WO2001023612A2

WO2001023612A2 - Determining mutations by selective reaction of the 2'-ribose position in hybridized oligonucleotides

Info

Publication number: WO2001023612A2
Application number: PCT/US2000/026320
Authority: WO
Inventors: Kevin Weeks; Stacy I. Chamberlin; Deborah M. John
Original assignee: University of North Carolina at Chapel Hill
Current assignee: University of North Carolina at Chapel Hill
Priority date: 1999-09-25
Filing date: 2000-09-25
Publication date: 2001-04-05
Anticipated expiration: 2002-03-25
Also published as: WO2001023612A3; AU7613500A; WO2001023612A9

Abstract

New chemical tagging methods and kits of the present invention utilize the discovery that chemical modification (e.g., acylation) of 2'-substituted ribonucleotides or deoxyribonucleotides by reactive compounds (e.g., activated esters) is sensitive to the base-paired state of the nucleotide. Perfectly base-paired positions are generally unreactive, while mismatched or unmatched bases are reactive under a wide variety of reaction conditions. The methods of the invention include a method of detecting a mutation in a nucleic acid molecule suspected of containing a mutation. The nucleic acid is hybridized to an oligonucleotide having a sequence complementary to the sequence the nucleic acid would have if a mutation were not present. The oligonucleotide comprises at least one nucleotide with a substitution at the 2'-ribose position. After the oligonucleotide is hybridized to the nucleic acid molecule, the hybridized oligonucleotide is then contacted with a reactive compound comprising a reporter moiety. Detection of the binding of the reporter moiety to the hybridized oligonucleotide indicates a mismatch i.e., a mutation) in the nucleic acid molecule. The methods and kits of the present invention are useful in detecting point mutations and other defects in nucleic acid sequences. These methods and kits are also useful for detecting single nucleotide polymorphisms (SNPs) and mutations responsible for cancer and other genetic diseases in humans; for quantifying amounts of nucleic acids; and for detecting conformational changes in nucleic acid structures, including those found in the products of in vitro selection experiments i.e., aptamers).

Description

METHODS AND KITS FOR DETERMINING MUTATIONS, LOCAL

CONFORMATIONAL CHANGES, AND AMOUNTS OF NUCLEIC ACIDS

BY SELECTIVE REACTION OF THE 2'-RIBOSE POSITION

IN HYBRIDIZED OLIGONUCLEOTIDES

Related Applications

This application claims the benefit of U.S. Provisional Application No. 60/156,124, filed 25 September 1999, which is hereby incorporated by reference in its entirety.

Statement of Federal Support

This invention was made with the support of the United States Government under Grant Number RO1 GM56222 from the National Institutes of Health. The United States Government has certain rights in this invention.

Field of the Invention

The present invention relates to methods and kits for detecting positions of local conformational flexibility in nucleic acid molecules and the application of these methods for detecting mutations in nucleic acid molecules. The present invention also relates to methods and kits for quantifying amounts of nucleic acid molecules, and for detecting conformational changes in nucleic acid molecules that bind ligand molecules.

Background of the Invention The ability to detect single base changes, insertions, or deletions is of fundamental importance for the detection of genetic mutation. Significant effort has focused on developing methods for detecting and scoring the thousands of small genetic differences, including single nucleotide polymorphisms (SNPs), that ultimately distinguish one individual from another. See, e.g, A. J. Schafer et al., Nature Biotech. 16, 33-39 (1998) and W. E. Evans et al., Science 286, 487-491 (1999).

