about 8000 bp, e.g., about 6000 bp.
The dsRNA substrate can comprise two hybridized strands of polymerized ribonucleotide. The dsRNA substrate can include modifications to either the phosphate-sugar backbone or the nucleoside. For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or a sulfur heteroatom. Modifications in RNA structure may be tailored to allow specific genetic inhibition while avoiding an adverse response in the cell harboring the RNA. Likewise, bases may be modified to block the activity of adenosine deaminase. The dsRNA substrate may be produced enzymatically or by partial/total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.
The dsRNA substrate is formed by a single self-complementary RNA strand or by two complementary RNA strands. dsRNA substrates comprising a nucleotide sequence identical to a portion of a target gene may be employed. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence are also of interest. Thus, sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). In some embodiments, there is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, sequence identity between the siRNA or miRNA and the portion of a target gene may be of interest. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript under stringent conditions (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed by washing; or conditions that are at least as stringent as these representative conditions). The length of the identical nucleotide sequences may be, for example, at least about 25, about 50, about 100, about 200, about 300 or about 400 bases or longer. In certain embodiments, the dsRNA substrate is from about 400 to about 800 bases in length. In certain embodiments 100% sequence identity between the RNA and the target gene is not required to practice inhibition applications of the invention. Thus the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
The dsRNA substrate can be synthesized either in vivo or in vitro. Furthermore, the dsRNA substrate can be synthesized in vitro in a living cell, or in a cell-free in vitro system. Endogenous polymerase of the cell can mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro. For transcription from a transgene in vivo or an expression construct, a regulatory region (e.g., promoter, enhancer, silencer, splice donor and acceptor, polyadenylation) may be used to transcribe the dsRNA strand (or strands). In some embodiments, the RNA strands of the dsRNA substrate are polyadenylated. In other embodiments, the RNA strands of the dsRNA substrate are not polyadenylated. In some embodiments, the RNA strands of the dsRNA substrate are capable of being translated into a polypeptide by a cell's translational apparatus or in a cell-free in vitro translation system. In some embodiments, the RNA strands of the dsRNA substrate are not capable of being translated into a polypeptide by a cell's translational apparatus or in a cell-free in vitro translation system.
The dsRNA substrate can be chemically or enzymatically synthesized by manual or automated reactions. The dsRNA substrate can be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, or SP6), e.g., using an expression construct encoding the dsRNA as template. The use and production of expression constructs are known in the art (see WO 97/32016; U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693; and the references cited therein). If synthesized chemically or by in vitro enzymatic synthesis, the RNA can be purified prior to introduction into the cell. For example, RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography or a combination thereof. Alternatively, the dsRNA construct may be used with no or a minimum of purification to avoid losses due to sample processing. The dsRNA construct may be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to promote annealing, and/or stabilization of the duplex strands.
In some embodiments, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, of the substrate dsRNA is cleaved to produce an miRNA or siRNA product.
In the reaction composition (e.g., the composition comprising a subject modified Dicer polypeptide and a dsRNA substrate), the amount of Dicer present in the composition can vary, and can be in a range of from about 20 ng/μl to about 160 ng/μl, e.g., from about 20 ng/μl to about 40 ng/μl, from about 40 ng/μl to about 60 ng/μl, from about 60 ng/μl to about 80 ng/μl, from about 80 ng/μl to about 100 ng/μl, from about 100 ng/μl to about 120 ng/μl, from about 120 ng/μl to about 140 ng/μl, or from about 140 ng/μl to about 160 ng/μl.
In some embodiments, the reaction composition (e.g., the composition comprising a subject modified Dicer polypeptide and a dsRNA substrate) is an aqueous composition, where the composition may include one or more additional components, e.g., buffers; salts such as NaCl, MgCl2, and the like; EDTA; DTT; ATP; and the like.
As discussed above, a subject method comprises contacting a subject modified Dicer polypeptide with a substrate dsRNA in a reaction composition that is then maintained under conditions sufficient to produce the desired siRNA or miRNA product. In some embodiments, a subject method is a cell-free in vitro method, by which is meant that the method occurs in a cell free environment, e.g., not inside of a cell or in the presence of cells. As such, in some embodiments, a subject method involves producing a product composition comprising an siRNA product or a miRNA product, where the product composition is produced by contacting a substrate dsRNA and a subject modified Dicer polypeptide, as described above, where the product composition is produced in a cell-free in vitro reaction, i.e., in vitro and outside of a cell.
In some embodiments, a subject modified Dicer polypeptide and a substrate dsRNA are contacted in reaction composition that includes a sufficient amount of Mg2+ to ensure adequate Dicer activity, where the amount of Mg2+ can range from about 0.5 mM to about 1.0 mM, or from about 2.5 mM to about 5.0 mM. In some embodiments, the reaction composition is free of ATP, and in other embodiments, 1 mM ATP is used in the reaction composition.
The reaction mixture is typically maintained under incubation conditions sufficient to produce the desired small regulatory RNA product. The reaction mixture is typically maintained at a temperature that ranges from about 30° C. to about 37° C., e.g., from about 35° C. to about 37° C. The reaction is carried out for a period of time ranging from about 15 minutes to about 24 hours, e.g., from about 15 minutes to about 30 minutes, from about 30 minutes to about 60 minutes, from about 1 hour to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 8 hours, from about 8 hours to about 12 hours, from about 12 hours to about 16 hours, or from about 16 hours to about 24 hours.
The small regulatory RNA product, e.g., the siRNA product or the miRNA produce, produced by a subject method may be used as is or further processed prior to use, e.g., separated from other components of the reaction mixture, e.g., the modified Dicer protein, any remaining dsRNA substrate, salts, buffers, etc. Any convenient separation protocol may be employed, including gel purification, chromatographic separation based on molecular weight or affinity resins, and classical precipitation, and the like.
