NUCLEIC ACID BINDING OLIGONUCLEOTIDES

The present application pertains to products and methods related to the ability of short nucleotide oligomers to bind the tertiary or globular structure of nucleic acids. This application discloses libraries of short oligomers and methods for using these libraries.

DETAILED DESCRIPTION

The present invention provides libraries and arrays of short oligomers and methods for using libraries and arrays to identify short oligomers that bind target nucleic acid molecules. We have discovered that short oligomers are remarkably useful agents for targeting other nucleic acid molecules, such as RNA. In particular, short oligomers can selectively and stably bind to a folded region of an RNA using an interaction that does not completely depend on Watson-Crick or Hoogstein base-pairing, and typically involves predominantly, interactions other than such base-pairing interactions. The energetics of these interactions differ substantially from helix-forming base-pairing so that, although these short oligomers would generally fail to form stable helices, they are effective at binding to a folded target nucleic acid. Short oligomers with these and related binding properties can be used as therapeutics, lead compounds, or reagents for detecting a target.

Because RNA molecules more readily exhibit tertiary structures, the present invention will be especially useful in the identification of short oligomers that bind target RNA molecules that contain tertiary or globular structural features.

Libraries and Arrays

The libraries and arrays of the present invention contain a plurality of short oligomers. These oligomers in the library may be composed of RNA, DNA, a nucleic acid analog or some combination of RNA and/or DNA and/or a nucleic acid analog. The length of these short oligomers can be any length between a 2mer and a 15 met, inclusive.

Methods of manufacturing DNA, RNA, and nucleic acid oligomers for use in libraries and arrays are well known in the art. These methods include variety of chemical synthesis protocols. Apparatus for synthesizing oligomers are well known and commercially available from manufacturers such as ABI™ Norwalk, Conn. and BioAutomation™ Plano, Tex.

Oligomers in the present invention are DNA, RNA, and/or a Nucleic acid analog. By DNA is meant a deoxyribonucleic acid. RNA refers to a ribonucleic acid. Nucleic acid analog refers to any of the wide variety of molecules that are recognized by practitioners in the art as being molecules that are chemically similar to DNA or RNA, composed of chemical substituents that can be assembled into an oligomer, and which are capable of binding a nucleic acid. Examples of nucleic acid analogs include nucleotide analogs and peptide nucleic acids. Nucleotide analogs include chemically modified variants of DNA of RNA, including modifications to one or more of the following chemical structures of a DNA or RNA molecule: the base, the sugar, the internucleoside phosphate linkages, and further including molecules having added substitutents such as diamines, cholesterol, lipophilic groups. One notable DNA analog is known in the art as morpholinos. Types of modified internucleoside phosphate linkages that characterize examples of DNA and RNA nucleotide analogs include: phosphodiester, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate and/or sulfone internucleotide linkages, or 3′-3′, 5′-2′ or 5′-5′ linkages, and combinations of such similar so linkages (to produce mixed backbone modified oligonucleotides). Examples of additional nucleotide analogs are described in Gallo et al., Design and applications of modified oligonucleotides (2003) Brazilian Journal of Medical and Biological Research 36:143-151; Luyten I. and Herdewijn, P. (1998) Hybridization Properties of base modified oligonucleotides within the double and triple helix motif, Eur. J. of Medicinal Chemistry; Herdewijn, P. (2000) Heterocyclic modifications of oligonucleotides and antisense technology, Antisense and Nucleic Acid Drug Development, 10: 297-310; Seeberger, P H and Acaruthers K H (1997) Modified oligonucleotides as antisense therapeutics in Applied Antisense Oligonucleotide Technology Stein C A and Krieg A M, eds., Wiley-Liss Inc. New York 51-72. Freier S M and Altman K H (1997) The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically modified DNA:RNA duplexes, Nucleic Acids Research, 25:4429-4443.

Peptide nucleic acids (PNAs) are a class of oligonucleotide analogs wherein the entire deoxyribose phosphate backbone has been replaced by a chemically different, structurally homomorphous backbone composed of (2-aminoethyl)glycine units. Despite this dramatic change in chemical makeup, PNAs recognize complementary DNA and RNA by Watson-Crick base pairing. Furthermore, PNAs have been shown to have numerous advantages over, DNA and is RNA oligomers. For example, PNAs lack 3′ to 5′ polarity and thus can bind in either a parallel or an antiparallel orientation to DNA or RNA (Egholm, M. et al., Nature 365:566, 1993). It has been demonstrated that PNAs can bind double-stranded DNA by invading the DNA duplex and displacing one strand to form a stable D-loop structure (Peffer et al., Proc. Natl. Acad. Sci. USA 90:10648, 1993). A further advantage of PNAs is that they are less susceptible to enzymatic degradation (Demidov et al. Biochem. Pharmacol. 48:1310, 1994) and bind RNA with higher affinity than analogous DNA oligomers (Norton et al. Nature Biotechnology 14:615, 1996).