Known methods for scoring single nucleotide changes in nucleic acid sequences generally utilize the detection of a signal from the hybridization of a probe oligonucleotide with the target nucleic acid sequence. The presence of a mutation at a given position produces a mismatch or bulge upon probe hybridization. These imperfect duplexes can be subsequently detected by their reduced thermal stability; by selective amplification; by using the duplexes as substrates for mismatch repair and endonuclease enzymes; by using differential chemical cleavage; and by using DNA "chips." See, e.g., M. Stoneking et al., Am. J. Hum. Genet. 48, 370-382 (1991); W. M. Howell et al., Nature Biotech. 17, 87-88 (1999); S. Tyagi et al., Nature Biotech. 14, 303-308 (1996) (reduced thermal stability); C. R. Newton et al, Nucl. Acids Res. 17, 2503-2516 (1989) (selective amplification); A. Lishanski et al., Proc. Natl. Acad. Sci. USA 91, 2674-2678 (1994); K. Okano et al., Anal. Biochem. 228, 101-108 (1995); B. J .Del Tito, Jr. et al., Clin. Chem. 44, 731-739 (1998)(mismatch repair and endonuclease enzymes); (R. G Cotton et al., Proc. Natl. Acad. Sci. USA 85, 4397- 4401(1988)); B. A. Jackson et al., J. Am. Chem. Soc. 119, 12986-12987 (1997); T. P Ellis et al., Hum. Mutat. 11, 345-353 (1998) (differential chemical cleavage); A. Marshall et al, Nature Biotech. 16, 27-31 (1998); D. G. Wang et al, Science 280, 1077-1082 (1998) (DNA chips). When the foregoing methods are used, mutation readout is generally carried out by chromatographic, fluorescent or electrochemical methods. See, e.g., Schafer et al., Cotton et al.; Jackson et al.; Howell et al.; and Marshall et al., supra. See also D. H. Johnston et al., J. Am. Chem. Soc. Ill, 8933- 8938 (1995); D. J. Caruanaet al., J. Am. Chem. Soc. 121, 769-774(1999); S. O. Kelley et al, Nucl Acids Res. 27, 4830-4837 (1999).

The chemical cleavage of mismatches and enzymatic cleavage approaches set forth above are sensitive to local disruptions in duplex structure. See Ellis et al., Lishanski et al., Okano et al., and Del Tito et al., supra. These methods can, in principle, detect any mismatch in the hybridized substrate, which is an advantage when screening for SNPs, but a disadvantage when attempting to interrogate a single site in a sequence. Another challenge in mutation detection lies in defining discriminatory hybridization conditions. For example, many perfect helices containing a majority of A-T base pairs have a lower stability than G-C rich duplexes with one or more mismatches. See N. Peyret et al., Biochemistry 38, 3468-3477 (1999); J. SantaLucia, Jr. Proc. Natl. Acad. Sci. USA 95, 1460-1465 (1998); H. K. et al., Nucl. Acids Res. 25, 3059-3065 (1997). This problem of thermodynamic stringency is a significant consideration in the application of hybridization approaches to SNP scoring. Despite advances in mutation detection technology, it is presently not always practical to sequence diagnostically many medically important genes including, for example, the human dystrophin, BRCA1, or BRCA2 genes. Direct sequencing is a common method for analyzing short stretches of DNA. See, e.g., U.S. Patent No. 5,595,890 to Newton et al. (the disclosures of all U.S. Patents cited herein are incorporated in their entirety). Screening for gene mutations in a semi-high throughput mode for long sequences is currently accomplished using methods such as nuclease digestion (see U.S. Patent No. 5,757,439 to Smith et al); chemical cleavage (see U.S. Patent No. 5,824,471 to Mashal et al. and U.S. Patent No. 5,698,400 to Cotton et al); selective PCR (see U.S. Patent No. 5,137,806 to LeMaistre et al); or electrophoretic shape polymorphisms (see R.G. Cotton, Mutat. Res. 285, 125-144

(1993)). Other methods are described in, e.g., U.S. Patent No. 5,879,886 to Meo et al; U.S. Patent No. 5,811,239 to Frayne et al., and U.S. Patent No. 5,871,902 to Weininger et al. Unfortunately, these methods generally rely on labor-intensive techniques, including time-consuming resolution by polyacrylamide gel electrophoresis. Moreover, many of these methods are incapable of specifically detecting a mutation in the presence of high concentrations of background wild type sequence as would be required, for example, when detecting cancerous tissue at the tumor-normal tissue border. See J. Prosser, Tibtech 11, 238-246 (1993). In view of the foregoing, general and accurate methods for genetic mutation detection that is readily adaptable to high throughput screening is desirable. Methods that selectively tag, in a site-specific way, single base mismatches in nucleic acids without the requirement to identify discriminatory hybridization conditions are particularly desired. Ideally, these methods would also be generically compatible with the most appropriate detection technology.