Research Applications
A small regulatory RNA can be used for modifying biological functions in a cell (e.g., a cell growing as a single-cell suspension in vitro; a cell in a multicellular organism; etc.), such as for example, RNA interference, gene knockdown or knockout, generating expression mutants, modulating cell growth, differentiation, signaling or a combination thereof. Thus, in some embodiments, a subject method involves: a) producing an siRNA using a subject method (i.e., using a subject modified Dicer polypeptide); and b) introducing the siRNA so produced into a cell (e.g., into a cell in vitro; or into a non-human cell in a multi-cellular organism in vivo).
One representative utility is a method of identifying gene function in an organism, e.g., higher eukaryotes comprising the use of the product siRNA to inhibit the activity of a target gene of previously unknown function. Instead of the time consuming and laborious isolation of mutants by traditional genetic screening, functional genomics using the subject product siRNA determines the function of uncharacterized genes by employing the siRNA to reduce the amount and/or alter the timing of target gene activity. The product siRNA can be used in determining potential targets for pharmaceutics, understanding normal and pathological events associated with development, determining signaling pathways responsible for postnatal development/aging, and the like. The increasing speed of acquiring nucleotide sequence information from genomic and expressed gene sources, including total sequences for mammalian genomes, can be coupled with use of the product siRNA to determine gene function in a cell or in a whole organism. The preference of different organisms to use particular codons, searching sequence databases for related gene products, correlating the linkage map of genetic traits with the physical map from which the nucleotide sequences are derived, and artificial intelligence methods may be used to define putative open reading frames from the nucleotide sequences acquired in such sequencing projects.
A simple representative assay involves inhibition of gene expression according to the partial sequence available from an expressed sequence tag (EST). Functional alterations in growth, development, metabolism, disease resistance, or other biological processes would be indicative of the normal role of the EST's gene product.
The ease with which the product siRNA construct can be introduced into an intact cell/organism containing the target gene allows the siRNA products to be used in high throughput screening (HTS). For example, individual clones from the library can be replicated and then isolated in separate reactions, but preferably the library is maintained in individual reaction vessels (e.g., a 96-well microtiter plate) to minimize the number of steps required to practice the invention and to allow automation of the process. Solutions containing the product siRNAs that are capable of inhibiting the different expressed genes can be placed into individual wells positioned on a microtiter plate as an ordered array, and intact cells/organisms in each well can be assayed for any changes or modifications in behavior or development due to inhibition of target gene activity.
The siRNA can be fed directly to, injected into, the cell/organism containing the target gene. The siRNA may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing an organism in a solution containing the siRNA. Methods for oral introduction include direct mixing of RNA with food of the organism. Physical methods of introducing nucleic, acids include injection directly into the cell or extracellular injection into the organism of an RNA solution. The siRNA may be introduced in an amount that allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of siRNA material may yield more effective inhibition; lower doses may also be useful for specific applications Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.
The function of the target gene can be assayed from the effects it has on the cell/organism when gene activity is inhibited. This screening could be amenable to small subjects that can be processed in large number, for example, tissue culture cells derived from invertebrates or vertebrates (e.g., mammals, such as murines, non-human primates, and humans).
If a characteristic of an organism is determined to be genetically linked to a polymorphism through RFLP or QTL analysis, the present invention can be used to gain insight regarding whether that genetic polymorphism might be directly responsible for the characteristic. For example, a fragment defining the genetic polymorphism or sequences in the vicinity of such a genetic polymorphism can be amplified to produce a dsRNA from which siRNA is prepared according to the subject methods, which siRNA can be introduced to the organism or cell, and whether an alteration in the characteristic is correlated with inhibition can be determined.
The present invention is useful in allowing the inhibition of essential genes. Such genes may be required for cell or organism viability at only particular stages of development or cellular compartments. The functional equivalent of conditional mutations may be produced by inhibiting activity of the target gene when or where it is not required for viability. The invention allows addition of siRNA at specific times of development and locations in the organism without introducing permanent mutations into the target genome.
In situations where alternative splicing produces a family of transcripts that are distinguished by usage of characteristic exons, an siRNA can target inhibition through the appropriate exons to specifically inhibit or to distinguish among the functions of family members.
Therapeutic Applications
An siRNA produced using a subject method also finds use in a variety of therapeutic applications in which it is desired to selectively modulate one or more target genes in a host, e.g., a whole animal, or a portion thereof, e.g., a tissue, an organ, etc, as well as in cells present such an animal, tissue, or organ. In such methods, an effective amount of an siRNA is administered to the host or target portion thereof. By “effective amount” is meant a dosage sufficient to selectively modulate expression of the target gene(s), as desired. As indicated above, in many embodiments of this type of application, methods are employed to reduce/inhibit expression of one or more target genes in the host or portion thereof in order to achieve a desired therapeutic outcome.
In some embodiments, a subject method comprises: preparing an siRNA according to a subject method (i.e., using a subject modified Dicer polypeptide); and administering an effective amount of the siRNA to an individual in need thereof.
Depending on the nature of the condition being treated, the target gene may be a gene derived from the cell, an endogenous gene, a pathologically mutated gene, e.g. a cancer-causing gene, one or more genes whose expression causes or is related to heart disease, lung disease, Alzheimer's disease, Parkinson's disease, diabetes, arthritis, etc.; a transgene, or a gene of a pathogen which is present in the cell after infection thereof, e.g., a viral (e.g., HIV-Human Immunodeficiency Virus; Hepatitis B virus; Hepatitis C virus; Herpes-simplex virus-1 and -2; Varicella Zoster (Chicken pox and Shingles); Rhinovirus (common cold and flu); any other viral form); or bacterial pathogen. Depending on the particular target gene and the dose of siRNA delivered, the procedure may provide partial or complete loss of function for the target gene. Lower doses of injected material and longer times after administration of siRNA may result in inhibition in a smaller fraction of cells.