In one embodiment libraries can be made according to a randomized DNA library strategy. (for examples see “Design Synthesis, and Amplification of DNA Pools for Construction of Combinatorial Libraries Pools and Libraries” (2000) in Current Protocols in Molecular Biology, Vol. 4, Unit 24.2, Ausubel et al., eds., John Wiley & Sons Inc., New York; Davis, P., and Ecker, D. J. (1996) in Methods in Molecular and Cellular Biology 40, Pinilla, C., and Houghton, R. A., eds., p. 23-33, John Wiley & Sons Inc., New York; Lima et al., Combinatorial Screening and Rational Optimization for Hybridization to Folded Hepatitis C Virus RNA of Oligonucleotides with Biological Antisense Activity, Journal of Biological Chemistry; 272: 626-38; 1997). A mixture of oligomers can be made on an oligonucleotide synthesizer (e.g. ABI model 394) using experimentally determined adjusted proportions of phosphoramidites of each of the four nucleotide bases (assayed by ratio of incorporation into all possible dimers) such that, when mixed into a single vial, equimolar incorporation of all four bases at each sequence position is reproducibly obtained, thus ensuring equimolar representation of all possible sequence oligonucleotides. Limited hydrolyses using snake venom phosphodiesterase I and examination of products by uv absorption on RP-HPLC can be used to confirm the equimolar representation of bases.

Alternatively, a completely randomized library composed of short oligomers may be prepared by manually or automatically directing the synthesis of each individual oligomer that is to be represented in the library. Such a strategy can be more time consuming but insures equimolar representation of every oligomer sequence. Again commercially available synthesizers may be used for this purpose.

A preferred way of building a short oligomer library uses nucleic acid array technologies. Arrays generally refer to any support that can contain a plurality of addresses suitable for the synthesis or deposition of nucleic acid or nucleic acid analogue oligomers. The support can be rigid or flexible and will contain a substrate suitable for depositing or synthesizing oligomers. The substrate can be made of glass, plastic, polymer, biological, non-biological, organic, inorganic materials suitable for depositing or synthesizing oligomers. Arrays can take the forms of multiwell plates, microtiter plates, microarray plates, particles, strands, gels, tubing, spheres, containers, capillaries, pads, slices, films, or slides. The substrate and its surface may also be chosen to provide appropriate light-absorbing characteristics. For instance, the substrate may be a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO2, SIN4, modified silicon, or any one of a wide variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinations thereof. Other suitable substrate materials will be readily apparent to those of skill in the art upon review of this disclosure.

Oligomers may be synthesized on an array using a variety of techniques known to those skilled in the art of oligomer synthesis on solid supports, e.g. the methods described in U.S. Pat. No. 5,143,854 and U.S. Pat. No. 5,510,270 and U.S. Pat. No. 5,527,681. These methods, involve activating predefined regions of a solid support and then contacting the support with a preselected monomer solution. These regions can be activated with a light source, typically shown through a mask (much in the manner of photolithography techniques used in integrated circuit fabrication). Other regions of the support remain inactive because illumination is blocked by the mask and they remain chemically protected. Thus, a light pattern defines which regions of the support react with a given monomer. By repeatedly activating different sets of predefined regions and contacting different monomer solutions with the support, a diverse array of polymers is produced on the support. Other steps, such as washing unreacted monomer solution from the support, can be used as necessary. Other applicable methods include mechanical techniques such as those described in PCT No. 92/10183, U.S. Pat. No. 5,384,261.

Additional methods applicable to array synthesis on a single support are described in U.S. Pat. No. 5,384,261. In these methods reagents are delivered to the support by either (1) flowing within a channel defined on predefined regions or (2) “spotting” on predefined regions. Other approaches, as well as combinations of spotting and flowing, may be employed as well. In each instance, certain activated regions of the support are mechanically separated from other regions when the monomer solutions are delivered to the various reaction sites.

Another method which is useful for the preparation of the immobilized arrays of single-stranded DNA molecules X of the present invention involves “pin-based synthesis.” This method, which is described in detail in U.S. Pat. No. 5,288,514, utilizes a support having a plurality of pins or other extensions. The pins are each inserted simultaneously into individual reagent containers in a tray. An array of 96 pins is commonly utilized with a 96-container tray, such as a 96-well microtitre dish. Each tray is filled with a particular reagent for coupling in a particular chemical reaction on an individual pin. Accordingly, the trays will often contain different reagents. Since the chemical reactions have been optimized such that each of the reactions can be performed under a relatively similar set of reaction conditions, it becomes possible to conduct multiple chemical coupling steps simultaneously. The invention provides for the use of support(s) on which the chemical coupling steps are conducted. The support is optionally provided with a spacer, S, having active sites. In the particular case of oligonucleotides, for example, the spacer may be selected from a wide variety of molecules which can be used in organic environments associated with synthesis as well as aqueous environments associated with binding studies such as may be conducted between the nucleic acid members of the array and other molecules. These molecules include, but are not limited to, so proteins (or fragments thereof), lipids, carbohydrates, proteoglycans and nucleic acid molecules. Examples of suitable spacers are polyethyleneglycols, dicarboxylic acids, polyamines and alkylenes, substituted with, for example, methoxy and ethoxy groups. Additionally, the spacers will have an active site on the distal end. The active sites are optionally protected initially by protecting groups. Among a wide variety of protecting groups which are useful are FMOC, BOC, t-butyl esters, t-butyl ethers, and the like.