These methods could find particular application in situations where detection of genetic variation (i.e., mutation) is desirable, as in the detection or diagnosis of disorders related to genetic abnormality. For example, such a method could be useful in detecting disorders caused by germline or somatic mutations, detecting genetic variation in populations, and detecting genetic polymorphisms, including single nucleotide polymorphisms (SNPs). In particular, a desirable method could find particular use in the detection of genetic mutations that are associated with certain cancers or susceptibility of developing certain cancers, thus providing a means for detecting the cancer itself, or the likelihood of developing the cancer. For example, point mutations in the human K-ras gene are frequently observed in many human cancers, especially pancreatic, colon and lung cancers. See J. L. Bos, Cancer Res. 49, 4682-4689 (1989). Clinical studies corroborate that K-ras activation occurs early in the development of many tumors. See generally, P.J. Browett and J.D. Norton, Oncogene 4, 1029-1036 (1989); R. Rosell, et al, Clin. Cancer Res. 2, 1083-1087 (1996); R.H. Hruban, et al., Am. J. Pathology 143, 545-554 (1993); L. Mao, et al, Cancer Res. 54, 1634-1637 (1994); D. Sidransky, et al., Science 256, 102-105 (1992). Accordingly, early detection of K-ras and other mutations would make possible both detection and early treatment of cancer. In particular, given the connection between point mutations and disorders of genetic abnormality (such as cancer), it would be desirable to have a general and practical method for the screening, analysis, detection and susceptibility prediction of such disorders. Such a method would advantageously utilize techniques that are adaptable to sensitive high throughput approaches, and preferably techniques that avoid gel electrophoresis and radioisotopes.

RNA folding is driven by several favorable processes including hydrogen bonding, base stacking, specific ion binding, and water and ion release. See M. J. Serra et al, Methods Enzymol 259, 242-261 (1995); J. H. Cate et al., Nat. Struct. Biol 4, 553-558 (1997); G. L. Conn et al., Curr. Opin. Struct. Biol. 8, 278-285 (1998). Although there is a net global loss in nucleotide configurational entropy to achieve the folded state, individual RNA regions vary significantly in their local stability. See R. Rigler et al., Ann. Rev. Biophys. Bioeng. 12, 475-505 (1983). For example, base paired helices and individual nucleotides involved in tertiary interactions are conformationally restrained, while hairpin loops may be locally dynamic. See

J. A. Jaeger et al., Biochemistry 3, 12522-12530 (1993). Conformational changes upon ligand binding and assembly with protein cofactors can further modulate local RNA stability. See, e.g., J.Woodcock et al., EMBO J. 10, 3099-3103 (1991); L. G. Laing et al., J. Mol. Biol. 237, 557-587 (1994); J. Feigon et al., Chem. Biol. 3, 611- 617 (1996); F. H. Allain et al., Nature 380, 646-650 (1996); K. M. Weeks, Curr. Opin. Struct. Biol. 7, 336-342 (1997); D. J. Patel, Curr. Opin. Struct. Biol. 9, 74-87 (1999). A method capable of detecting changes in local stability would be useful in understanding RNA folding and for de novo RNA structure prediction. Chemical and enzymatic footprinting techniques remain the principal methods for monitoring RNA conformational changes. Structural interactions can be inferred by monitoring reaction of individual bases with enzymatic, electrophilic or oxidative reagents. See generally, D. J. Patel, Curr. Opin. Struct. Biol. 9, 74-87 (1999); C. Ehresmann et al., Nucleic Acids Res. 15, 9109-9128 (1987); G. Knapp, Methods Enzymol. 180, 192-212 (1989); D. A. Peattie et al., Proc. Natl. Acad. Sci. USA 78, 2273-2277 (1981); D. Moazed et al., J. Mol. Biol. 187, 399-416 (1986); X. Chen et al., Biochemistry 32, 7610-7616 (1993); H. H. Thorp, Adv. Inorg Chem. 43, 127-177 (1995). Many reagents and enzymes are selective for one or a few nucleotides; accordingly, multiple and individually optimized experiments are required to monitor all positions within an RNA molecule. The solvent accessibility of the RNA backbone can be monitored at all residues, using hydroxyl radical cleavage. See T. D. Tullius et al., Proc. Natl. Acad. Sci. USA 83, 5469-5473 (1986); J. A. Latham, Science 245, 276-282 (1989); B. Sclavi et al., Science 279, 1940-1943 (1998). Pb²⁺ also cleaves the RNA backbone in a reaction governed by both backbone flexibility and sequence selective factors. See J. Ciesiolka et al., J. Mol. Biol. 275, 211-220 (1998; V. Perret, et al., Biochimie 72, 735- 744 (1990).