An siRNA produced using a subject method finds use in the treatment of a variety of conditions in which the modulation of target gene expression in a mammalian host is desired. By treatment is meant that at least an amelioration of the symptoms associated with the condition afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g. prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition.
A variety of hosts are treatable using an siRNA. Generally such hosts are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, and non-human primates such as chimpanzees and monkeys). In some embodiments, the hosts will be humans.
The present invention is not limited to modulation of expression of any specific type of target gene or nucleotide sequence. Representative classes of target genes of interest include but are not limited to: developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors); oncogenes (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETS1, ETV6, FOR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM 1, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor genes (e.g., APC, BRCA 1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, TP53, and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases, cellulases, chalcone synthases, chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, granule-bound starch synthases, GTPases, helicases, hemicellulases, integrases, inulinases, invertases, isomerases, kinases, lactases, Upases, lipoxygenases, lysozymes, nopaline synthases, octopine synthases, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, phytases, plant growth regulator synthases, polygalacturonases, proteinases and peptidases, pullanases, recombinases, reverse transcriptases, RUBISCOs, topoisomerases, and xylanases); chemokines (e.g. CXCR4, CCR5); the RNA component of telomerase; vascular endothelial growth factor (VEGF); VEGF receptor; tumor necrosis factors nuclear factor kappa B; transcription factors; cell adhesion molecules; Insulin-like growth factor; transforming growth factor beta family members; cell surface receptors; RNA binding proteins (e.g. small nucleolar RNAs, RNA transport factors); translation factors; telomerase reverse transcriptase); etc.
The siRNA can be introduced into the target cell(s) using any convenient protocol, where the protocol will vary depending on whether the target cells are in vitro or in vivo.
Where the target cells are in vivo, the siRNA can be administered to the host comprising the cells using any convenient protocol, where the protocol employed is typically a nucleic acid administration protocol, where a number of different such protocols are known in the art. The following discussion provides a review of representative nucleic acid administration protocols that may be employed. The nucleic acids may be introduced into tissues or host cells by any number of routes, including microinjection, or fusion of vesicles. Jet injection may also be used for intra-muscular administration, as described by Furth et al. (1992), Anal Biochem 205:365-368. The nucleic acids may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or “gene gun” as described in the literature (see, for example, Tang et al. (1992), Nature 356:152-154), where gold microprojectiles are coated with the DNA, then bombarded into skin cells.
For example, the d-siRNA agent can be fed directly to, injected into, the host organism containing the target gene. The agent may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, etc. Methods for oral introduction include direct mixing of RNA with food of the organism. Physical methods of introducing nucleic acids include injection directly into the cell or extracellular injection into the organism of an RNA solution.
In certain embodiments, a hydrodynamic nucleic acid administration protocol is employed. Where the agent is a ribonucleic acid, the hydrodynamic ribonucleic acid administration protocol described in detail below is of particular interest. Where the agent is a deoxyribonucleic acid, the hydrodynamic deoxyribonucleic acid administration protocols described in Chang et al., J. Virol. (2001) 75:3469-3473; Liu et al., Gene Ther. (1999) 6:1258-1266; Wolff et al., Science (1990) 247: 1465-1468; Zhang et al., Hum. Gene Ther. (1999) 10:1735-1737: and Zhang et al., Gene Ther. (1999) 7:1344-1349; are of interest.
Additional nucleic acid delivery protocols of interest include, but are not limited to: those described in U.S. Pat. Nos. 5,985,847 and 5,922,687 (the disclosures of which are herein incorporated by reference); Acsadi et al., New Biol. (1991) 3:71-81; Hickman et al., Hum. Gen. Ther. (1994) 5:1477-1483; and Wolff et al., Science (1990) 247: 1465-1468; etc.
An siRNA (also referred to as an “agent” or an “active agent”) can be administered to the host using any convenient means capable of resulting in the desired modulation of target gene expression. Thus, the agent can be incorporated into a variety of formulations for therapeutic administration. More particularly, the agents can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration.
In pharmaceutical dosage forms, the agents may be administered alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.
Suitable delivery reagents for administration of an siRNA include the Mints Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine); and liposomes.
For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
The agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
The agents can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. An active agent can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more agents Similarly, unit dosage forms for injection or intravenous administration may comprise the agent(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and non-human animal subjects, each unit containing a predetermined quantity of an active agent calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
Those of skill in the art will readily appreciate that dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Preferred dosages for a given active agent are readily determinable by those of skill in the art by a variety of means.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
Example 1
Generation and Characterization of a Modified Dicer
Materials and Methods
RNA Substrates
A 73 nucleotide (nt) human let-7 (hlet-7) hairpin RNA was transcribed by T7 RNA polymerase from a construct built in a hammerhead-hepatitis delta virus double ribozyme system. All of other RNA substrates used in this study were synthesized by IDT (Integrated DNA Technologies, Inc, Coralville, Iowa). All of the RNAs were purified by 16% urea-PAGE. For both filter binding and dicing assays, the purified RNA substrates were 5′-end 32P labeled with T4 polynucleotide kinase (New England Biolab, Inc. Beverly, Mass.). The RNA substrates used in this study are:







hlet-7,


(SEQ ID NO: 7)


5′-UGAGGUAGUAGGUUGUAUAGUUUUAGGGUCACACCCACCACUGGGA


 


GAUAACUAUACAAUCUACUGUCUUACC-3′;


 


37a,


(SEQ ID NO: 5)


5′-UGAGGUAGUAGGUUGUAUAGUUUGAAAGUUCACGAUU-3′;


and


 


37b,


(SEQ ID NO: 6)


5′-UCGUGAACUUUCAAACUAUACAACCUACUACCUCAUU-3′.






Generation of hDicer Proteins

To generate human Dicer (hDcr) recombinant proteins, a full length wild-type and ΔDEAD (deletion of DEAD domain) hDcr cDNAs (accession number NP—803187) was generated by PCR with primer sets of hDcr-F/hDcr-R and DEAD-F/hDcr-R, respectively (see below). The PCR products were cloned into pFastBac plasmid (Invitrogen) after Sfo I and Xho I digestions. All of other constructs to produce hDcr-derivative proteins were generated by PCR using QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene) with primer set of DUF-F/DUF-R to delete DUF283, and with primer set of dsRBD-F/dsRBD-R to delete dsRBD domain (see the sequences below). For generation of double-deletion of DEAD and dsRBD domains, ΔDEAD hDcr cDNA was used as template in a PCR reaction with primer set of dsRBD-F/dsRBD-R. For generation of hWalker (a point-mutation in ATP-binding motif in DEAD domain), a PCR was performed with the primers of hW-F and hW-R in the presence of wild-type hDcr cDNA. The corresponding recombinant Bacmid DNAs were obtained by transforming pFastBac plasmids into competent DH10Bac E. Coli cells (Invitrogen) and these Bacmid DNAs were transfected into Sf9 cells with FuGene Transfection Reagent (Roche Applied Science) for generating baculovirus, which is used to produce of recombinant hDcr proteins in Sf9 cells. The hDcr proteins were purified through Ni2+-affinity followed by gel filtration column of HiLoad 16/60 Superdex 200 (GE Healthcare).
The PCR oligos (primers) are shown below (restriction sites for Sfo I and Xho I are underlined):









hDcr-F:



(SEQ ID NO: 12)



5′-GGGGGCGCCATGAAAAGCCCTGCTTTGCAACCCCTCAG



 



CATGGCAG-3′;



 



hDcr-R:



(SEQ ID NO: 13)



5′-CCCCTCGAGTCAGCTATTGGGAACCTGAGGTTGATTAGC-3′;



 



DEAD-F:



(SEQ ID NO: 14)



5′-GGGGGCGCCATGGATGATGATGACGTTTTCCCACCATA



 



TGTGTTG-3′;



 



DUF-F:



(SEQ ID NO: 15)



5′-CGAGTCACAATCAACACGGACCATTTGATGCCAGTTGG



 



GAAAGAG-3′;



 



DUF-R:



(SEQ ID NO: 16)



5′-CCCAACTGGCATCAAATGGTCCGTGTTGATTGTGACTC



 



GTGGACC-3′;



 



dsRBD-F:



(SEQ ID NO: 17)



5′-GAAAAGTTTTCTGCAAATAATCAACCTCAGGTTCCCAA



 



TAGCTG-3′;



 



dsRBD-R:



(SEQ ID NO: 18)



5′-GGGAACCTGAGGTTGATTATTTGCAGAAAACTTTTCTA



 



TTAGTGGC-3′;



 



hW-F:



(SEQ ID NO: 19)



5′-AACACTGGCTCAGGGGCGACATTTATTGCAGTAC-3′;



 



hW-R:



(SEQ ID NO: 20)



5′-GTACTGCAATAAATGTCGCCCCTGAGCCAGTGTT-3′.