Various exemplary protecting groups are described in, for example, Atherton et al., 1989, Solid Phase Peptide Synthesis, IRL Press at Oxford University Press, New York. In some embodiments, the spacer may provide for a cleavable function by way of, for example, exposure to acid or base.

Yet another method which is useful for synthesis of compounds and arrays of the present invention involves “bead based synthesis.” A general approach for bead based synthesis is described in PCT/US93/04145 (filed Apr. 28, 1993).

For the synthesis of molecules such as oligonucleotides on beads, a large plurality of beads are suspended in a suitable carrier (such as water) in a container. The beads are provided with optional spacer molecules having an active site to which is complexed, optionally, a protecting group. At each step of the synthesis, the beads are divided for coupling into a plurality of containers. After the nascent oligonucleotide chains are deprotected, a different monomer solution is added to each container, so that on all beads in a given container, the same nucleotide addition reaction occurs. The beads are then washed of excess reagents, pooled in a single container, mixed and re-distributed into another plurality of containers in preparation for the next round of synthesis. It should be noted that by virtue of the large number of beads utilized at the outset, there will similarly be a large number of beads randomly dispersed in the container, each having a unique oligonucleotide sequence synthesized on a surface thereof after numerous rounds of randomized addition of bases. An individual bead may be tagged with a sequence which is unique to the double-stranded oligonucleotide thereon, to allow for identification during use.

For some applications it is desirable to conjugate the oligomers of the library or array to a carrier protein, e.g., a serum albumin, or a protein that has some affinity for the target molecule, e.g. an nucleic acid binding protein, e.g. an RNA binding protein, or a basic protein. This strategy can be used to identify oligomers whose binding of the target molecule is enhanced by the presence of the conjugated protein. The use of trimeric protein complexes to enhance the binding of a small protein ligand has been described, e.g., in Briesewitz, R. et al. (1999), Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces, Proc. Natl. Acad. Sci. USA, Vol. 9c, pp. 1953-1958.

Target Molecules

One embodiment of the present invention is a library or array of short oligomers, between 2mers and 15 ms, inclusively, contacted with a target nucleic acid molecule that contains tertiary or globular structure. As used in this application, the tertiary or globular structure of a nucleic acid is the non-linear spatial organization of the nucleic acid that allows the nucleic acid to bind to a short oligomer using an interaction that includes at least one non-canonical interaction. As used in this application, non-canonical binding refers to binding forces that are independent of Watson-Crick, Hoogstein base-pairing rules.

Typically, the target RNA molecule includes at least one region with a intramolecular tertiary or globular structure. Exemplary tertiary structures include hairpins, bulges, G-quartets, non-helical structures, and structures stabilized by interactions between non-contiguous nucleotides.

Target RNA molecules include molecules that contain at least a portion of a viral genomic sequence with tertiary or globular structure. Examples of RNA virus genomes that have been reported as exhibiting tertiary or globular structure include: Hepatitis C virus, Human Immnuodeficiency virus, Herpes virus, Kaposi's sarcoma-associated herpesvirus, Coronavirus, Bovine Coronavirus, Bovine viral diarrhea virus, GB virus-B, GB virus-C, Classic swine fever virus, foot-and-mouth disease virus, Friend murine leukemia virus, Moloney murine leukemia virus, Rous' sarcoma virus, Harvey sarcoma virus, Rhopalosiphum padi virus, Cricket paralysis virus, poliovirus, rhinovirus, encephalomyocarditis virus, and hepatitis A virus, Plautia stali intestine virus (PSIV). Additional suitable target RNA molecules include sequences from at least a portion of the genome with tertiary or globular structure for any of the following RNA viruses: Retroviruses (e.g. HIV, SIV, Avian leukemia virus, Human spumavirus) double stranded RNA viruses (e.g. rotavirus, blue tongue virus, Colorado tick fever virus), (+)sense virus (e.g. Hepatitis C virus, Hepatitis E virus, Hepatitis A virus, Bovine diarrhea virus1, poliovirus, human rhinovirus A, Norwalk virus, Tobacco mosaic virus), (−)sense RNA virus (e.g. Marburg virus, Ebola virus, Measles virus, Mumps virus, Sendai virus, Human respiratory syncytial virus, Rabies virus, Influenza A, B, or C virus). The target nucleic acid can also be an mRNA from a DNA virus or an integrated virus (e.g., an integrated retrovirus), etc.