Biophysical approaches for detecting the extent of local residue flexibility in nucleic acids are also known. Temperature factors, obtained from crystallographic refinements, can be related to local residue flexibilities. See S. H. Northrup et al., Nature 287, 659-660 (1980);

J. Kuriyan et al., Proc. Natl. Acad. Sci. USA 88, 2773-2777 (1991). These methods, however, have certain disadvantages. Detailed interpretation of crystallographic data is limited to high resolution structures and can be complicated by crystal packing effects. Nuclear magnetic resonance techniques, such as imino proton exchange experiments, are sensitive to local motions including base flipping and helix-coil equilibria. See B. R Reid., Ann. Rev. Biochem. 50, 969-996 (1981); N. Figueroa et al., Proc. Natl. Acad. Sci. USA 80, 4330-4333 (1983). NMR studies are limited to small RNA systems or truncated versions of large RNAs. Tritium exchange experiments have been used to analyze structural changes upon ligand binding at purine residues. See R. C. Gamble et al., Biochemistry 15, 2791-2799 (1976).

A simple and accurate method for detecting conformational changes and mapping local flexibility of nucleic acids continues to be desirable in light of the limitations of known methodologies described above.

Several research groups have previously shown that 2'-amine positions in RNA can be selectively modified by various reagents, including S-ethyl trifluoroacetate (M. Imazawa et al.,

J. Org. Chem. 44, 2039-2041 (1979)); isothiocyanates (H. Aurup, et al., Nucl. Acids Res. 22, 20-24 (1994)), and succinimidyl esters (S. B. Cohen and T.R. Cech, J. Am. Chem. Soc. 119, 6259-6268 (1997)). However, it has not been previously shown that reaction at the 2'-ribose position is sensitive to the local flexibility in nucleic acid molecules or the presence of nucleic acid mismatches, insertions or deletions.

Summary of the Invention

The present invention relates to the inventors' surprising discovery that chemical modification (e.g., acylation) of 2'-substituted ribonucleotides or deoxyribonucleotides by reactive compounds (e.g., activated esters) is sensitive to the base-paired state of the nucleotide. Chemical reaction at the ribose 2'-position is also more generally sensitive to local nucleotide flexibility such that involvement of an individual base in stable base pairing or other tertiary interaction renders the bases resistant to reaction at the 2'-ribose position. Perfectly base-paired positions are generally unreactive, while mismatched or unmatched bases are reactive under a wide variety of reaction conditions. In light of this discovery, the present inventors have developed new chemical tagging methods to detect mutations (e.g., point mutations, deletion mutations, insertion mutations) and other defects in nucleic acid sequences. These novel methods are also useful for quantifying nucleic acid hybridization; for detecting local flexibility of nucleic acid molecules; and for detecting conformational changes induced by ligand binding to the nucleic acids. These methods can be used to detect single nucleotide polymorphisms (SNPs) and mutations responsible for cancer and other genetic diseases in humans; to quantify absolute RNA and DNA amounts; and to detect conformational changes in RNA and DNA structures, including those found in the products of in vitro selection experiments (i.e., aptamers). The methods of the present mvention employ oligonucleotide probes in which the 2'-ribose position (normally, H in DNA, and OH in RNA) is substituted with, for example, an amino (NH₂) group. Modification (e.g., acylation) of 2'-amine substituted nucleotides offers an approach for site-specifically querying and tagging a mismatched nucleotide in a hybridized duplex. 2'-amine substituted nucleotides in RNA or DNA react with and are modified by a variety of electrophilic reagents including S-ethyl trifluoroacetate, isothiocyanates, and succinimidyl esters, as described in more detail herein. Reaction with a succinimidyl ester yields the 2'-amide product (see Scheme 1, below). Although the inventors do not wish to be bound to any particular theory of the invention, it appears that this reaction is much more rapid when the 2'-modified nucleotide forms a mismatch or is conformationally flexible than when it forms a canonical base pair.