Filter Binding Assay

A serial dilution of proteins were incubated in a buffer containing 20 mM Tris-Cl (pH 7.5), 25 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol (DTT), 1% glycerol and  ̃0.5-1 nM (1500 c.p.m.) of 32P-labeled duplex RNA substrate (one strand was labeled) at room temperature for 60 min in a volume of After this incubation, 25 μl of each reaction were used for dot-blotting, which was conducted in a dot-blot apparatus equipped with 3 membranes of Tuffryn, Protran and Nytran (from top to bottom). After dried, the bound (on Protran) or free (on Nytran) RNAs were detected and quantified with Phosphoimager (GE Healthcare). Percentage of bound RNAs was calculated by 100 multiplied by the ratio of radioactivity (volumes) detected on the Protran membrane over the total input volumes (radioactivity). The bound RNAs (%) (Y-axle) were plotted against protein concentrations (X-axle). Kd was determined by global fitting to the equation of kobsd=(kmax×[Dicer]) (K1/2+[Dicer])−1, here kobsd is the observed rate constant at a particular protein concentration, kmax is the maximal rate constant with saturating protein, and K1/2 (or Kd) is the protein concentration that provides half the maximal rate. Curve fitting was conducted with KleidaGraph (Synery Software).
In Vitro dsRNase Activity Assay
For a dsRNase assay (dicing assay), a high specific radioactivity dsRNA substrate was 5′-end labeled with (γ-32P)ATP. The labeled RNA was either self-annealed (forming a hairpin RNA substrate) or annealed with its complementary oligo. An in vitro dsRNase assay was performed with certain amount of hDcr protein and certain amount of RNA substrate (indicated in figure legends) at 30° C. for certain period of time in a volume of 10 μl (unless otherwise indicated) of 20 mM Tris-Cl (pH 6.5), 1.5 mM MgCl2, 25 mM NaCl, 1 mM DTT and 1% glycerol. Reactions were stopped by addition of 1.2 volume of loading buffer (95% formamide, 18 mM EDTA, 0.025% SDS, 0.1% xylene cyanol and 0.1% bromphenol blue). After heating at 75° C. for 10 min, the samples were analyzed by electrophoresis on a 15% polyacrylamide-7M urea gel run in TBE buffer. The gel was dried and the products were detected and quantified by Phosphoimager (GE Healthcare).
Kinetic Analysis of Human Dicer Proteins
A single-turnover experiment was performed in 90 μl reaction mixture containing 1-2 nM of label duplex RNA substrate and 60 nM of protein. 10 μl of reaction mixture were taken out and put into 12 μl of RNA loading buffer at the time points of 0, 0.5, 1, 2, 2.5, 5, 10, 20, 40, and 80 min, respectively. The RNAs in the aliquots were fractioned by 15% urea-polyacrylamide gel electrophoresis and images were quantified by Phosphorimager. Graphs were made by using KleidaGraph.
In order to obtain serial initial rates of human Dicer proteins, multiple-turnover experiments were performed in 90 μl reaction mixture containing 5 nM protein and duplex RNA at concentration of 25, 50, 75, 100, 150, 225, and 375 nM, respectively. Aliquots were taken at the time points of 0, 0.5, 1, 2, 2.5, 5, 10, 20, 40, and 80 min, respectively and analyzed by gel electrophoresis. After quantification, initial rates were determined by linear regression (from 0 to 10 min) using Excel. Km was determined by KleidaGraph global fitting to the equation of V=(Vmax×(Km+S)−1, where V is the velocity or initial rate, S is target RNA concentration.
Results
Dicer's DExD/H-Box Domain Inhibits Single-Turnover dsRNA Cleavage Rates
To investigate dsRNA recognition and cleavage by human Dicer, the wild-type (accession no. NP—803187) and five mutant forms of recombinant human Dicer (hDcr) protein were prepared (FIG. 1A). Specifically, a point mutation of lysine to alanine at position 70 (K70A) in the ATP-binding motif (hWalker) and a deletion of amino acids 1-604 spanning the entire DExD/H-box domain (ΔDEAD) were created to analyze the functional contributions of the DExD/H-box domain To explore the role of the C-terminal double-stranded RNA binding domain (dsRBD) and the domain of unknown function (DUF283) domains, hDcr variants lacking these domains were prepared (ΔdsRBD1844-1922 and ΔDUF630-709, respectively). A more substantially truncated form of hDcr (2DD) was prepared lacking both the DExD/H-box and the dsRBD domains. Each Dicer variant was produced using a baculovirus expression system and purified by Ni2+-affinity chromatography followed by gel filtration chromatography after site-specific protease cleavage to remove the N-terminal His6 affinity tag. In each case, 0.5-1.0 mg of purified recombinant protein was obtained routinely from one liter of cell culture (FIG. 1B). Activity assays showed that all five hDcr variants were able to cleave double-stranded RNA substrates (FIG. 1C).
FIGS. 1A-C. Domain structures and expression of human Dicers (hDcrs). A, Schematic illustration of the domain structure of different hDcrs; B, Polyhistidine-tagged recombinant proteins, expressed in a baculovirus expression system and purified by gel filtration after N2+-affinity chromatography and analyzed by 10% SDS-PAGE; C, A dsRNase activity assay (with 60 nM protein and 2-4 nM 32P-labeled duplex RNA 37ab), showing all of recombinant proteins are active.