In another embodiment, the target nucleic acid includes at least a segment of an mRNA containing tertiary or globular structure, e.g., a coding regions, a 5′ non-coding region, or a 3′ non-coding region. Any mRNA can be a target nucleic acid. For example, the mRNA may be a mammalian mRNA, e.g., an mRNA encodes a nucleic acid that contributes to a neoplastic disorder, e.g., a cancer, e.g., a metastatic cancer. For example, the mRNA may encode an oncogene, a signal transduction protein, a transcription factor, or a cell adhesion molecule. In another embodiment, the mRNA may be a bacterial, plant, or fungal mRNA. For example, the mRNA may contribute to pathogenicity.

Another class of target RNAs include non-coding RNAs, e.g., non-coding RNAs that have a function, e.g., a catalytic, structural, or regulatory function. Exemplary non-coding RNAs include RNA components of telomerase, signal recognition particle, the splicesomes (e.g., the U1, U2, U3, U5, U9, etc. RNAs), ribosome (e.g., components of the SS, and 16S RNAs), guide RNAs (e.g., that participate in RNA editing), snRNAs, SsrA RNA, and so forth. Still other functional RNAs can participate in nuclear and cytoplasmic transport, and viral packaging.

Additional examples of RNA containing tertiary or globular structure include mRNAs that encode the following: translation initiation factors, e.g. eIF4G or DAP5; transcription factors e.g., c-myc, NP-B repressing factor (NRF); growth factors e.g. Vascular is endothelial growth factor (VEGF), Fibroblast growth factor 2 (FGF-2), Platelet-derived growth factor B (PDGF-B); homeotic genes e.g. Antennapedia; Survival Proteins e.g. X-linked inhibitor of apoptosis (XIAP), Apaf-1; and BiP. Other RNA molecules will be recognized as containing tertiary or globular structures, for example, mRNAs containing Internal Ribosome Entry Sites (IRES) as well as RNAs referred to as Viroids (Avocado sun-blotch viroid, potato spindle tuber viroid) or Virusoids (e.g. Barley yellow dwarf virusoid, Tobacco ringspot virusoid)

The target viral nucleic acid sequences or target mRNAs of the present invention may be produced or isolated using a variety of means known to persons skilled in the production of RNA molecules. Such means include, but are not limited to, the synthesis of RNA molecules using RNA synthetic chemistry, production of RNA by in vitro transcription, the recombinant production of RNA and harvesting of RNA from a host cell, or by harvesting RNA from cells infected with an active virus particle.

Examples of DNA targets include telomeres, e.g., G-quartet structures, slippage complexes, e.g., slippage complexes formed by a trinucleotide repeat (e.g., CAG), a DNA replication complex, a transcription complex, DNA mismatches, DNA adducts, mutagenic lesions, and so forth.

In one embodiment of the invention target nucleic acid is labeled. Such labeling facilitates detection of a target that is bound to an oligomer in a library or on an array. Examples of methods for labeling a target nucleic acid include but are not limited to: radiolabeling the target, conjugating the target to a reporter molecule or conjugating the target to molecule capable of binding to a secondary reporter molecule. In one embodiment, the target nucleic acid is prepared by transcribing a DNA template. The transcription reaction can include one or more labeled nucleotides. For example, the nucleotide can be radiolabeled or can include a moiety that can be used to attached to a label. It is also possible to label the target nucleic acid after contacting the target nucleic acid to one or more members of the library.

Radiolabels suitable for use in the present invention include radionucleotides which can be readily incorporated into a target molecule. Examples of radionucleotides include standard32P,33P and35S labeled dATP, dTTP, dCTP, dGTP, ATP, UTP, CTP or GTP which are commercially available, e.g., Perkin-Elmer® Wellesley, Mass., Amersham Biosciences® Piscataway, N.J. Radionucleotides may be presented to cells infected with a virus, whereupon replicating virus incorporates the radiolabeled into its genomic material, which may be harvested for use in the present invention. Alternatively radionucleotides may be added to in vitro transcriptions systems such as commercially available SP6, T3, and/or T7 phage polymerase systems, which are well known in the art and also available commercially from vendors such as Promega® (Riboprobe®) Madison, Wis. or Ambion® (Megascript®) Austin, Tex.