Accordingly, an aspect of the present invention is a method of detecting a mutation in a nucleic acid molecule suspected of containing a mutation. The nucleic acid molecule is hybridized to an oligonucleotide having a sequence complementary to the sequence the nucleic acid would have if a mutation were not present therein. Alternatively, the nucleic acid molecule is hybridized to an oligonucleotide having a sequence complementary to the sequence the nucleic acid would have if a particular mutation were present. The oligonucleotide comprises at least one nucleotide with a substitution at the 2'-ribose position. After the oligonucleotide is hybridized to the nucleic acid molecule, the hybridized oligonucleotide is then contacted with a reactive compound comprising a reporter moiety. In one embodiment of the invention, the substitution at the 2'-ribose position is an amine (NH₂) substitution, and the 2'-NH₂ ribonucleotides or deoxyribonucleotides are contacted with a reactive compound such as an activated ester (e.g., a succinimidyl ester). Reaction of the 2'-NH₂ group with the activated ester forms a 2'-amide product. Detection of this reaction by detecting the binding of the reporter moiety to the hybridized oligonucleotide indicates a mismatch between the nucleic acid molecule and the oligonucleotide. Ifthe oligonucleotide initially hybridized to the nucleic acid molecule had a sequence complementary to the sequence the nucleic acid molecule would have if a mutation were not present therein, then the detection of the reaction (i.e., the detection of the mismatch) is indicative that a mutation is present in the nucleic acid molecule. Ifthe oligonucleotide initially hybridized to the nucleic acid molecule had a sequence complementary to the sequence the nucleic acid molecule would have if a particular mutation were present therein, then the detection of the reaction (i.e., the detection of the mismatch) is indicative that the mutation is not present in the nucleic acid molecule.

As used herein, "binding" of the reporter moiety to the hybridized oligonucleotide means that the reactive compound comprising the reporter moiety has reacted with and covalently modified the substituted 2'-ribose position of the hybridized oligonucleotide. The nucleic acid molecule and the oligonucleotide may each independently be selected from the group consisting of RNA, DNA, PNA, and the analogs and modified forms thereof. The nucleic acid molecule or the oligonucleotide may be affixed to a solid support.

Those skilled in the art will recognize that the basic methodology described above is also useful in methods of determining the specificity of hybridization between nucleic acid molecules; methods of quantitatively determining the extent of specific hybridization in a population of nucleic acid molecules; methods of quantitatively differentiating between the amounts of double-stranded and single- stranded nucleic acid in a nucleic acid population (and thus determining the amount of, for example, cellular RNA in a sample); methods of detecting disorders related to genetic abnormality (i.e., mutation), including those disorders caused by germline or somatic mutation; methods of detecting genetic variation in populations, and methods for detecting genetic polymorphisms, including single nucleotide polymorphisms

(SNPs). In addition, the method is useful for detecting other conformational changes in a nucleic acid molecule including, for example, induced fit binding of a nucleic acid aptamer to a protein, to other cellular components, or to small or large molecule ligands. These methods are all aspects of the present invention. Kits useful in the practice of the present invention are also an aspect of the invention.