To assess the contributions of various hDcr domains to dicing activity, we first tested the catalytic activity of each hDcr variant under single-turnover conditions using two different substrates. One substrate was a double-stranded RNA substrate containing 2-nucleotide 3′ overhangs on either end (37ab), whereas the other was the human pre-let7 hairpin RNA (FIG. 2A). In the presence of excess protein, the wild-type hDcr enzyme catalyzed cleavage of the 37ab substrate to yield 22-nt. products at an initial rate of 0.18 fmol min−1 (FIG. 2B, D). Deletion of either the dsRBD or the DUF domain significantly reduced the observed cleavage rate under these single-turnover conditions. These results are consistent with previous data showing that deletion of the dsRBD resulted in decreases of 1.9- and 2.5-fold cleavage rate for dsRNA and hairpin RNA, respectively (Zhang et al, 2004). Surprisingly, however, deletion or mutation of the DExD/H-box domain significantly enhanced the cleavage rate relative to that observed for the wild-type enzyme (FIG. 2B, D). Deletion of the DExD/H-box domain had the most pronounced effect, with an  ̃8-fold faster rate of dicing as measured for the 37ab dsRNA substrate.
More modest differences were observed for the pre-let7 substrate, although in general this RNA was cleaved 6-18 fold more rapidly than 37ab (FIG. 2C, D). Different cleavage efficiencies by hDcr on pre-miRNAs and generic dsRNA substrates were observed previously (Provost et al, 2002), implying that miRNAs and siRNAs are recognized or processed differently in vivo. The lack of pronounced stimulation of pre-let7 cleavage by the hDcr lacking DExD/H-box domain is consistent with the finding that deletion of the DExD/H-box domain of Drosophila Dicer1 (Dcr1) does not significantly affect pre-miRNA processing activity (Lee et al, 2006; Ye et al, 2007). Taken together, our data show that the activity of hDcr is substrate-dependent, and its DExD/H-box domain inhibits cleavage, particularly for a prefect-duplex dsRNA substrate.
FIGS. 2A-D. Single-turnover activity of hDcr proteins. A, Schematic illustration of a perfectly-matched dsRNA (37ab, right top panel) and a miniature pre-miRNA (pre-hlet-7, right bottom). The asterisks (*) in the substrates indicate that the substrate or strand was 5′-end labeled with 32P. Left panel is an example showing a time-course dicing assay of flhDcr (120 nM) with 2 nM 5′-end labeled 37ab dsRNA after 80 min incubation at 37° C. B and C, Graphs showing single-turnover dicing assays of hDcr proteins (60 nM) with 2-4 nM (3000 c.p.m.) duplex RNA substrate 37ab (2B) or pre-hlet-7 (2C). Value at each time point was averaged from 2 independent experiments. Fraction of duplex RNAs cleaved (%) (Y-axle) were plotted against incubation time (min) (X-axle), and cleavage fraction curves were obtained by global fitting with Kleidagraph (Synery Software) to the equation of S=(a−b)exp(−kobsdt)+b, here S is the fraction of dsRNA cleaved at each time point, a is the fraction of dsRNA at the beginning of the reaction, b is the fraction of dsRNA at the reaction plateau (t-->∞), and kobsd is the observed rate constant of the reaction; D, A summary of initial rates of hDcr proteins with RNA substrates 37ab and pre-hlet-7, calculated from first 10 min data.
Dicer's DExD/H-Box Domain Does Not Significantly Alter Substrate Binding Affinity
The substrate-dependent differences in dicing activity observed for the different human Dicer variants might reflect differences in substrate recognition. To test this possibility, we measured the binding affinity of the hDcr recombinant proteins to either the perfect duplex (37ab) or pre-hlet-7 RNA. Under conditions in which free magnesium ions are chelated and hence dicing is inhibited, the affinities of four of the mutant hDcr enzymes for either substrate were within three-fold of those measured for the full-length enzyme (75 nM and 30 nM, respectively) (FIG. 3) Similar Kds were obtained using a 64 nt duplex RNA. These values are similar to those measured previously (Provost et al, 2002; Vermeulen et al, 2005). A catalytically inactive hDcr protein showed similar binding affinities for each RNA in the presence of 10 mM Mg2+, confirming that there are no measurable effects of magnesium on RNA binding (Provost et al, 2002; Zhang et al, 2002). Notably, deletion of the dsRBD did not significantly affect RNA binding affinity, in contrast to a previous report showing that a segment of the dsRBD alone binds to dsRNA (Zhang et al, 2004) (FIG. 3A, B). Furthermore, our results do not support the prediction that the DUF283 domain is a dsRNA-binding domain as suggested previously (Dlakic, 2006). The protein lacking both the DExD/H-box domain and the dsRBD (2DD) bound  ̃3-4 fold more weakly to either substrate relative to full-length Dicer (FIG. 3A-C). It is possible that this severely truncated protein is generally destabilized, or that the DExD/H-box domain plays a small but measurable role in substrate binding.
FIGS. 3A-C. Binding affinity of hDcr proteins to duplex RNAs. A and B, KaleidaGraph global fitting results from filter binding assays of hDcr proteins to duplex RNAs of 37ab (3A) or pre-hlet-7 (3B). Value at each protein concentration was averaged from 2 independent filter binding assays. C, A summary of dissociation constants (Kd, nM) resulted from A.
DEAD-Box Domain Deletion Enhances the Catalytic Rate Constant for Dicer