A target molecule may be labeled by conjugating the target to a reporter molecule such as a fluorescent molecule. Fluorescent molecules are well known in the art and include: chromophores, fluorophores, and chemiluminescent moieties. More specifically such molecules include green fluorescent proteins, cyanine dyes (e.g., CYA, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5) coumarin and its derivatives, e.g. 7-amino-4-methylcoumarin, aminocoumarin and hydroxycoumarin, BODIPY dyes, such as BODIPY FL, cascade blue, Cascade Yellow, fluorescein and its derivatives, e.g. fluorescein isothiocyanate, Oregon green, Marina Blue, rhodamine dyes, e.g. rhodamine red, tetramethylrhodamine and rhodamine 6G, Texas Red, eosins and erythrosins, FITC, DAPI etc. Methods for conjugating reporter molecules to nucleic acids and nucleic acid analogues are well known, see for example: Hughes, I. R. et al. Expression profiling using arrays fabricated by an ink-jet oligonucleotide synthesizer. (2001) Nat Biotechnol 19(4), 342-7; Randolph, J. B., and Waggoner, A. S. Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes. (1997) Nucleic Acids Res 25(14), 2923-9; Brumbaugh, J. A., et al. Continuous, online DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores. (1988) Proc Natl Acad Sci USA 85(15), 5610-4; Wilkerson, D., The Scientist 12[10]:20, May 11, 1998.

The target molecule can alternatively be conjugated to a moiety suitable for secondary labeling. A moiety suitable for secondary label is a primary label that allows indirect detection of the molecule to which it is conjugated. For example, a secondary label can bind or react with a primary label for detection, can act on an additional product to generate a primary label (e.g. enzymes), or may allow the separation of the compound comprising the secondary label from unlabeled materials, etc. Secondary labels include, but are not limited to, one of a binding partner pair; chemically modifiable moieties; nuclease inhibitors, enzymes such as horseradish peroxidase, alkaline phosphatases, luciferases, etc. Preferably, the moiety suitable for secondary label and the secondary label are binding partners. For example, the label may be a hapten or antigen, which will bind its binding partner. For example, suitable binding partner pair include, but are not limited to: antigens and antibodies (including fragments thereof (FAbs, etc.)); proteins and small molecules, including biotin and streptavidin; enzymes and substrates or inhibitors; other protein-protein interacting pairs; receptor-ligands; and carbohydrates and their binding partners. Preferred binding partner pairs include, but are not limited to, biotin (or imino-biotin) and streptavidin, digoxygenin and Abs, and Prolinx™ reagents. Biotinylated nucleotides are commercially available, and may be incorporated into target molecules to make them suitable for secondary labeling or identification using streptavidin conjugates, such as Dynabeads™.

In some applications the target molecule is conjugated to a protein that has some affinity for the target molecule. Alternatively, the target RNA is contacted to an oligomer library or array in the presence of a non-conjugated protein that has an affinity for the target RNA. This strategy can be used to identify oligomers whose binding of the target molecule is enhanced by the presence of the conjugated protein See, e.g., Briesewitz, R. et al. (1999), Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces Proc. Natl. Acad. Sci. USA, Vol. 96, pp. 1955-1958 (describing trimeric protein complexes to enhance the binding of a small protein ligand).

Identifying Oligomers that Bind Target Molecules

Methods for detecting a binding interaction between an oligomer and a target molecule varies depending on the type of oligomer library or array that is utilized and/or on the type of labeling that is employed. One method for identifying an oligomer that interacts with a target molecule involves immobilizing the target molecule, contacting the target molecule with oligomers, washing oligomers that do not bind the target, capturing and identifying the oligomer that bind to the target. The target molecule may be immobilized covalently or non-covalently on a column, on a plate, on a bead, or on any other material suitable for immobilizing a target nucleic acid. Once the oligomer that binds is separated from library members that do not bind, the binding oligomer is identified. For identification purposes it is preferable that the small oligomers themselves be tagged with a unique identifier, such uniquely identified oligomers may be made according to the method of U.S. Pat. No. 6,620,584. In another embodiment, mass spectroscopy can be used. Once an accurate mass is known, it is possible to determine which possible sequences are bound. Ambiguities can be resolved by re-testing oligomers individually. In still another embodiment, the oligomer includes a tag that can be used to amplify the oligomer using PCR, LCR, or other nucleic acid amplification method. The amplified oligomer can be identified, e.g., by sequencing.

Another method for identifying oligomers that bind target nucleic acid utilizes a library oligonucleotides immobilized on the substrate of a array. In this embodiment the target nucleic acid, e.g. RNA, is labeled. The labeled target RNA is incubated with the array in such a way that the RNA molecule has an opportunity to come in contact with the oligomers immobilized on the array. After an incubation period, the array may optionally be washed, then the array is analyzed to determine where on the array, if anywhere, the target RNA binds. The address where the target RNA binds is then correlated with an oligomer that is known to be located at the address where the target RNA binds, thereby identifying the oligomer that binds the target RNA.