The present invention finds particular advantage in that it is a chemical method of detecting single base pair mutations and conformational changes in nucleic acids, in comparison with the more cumbersome enzyme-based methods currently used. In a method of the present invention, modified oligonucleotides may be detected with a wide variety of technologies, including high throughput formats. For example, the mismatch detection methods of the present invention are compatible with other technologies, including allele specific hybridization (ASH) and DNA chip technology. Such high throughput methods avoid the need for time-consuming and expensive electrophoresis methods. The present invention provides methods for detecting mutations (e.g., single base pair mutations, deletion mutations, insertion mutations) in nucleic acids that are potentially faster, simpler and less expensive than the techniques that are currently used. The present invention advantageously utilizes bi-functional reagents in which one functionality reacts with the 2'-modified group, while the other functionality is a reporter or "visualization" moiety. Accordingly, a broad range of detection methodologies (including avidin-biotin detection, fluorescent detection, electrochemical detection, fluorescent resonance energy transfer, fluorescence quenching, and mass spectroscopy detection) may be utilized with the present invention.

The present methods also allow for the detection of a wide range of mutations. Additionally, the methods of the present invention may advantageously detect specific mutations in a background of a large amount of wild type sequences. The methods of the present invention are sensitive to local (as opposed to global) differences in nucleic acid duplex stability. Thus, the present invention allows the practitioner to avoid the painstaking optimization of hybridization conditions required of existing allele-specific methods of mutation detection. Finally, the present invention provides a general method for mapping local RNA stability that requires minimal optimization and can be used to monitor RNAs of any size at single nucleotide resolution. The foregoing and other aspects of the present invention are explained in detail in the specification set forth below.

Brief Description of the Drawings

FIG 1. is a graphical illustration of a mismatch detection model of the present invention. A 20-nucleotide probe oligonucleotide (labeled "oligonucleotide" in the Figure) contains a ribose 2'-NH₂ substitution at position 10. Residue 10 is shown as 2'-deoxy-2'-amino-cytidine. A nucleic acid molecule complementary to the probe oligonucleotide hybridizes to the probe oligonucleotide to form a perfect duplex. A nucleic acid molecule not complementary to the modified oligonucleotide at position 10 yields a duplex containing a single mismatch. The example illustrated in the

Figure is for a DNA oligonucleotide probe hybridizing to a DNA target. In the cases where either of these strands is RNA, uridine (U) is substituted for thymidine (T).

FIGS. 2A, 2B, and 2C illustrate that the modification of site-specific 2'-NH₂ substituted DNA oligonucleotide detects base-pairing. FIG. 2A is a schematic of the structure of a 2'-amido modified product. FIG. 2B is the sequence of the oligonucleotide probe, with "X" indicating the position of either a 2'-NH₂ or a 2'-OH group. FIG. 2C illustrates the time course of the modification of the oligonucleotide as a function of hybridization whether the 2'-NH₂ containing position forms a perfect base pair or not. The Figure also shows that the chemical reaction is selective for the 2'-NH group. Modified oligonucleotides were resolved by denaturing electrophoresis and detected using a radiolabel. The oligonucleotide shown in FIG. 2B was 5'-³²P- end labeled. Lanes marked "NH₂" and "2'-OH" indicate reactions in which the oligonucleotide shown in FIG. 2B contained a 2'-NH₂ or 2'-OH, respectively. "P" indicates reactions pre-quenched with DTT. Reaction of the mismatch-containing duplexes is much faster than reaction of the perfect duplex. In this Figure, both strands shown are DNA.