The lack of substantial differences in hDcr substrate binding affinities, particularly for the DExD/H-box deletion protein, led us to wonder whether the enhanced rate of single-turnover catalysis by this enzyme resulted from a direct effect on the catalytic rate constant. To test this, we measured the rate of 37ab substrate cleavage using a sub-saturating amount of enzyme and various substrate concentrations. Plots of initial reaction rate (vo) versus substrate concentration showed that both full-length Dicer and the DExD/H-box deletion enzyme demonstrate classical Michaelis-Menten kinetics (FIG. 4A, B). This analysis enabled determination of apparent Km, Vmax and kcat values, as well as comparison of enzyme efficiency as revealed by kcat/Km (FIG. 4C).
Km and kcat, determined from nonlinear least squares fits of vo versus substrate concentration, showed that Km values for full-length hDcr and ΔDEAD were 18.8 and 11.6 nM, respectively (FIG. 4C). In contrast, the turnover number, kcat, increased  ̃40-fold as a result of DExD/H-box deletion (FIG. 4C). Thus, the ratio of kcat to Km (kcat Km−1) of the ΔDEAD protein is 65-fold greater than that of full-length hDcr protein (FIG. 4C). Since this ratio is a classical measure of enzyme efficiency and corresponds to the second-order rate constant for the reaction when the concentration of substrate is much less than Km (Haley & Zamore, 2004), the DExD/H-box domain apparently plays a role in the rate of conformational changes required for formation of the enzyme-substrate complex. In support of this idea, the Walker-motif point mutant Dicer (hWalker) showed significantly increased binding to the hydrophobic interaction reagent 1-anilino-naphthalene-8-sulfonate (ANS) (FIG. 5). Because ANS binds more strongly to partially unfolded or “molten-globule” proteins (Semisotnov et al, 1991), this finding supports the conclusion that structural destabilization or rearrangement of the DExD/H-box domain triggers catalytic activation of human Dicer.
FIGS. 4A-C. Kinetic analysis of wild-type and ΔDEAD hDcr proteins. A (flhDcr) and B (ΔDEAD), plots of initial velocity versus substrate concentration. DEAD domain inhibits multiple-turnover of hDicer cleavage of the RNA target (compare A to B). C, A summary of kinetics of flhDcr and ΔDEAD. Deletion of DEAD domain made the protein about 65 folds more efficient (kcat Km−1 of ΔDEAD over that of flhDcr).
FIG. 5. ANS binding assay. Folding status of a protein can be monitored by its binding ability to a fluorescent hydrophobic probe, ANS (1-anilino-naphthalene-8-sulfonate) since ANS has much stronger affinity to the “molten” globule intermediate state of a protein. In a 70 μl binding reaction, 5 μM of ANS and 1 μM protein were used. After 30 min incubation at room temperature, fluorescence intensity was scanned from 425 to 545 nM in the machine of FluoroMax-3 (Jobin Yvon Inc) after excited at 460 nM.
TRBP Binding Stimulates Substrate Cleavage by Full-Length Dicer
Previous experiments using immunoprecipitation showed that the DExD/H-box domain of Dicer is critical for interaction with human partner proteins TRBP and PACT (Lee et al, 2006) and the fly protein Loquacious (Logs) (Ye et al, 2007). In each case these partner proteins share homology with known RNA binding motifs and have been thought to enhance the affinity and/or specificity of Dicer for its substrates. To test whether a direct physical interaction can be observed between purified hDcr and TRBP, we used size exclusion chromatography to examine the ability of these proteins to form a stable complex. Consistent with prior immunoprecipitation data, full-length human Dicer spontaneously formed a complex with TRBP, while Dicer lacking the DExD/H-box domain (ΔDEAD) did not (FIG. 6A). Thus, the helicase motif of human Dicer is sufficient and necessary for TRBP binding to Dicer without requiring other factors.
We wondered whether TRBP binding to the DExD/H-box domain might stimulate hDcr activity and thus serve as a trigger to activate dicing by the complex. To test this possibility, the dicing activity of the hDcr-TRBP complex purified by size-exclusion chromatography was compared to that of full-length hDcr or the DExD/H-box deletion protein in a multiple-turnover assay (FIG. 6B). Interestingly, we find that although not as dramatic as DExD/H-box deletion, binding of TRBP to full-length hDcr increased the cleavage rate of the resulting protein complex (FIG. 6B). It is possible that this change results from conformational changes induced in hDcr itself upon TRBP binding. This result supports our model that the presence of DExD/H-box domain makes the processing center of human Dicer less efficient/accessible, and that removal or conformational rearrangement alleviates this effect.
FIGS. 6A and 6B. Interaction of human Dicer with TRBP mediated by DEAD domain A, TRBP interacts with hDcr through DEAD domain. Prior to size exclusion chromatography, 2.5 nmol of each human Dicer protein and 9.0 nmol of TRBP were pre-incubated on ice for 60 minutes. Left panel, a chromatogram (top) of wild-type human Dicer (flhDcr) with TRBP and SDS-PAGE gel analysis of fractions (bottom); Right panel, a chromatogram (top) of ΔDEAD human Dicer (ΔDEAD) with TRBP and SDS-PAGE gel analysis of fractions (bottom). B, Binding of TRBP to hDcr increased its dicing activity. A multiple-turnover assay for 37ab cleavage using 100 nM dsRNA and 5 nM hDcr or hDcr-TRBP.
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