Typically the binding and wash conditions are non-denaturing. For example, the array and the target nucleic acid are maintained at a temperature at least 3, 5, or 10 degrees below the melting temperature of the target nucleic acid, particularly, the folded structure of interest. Exemplary binding and wash conditions have a physiological ionic strength, an ionic strength of between 0.1, 0.5, 0.8, 0.9, 1.1, 1.5, or 2 fold that of phosphate buffered saline (PBS), or an ion strength less than 2×, 1×, or 0.75×SSC sodium chloride/sodium citrate as described inCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Exemplary binding and wash conditions have a pH between 5-10.5, 6-9, 6-8.5, 6-8, or 6.5-7.5.

Methods for determining the address to which the target binds will vary depending on the array's substrate composition and also the type of label that is used. Persons of skill in the field of arrays will recognize that a large number of commercially available array scanners are suitable for use with the present invention (e.g. scanner manufacturers include Affymetrix® Santa Clara, Calif., Axon® Union City, Calif., GeneFocus Waterloo, Ontario Canada, and Packard Bioscience® San Jose, Calif.).

In some embodiments the array will not be so large as to exclude simpler methods like autoradiography which is suitable for use with radioactive or fluorescently labeled targets. The array is exposed to an X-ray film or a phosphorimager, which is developed and read or scanned to determine the address on the array to which the target molecule is bound. Scanners and systems appropriate for developing X-rays or phosphorimager screens are commercially available, e.g. from Kodak, Hewlett Packard and Molecular Dynamics. The address is then correlated to at least one oligomer, thereby identifying an oligomer that potentially binds a target nucleic acid.

Information derived from scanning the array directly or from scanning X-ray film or a phosphorimager can be stored on a database, thereby creating a database containing information that can be used to identify small oligomers that bind to a target nucleic acid. Preferably the database is an electronic database. The information in a an electronic database can be copied and/or transferred electronically to other databases. The contents of the database may also be manipulated using software which is readily available to persons in the art from manufacturers of array scanners.

Additional implementations for identifying a useful oligomer include the following. In one implementation, a target, e.g., a target nucleic acid, is contacted to addresses of an array. Interaction between the target and the addresses are detected. Quantitative or qualitative (e.g., binary—on/off, or generic—off/low/medium/high) evaluations of the interaction are made. The evaluations can be stored, e.g., in a database table that associates addresses to evaluation information or that associates address content (e.g., oligomer sequence) with evaluation information. In another example, the evaluation is stored in the form of an image, e.g., a rasterized image, e.g., from a CCD camera used to image at least a region of the array. Evaluation information is then used to identify one or more candidate oligomers that have a detectable interaction with the target. For example, it may be useful to order candidate oligomers using the qualitative information.

In one implementation, candidate oligomers are further evaluated for specificity. In one embodiment, specificity information is obtained by contacting a non-target, e.g., a non-target nucleic acid, to addresses of an array and evaluation interactions. For example, a non-target nucleic acid may be related to the target nucleic acid, but may differ in a region of interest. For example, the target nucleic acid may be a nucleic acid from a pathogen (e.g., a virus or bacteria), whereas the non-target nucleic acid may be a homologous nucleic acid of a host organism and oligomers are desired which specifically interact with the target. Specificity can be at least 0.5, 1.0, 1.2, 1.5, 2, 5, 10, 100, or 1000 fold preference. In another example, the non-target is a pool or population of nucleic acids, e.g., mRNA extracted from a cell, ribosomal RNA, and so forth. Interactions can be evaluated to determine if the candidate oligomer has a general interaction with all transcripts, or if it has heightened specificity. Other forms of testing include o e-to-one assays, e.g., a fluorescence assay that evaluates interaction between the candidate oligomer and the target or non-target. In another exemplary assay, the target and non-target are disposed on an array, and the candidate oligomer is labeled and contacted to the array. In still another exemplary assays, different RNA species are separated in a gel and blotted to a filter. The candidate oligomer is contacted to the filter and locations at which the binds to the filter are detected. Standards and controls can be used to determine the size or identify of the species with which the candidate oligomer interacts.

Designing Oligomers that Bind Target Nucleic Acids

The present invention provides a method for designing oligomers that bind a target nucleic acid. Identification of a short oligomer's sequence that binds a target nucleic acid can be used to direct the production of large quantities of that oligomer for use in pharmaceutical compositions, such as those described below. It may also be desirable to design modified oligomers based on the base sequence of the oligomer that was identified as binding the target. For example, in some diagnostic applications it is desirable to conjugate a primary label, or a moiety suitable for use as a secondary label, to an oligomer with the same base sequence as was identified as binding the target molecule. In other applications it is desirable to conjugate a therapeutically useful molecule to an oligomer that binds a specific target molecule. Examples of therapeutically useful molecules include radioisotopes, chemicals, ribozymes, and other molecules whose usefulness is enhanced by being targeted to a nucleic acid molecule.

The sequence information of an oligomer that binds a target can also be used to design an oligomer with the same base sequence but differs chemically from the oligomer identified as binding the target molecule. For example, a DNA oligomer identified as binding a target molecule can be used as the template for designing an RNA molecule that has the same base sequence as the DNA oligomer, except that any thymine residue on the DNA oligomer will be represented by uracil residue on the designed RNA oligomer. A DNA oligomer identified as binding a target molecule can be used to design a peptide nucleic acid or a phosphorothioate DNA analog oligomer that has an identical base sequence. Stated more generally a DNA, RNA or a nucleic acid analog oligomer identified as binding target nucleic acid can be used to design a DNA, RNA and/or nucleic acid oligomer that has an identical or equivalent base residue sequence but that is chemically distinct from the oligomer originally identified as binding the target molecule.

It is also possible to use one or more oligomers to form a compound that includes at least two oligomer sequences that each interact with a target (e.g., the same or different target). In one embodiment, the compound includes multimers of a single oligomer sequence that interacts with a particular target. Such a compound can be used to bring two molecules into proximity with one another. In another embodiment, the compound includes one oligomer that interacts with a first target and a second oligomer that interacts with a second target. The compound can be linear or can be branched, e.g., a dendrimer.

Characterizing Oligomers that Bind a Target Molecule

The present invention provides methods for evaluating the interaction between an oligomer and a target molecule. In one method, a short oligomer that has been identified as binding a target is further evaluated by contacting the oligomer and the target in solution and then evaluating the oligomer's ability to bind the target (e.g., to validate the interaction detected on the Methods for assaying the ability of an oligomer to bind a target will be readily apparent to one of ordinary skill in the field of binding interactions between oligomers and target molecules. Such methods include, but are not limited to performing gel shift assays, footprinting assays, affinity cleavage assays, Fluorescence Resonance Energy Transfer (FRET) experiments, surface plasmon resonance, X-ray crystallography and other methods suitable for revealing an interaction between an oligomer and the target molecule of the present invention.

In another embodiment, the oligomer is evaluated in a functional assay, e.g., an in vitro (e.g., cell-free or cell-based) functional assay or an in vivo functional assay. In an example where the target is an mRNA, a functional assay can be ability to translate the mRNA in the presence of the oligomer. The mRNA and the oligomer can be contacted to translation reagents, e.g., an translation extract. Ability of the mRNA to be translated can be evaluated, e.g., by detecting incorporation of amino acids into nascent proteins or by detecting formation of the encoded protein or fragments thereof. In an example, where the target is a catalytic RNA or a functional RNA, the functional assay can include evaluating the ability of the target to effects its function. For example, if the target is a spliceosome component, the assay can be an in vitro splicing assay to which the oligomer is added.

In some embodiments, it is possible to perform the functional assay on a library of oligomers, e.g., without first detecting binding interactions between the oligomer and the target. In one implementation, components of the assay can be contacted to an array that includes immobilized oligomers. For example, to identify an oligomer that modulates translation of an mRNA, a translation extract and the mRNA can be contacted to the array, and then the array is evaluated to identify addresses at which translation is altered (e.g., increased or decreased). For example, to identify an oligomer that modulates splicing, splicing components can be contacted to the array, and then the array is evaluated to identify addresses at which splicing is altered (e.g., increased or decreased). For example, to identify an oligomer that modulates telomerase, telomerase and a telomerase substrate can be contacted to the array, and then the array is evaluated to identify addresses at which telomere extension is altered (e.g., increased or decreased).

Another method of further evaluating an identified oligomer involves contacting the oligomer to a cell and then evaluating the cell. The identified oligomer may optionally be prepared in a lipophilic suspension before contacting a cell, e.g. suitable lipophilic solvents or vehicles can include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. The identified oligomer may alternatively be prepared in any one or more of the pharmaceutical compositions described below before the oligomer contacts the cell. In still other embodiments, the oligomer is delivered to the cell using a transfection reagent (e.g., LIPOFECTAMINE™), or a procedure that facilitates uptake (e.g., electroporation). Cell evaluations can very depending on the target molecule which the identified oligomer has been shown to bind. Examples of evaluations include but are not limited to assaying the expression of a gene product within the cell, assaying a physiological function that has been correlated with the target molecule to which the identified oligomer binds or assaying for infection or replication of a virus and/or assaying for expression of viral genes.

In some embodiments, it is possible to perform the functional cell-based assay on a library of oligomers, e.g., without first detecting binding interactions between the oligomer and the target. For example, cells can be contacted to an array, and then evaluated. In another example, the cells can be cultivated in separate containers (e.g., plates or wells of an array) and then contacted with members of a library of oligomers, e.g., individually or in a pool. The split-and-pool method can be used deconvolved a pool of oligomers to identify an individual oligomer that alters function in the assay. The split-and-pool method can also be used for cell-free assays and in vivo assays.