## Claims

1. A modified Dicer polypeptide, wherein said modified Dicer polypeptide lacks at least about 200 amino acids of the DExD/H-box domain set fourth in SEQ ID NO:2, generates miRNA or siRNA from a double-stranded RNA substrate, and exhibits a kcat that is at least 5-fold higher than the kcat of a Dicer polypeptide having the amino acid sequence set fourth in SEQ ID NO:1.

2. The modified Dicer polypeptide of claim 1, wherein the modified Dicer polypeptide lacks the DExD/H-box domain.

3. The modified Dicer polypeptide of claim 2, wherein the modified Dicer polypeptide lacks an amino acid sequence having at least about 75% amino acid sequence identity with a stretch of at least 100 contiguous amino acids of amino acids 1-604 of the amino acid sequence set fourth in SEQ ID NO:2.

4. The modified Dicer polypeptide of claim 1, wherein the modified Dicer polypeptide comprises an amino acid sequence having at least about 75% amino acid sequence identity to the amino acid sequence set fourth in SEQ ID NO:3.

5. The modified Dicer polypeptide of claim 1, wherein said modified Dicer polypeptide has a kcat of from about 0.5×10−1 s−1 to about 1.0×10−5 s−1.

6. A composition comprising: a) the modified Dicer polypeptide of claim 1; and b) a buffer.

7. A nucleic acid comprising a nucleotide sequence encoding a subject modified Dicer polypeptide of claim 1.

8. The nucleic acid of claim 7, wherein said nucleic acid is a recombinant expression construct.

9. A genetically modified host cell comprising the nucleic acid of claim 7.

10. The genetically modified host cell of claim 9, wherein said host cell is a prokaryotic host cell.

11. The genetically modified host cell of claim 9, wherein said host cell is a eukaryotic host cell.

12. A method of producing the modified Dicer polypeptide of claim 1, the method comprising:
a) culturing the genetically modified host cell of claim 9 in culture medium in vitro under conditions such that the cell produces the modified Dicer polypeptide; and
b) recovering the modified Dicer polypeptide produced by the cell.

13. A method of producing an siRNA, the method comprising contacting the modified Dicer polypeptide of claim 1 with a double-stranded RNA (dsRNA) substrate, wherein the modified Dicer polypeptide cleaves the dsRNA substrate, thereby producing an siRNA.

14. The method of claim 13, wherein the siRNA has a length of from about 21 to about 23 nucleotides.

15. The modified Dicer polypeptide of claim 1, wherein the modified Dicer polypeptide comprises an amino acid sequence having at least about 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:3.

16. The modified Dicer polypeptide of claim 15, wherein the modified Dicer polypeptide has a length of from about 1100 amino acids to about 1320 amino acids.

17. The modified Dicer polypeptide of claim 15, wherein the modified Dicer polypeptide exhibits a kcat that is at least 10-fold higher than the kcat of a Dicer polypeptide having the amino acid sequence set forth in SEQ ID NO:1.

18. The modified Dicer polypeptide of claim 15, wherein the modified Dicer polypeptide exhibits a kcat that is at least 40-fold higher than the kcat of a Dicer polypeptide having the amino acid sequence set forth in SEQ ID NO:1.