Pharmaceutical Compositions

Pharmaceutical compositions of this invention can include a short oligomer compound, e.g., an oligomer compound identified by a method or methods described herein, or a pharmaceutically acceptable salt thereof; optionally an additional agent selected from a protein that enhances the binding of a short oligomer to a target nucleic acid, an inhibitory agent (small molecule, polypeptide, antibody, etc.), an immunosuppressant, an anti-viral agent, anti-canter agent, anti-inflammatory agent, or an anti-vascular hyperproliferation compound, a compound to treat neurological disorders, and an anti-obesity compound; and any pharmaceutically acceptable carrier, adjuvant or vehicle. Alternate compositions of this invention comprise a short oligomer compound identified by a method or methods described herein or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier, adjuvant or vehicle.

In another embodiment, the composition includes a polymer whose monomer sequence is based on the sequence of monomers in an oligomer identified by a method described herein. For example, it is possible to make an oligomer with an altered backbone relative to an identified sequence, but including the same or similar bases. The altered backbone can have reduced negative charge, e.g., phosphates can be replaced by sulfur containing group, or phosphodiester, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate, sulfone internucleotide, and/or peptide linkages.

The term “pharmaceutically acceptable carrier or adjuvant” refers to a carrier or adjuvant that may be administered to a patient, together with a short oligomer compound identified by a method or methods described herein, and which does not destroy the pharmacological activity of the short oligomer compound and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical compositions of this invention may comprise formulations utilizing liposome or microencapsulation techniques. Such techniques are known in the art.

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a short oligomer compound of this invention with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the short oligomer compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene-polyoxypropylene compound, emulsifying wax, and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active short oligomer compound suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included in this invention.

Dosage levels of between about 0.01 and about 100 mg/kg body weight per day, alternatively between about 0.5 and about 75 mg/kg body weight per day of the target inhibitory compounds described herein are useful in a monotherapy and/or in combination therapy for the prevention and treatment of target mediated disease. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations contain from about 20% to about 80% active compound.]]

All references cited herein, whether in print, electronic, computer readable storage media or other form, are expressly incorporated by reference in their entirety, including but not limited to, abstracts, article, journals, publications, texts, treatises, internet web sites, databases, patents, and patent publications.

The phrase “15mer or shorter” as used in this application refers to any oligomer that has from 1 to 15, inclusive, monomers that can be nucleotides or nucleotide analogs, e.g. a 15mer, 14mer, 12mer, 11mer, 10mer, 9mer, 8mer, 7mer, 6mer, 5mer, 4mer, 3mer 2mer or 1mer.

Array

A short DNA 5mer array was made to order by Combimatrix® Mukilteo, Wash. The array contained 1,000 of the 1,025 possible DNA 5mer oligomers, and provided a separate address for each oligomer.

Target Sequence

The target nucleic acid sequence used was the RNA transcript of the internal ribosome entry site of the Hepatitis C virus, nucleotides 1-372 (HCV-IRES). The RNA was prepared using a Megascript™ T7 kit, nuclease free water and 5-(3-aminoallyl)-UTP, which were all provided by Ambion Inc. A DNA template encoding the HCV-IRES was constructed according to standard techniques and linearized. RNA synthesis reaction mixture contained the following:

The reaction mixture was incubated at 30° C. overnight and purified using Rneasy® kit from QIAGEN, Inc. Target Sequence was then labeled using a Cy3 monoreactive dye pack from Amersham Biosciences, Inc. Dried HCV-IRES RNA was added to 20 μL of sodium carbonate (0.1 M pH 8.5) followed by 20 μL Cy3 dye solution. The reaction mixture was mixed and incubated at room temperature for 1 hour in the dark and purified using RNeasy® mini-kit.

Hybridization of the HCV-IRES RNA to DNA 5Mers

The array was incubated with RNase free water for 30 minutes at 37° C. then washed three times with wash buffer (300 mM NaCl, 100 mM MgCl2). Hybridization was effected by adding a solution of HCV-IRES RNA (92.7 nM in 300 mM NaCl, 100 mM MgCl2 and 100 mM Tris pH 7.4) into the hybridization chamber. The array was incubated at 4° C. overnight in the dark and rinsed three times with wash buffer.

Identification of 5Mers that Bind HCV-IRES RNA

The array was scanned at 532 nm using a GenePix® 4000B scanner from Axon Instruments, Inc. Scanner data was analyzed using the GenePix® Pro 5.0 software provided by Axon Instruments, Inc. From each array analyzed the 40 addresses exhibiting the most fluorescence were, selected. If an address corresponding to the same oligomer was selected in 3 out of 5 arrays analyzed, that oligomer was scored as likely binding ligand for the HCV-IRES RNA.

Results

One sample experiment identified the following six 5mers as likely binding candidates for HCV-IRES. (Index number designates the oligomer's address on the array)

In this experiment, different hybridizations reactions were performed. The 40 addresses that exhibited the most intense fluorescence for each hybridization reaction are shown in Table 1.