FIGS. 3 A and 3B illustrate the visualization and quantification of mismatch- dependent modification for the reactions shown in FIG. 2. FIG. 3 A illustrates that specific modification adds a bulky biotin moiety to the oligonucleotide probe yielding a product that is retarded in a 20%, denaturing polyacrylamide gel. FIG. 3B is a graphical illustration of the kinetic analysis of the reaction illustrated in FIG. 2. The mismatched oligonucleotide probes react approximately 30-fold more rapidly than the perfectly base-paired oligonucleotide probe. Reaction conditions were 100 mM HEPES (pH 8.0), 50 mM succinimidyl ester. In FIG. 3B, data points shown as squares indicate a C:G (perfectly base-paired) duplex; data points shown as diamonds indicate a C:A mismatch; data points shown as upward-pointing triangles indicate a C:T mismatch; data points shown as downward-pointing triangles indicate a C:C mismatch; data points shown as a circle are probe-only. FIGS. 4 A and 4B illustrate that identification of mismatched bases can be performed under a variety of conditions. In FIG. 4A, mismatches are detected at 35°C in 100 mM HEPES (pH 8.0), 75 mM succinimidyl ester. In FIG. 4B, conditions are at 50°C using 50 mM ester. Individual kinetic analyses are identified in the legend at right. In the experiments illustrated by both Figures, 2'-NH₂- containing oligodeoxynucleotides were hybridized to test DNA nucleic acid molecules to form either a complementary or mismatched duplex. Data shown for each type of duplex represent the fraction of unreacted duplex (Y-axis) as a function of time over 60 minutes. In FIGS. 4A and 4B, data points shown as squares indicate a C:G (perfectly base-paired) duplex; data points shown as diamonds indicate a C:A mismatch; data points shown as upward-pointing triangles indicate a C:T mismatch; data points shown as downward-pointing triangles indicate a C:C mismatch; data points shown as circles are probe-only.

FIGS. 5 A and 5B illustrate the use of a DNA oligonucleotide probe to hybridize with either DNA complementary strands (FIG. 5A) or with RNA complementary strands (FIG. 5B). FIG. 5 A illustrates the method of the present invention with DNA-DNA duplexes. A 2'-NH uridine DNA oligonucleotide was hybridized to DNA nucleic acid molecules. FIG. 5B illustrates the method of the present invention with DNA-RNA duplexes. In FIG. 5B, the same 2'-NH₂ uridine DNA oligonucleotide as in FIG. 5A was hybridized to RNA nucleic acid molecules. The sequence of the oligonucleotide probe is the same as shown in FIG. 2B except that the 2'-NH₂ cytidine in position 10 is replaced by a 2'-NH₂ uridine nucleotide. In FIGS. 5A and 5B, data points shown as squares indicate a U:A (perfectly base- paired) duplex; data points shown as diamonds indicate a U:G mismatch; data points shown as upward-pointing triangles indicate a U:U or U:T mismatch; data points shown as downward-pointing triangles indicate a U:C mismatch; data points shown as circles are probe-only.

FIGS. 6A and 6B illustrate that the present invention is effective in using a 2'- NH DNA strand to detect mutations in RNA strands across from a 2'-NH₂ cytosine nucleotide and under different conditions. Conditions in FIG. 6A were 100 mM

HEPES (pH 8.0) 1.5 M NaCl, 75 mM succinimidyl ester at 35°C. Conditions in FIG. 6B were without NaCl at 50°C. In FIGS. 6A and 6B, data points shown as squares indicate a C:G (perfectly base-paired) duplex; data points shown as diamonds indicate a C:A mismatch; data points shown as upward-pointing triangles indicate a C:U mismatch; data points shown as downward-pointing triangles indicate a C:C mismatch; data points shown as circles are probe-only.

FIGS. 7 A and 7B illustrate the use of the present invention to use an RNA strand to detect mutations across from an RNA (FIG. 7A) or DNA strand (FIG. 7B). In FIGS. 7A and 7B, data points shown as squares indicate a C:G (perfectly base- paired) duplex; data points shown as diamonds indicate a C:A mismatch; data points shown as triangles indicate a C:T or C:U mismatch; data points shown as circles are probe-only.

FIGS. 8A and 8B show that reaction at the ribose 2'-position can be used to detect deletions. A schematic of the deletion is shown in FIG. 8A, and the kinetic analysis is shown in FIG. 8B. Kinetic analysis was performed as described in FIG. 3 except that the reaction temperature was 35°C and the succinimidyl ester was at 75mM. In FIG. 8B, data points shown as squares indicate a C:G (perfectly base- paired) duplex; data points shown as diamonds indicate a deletion; data points shown as circles are probe-only.

FIG. 9 is a schematic illustrating the use of chemical modification of a 2'- ribose position for quantification of target nucleic acids. In this Figure, the probe oligonucleotide and target nucleic acid are shown as thick and thin lines, respectively. FIG. 10 shows quantification of the absolute amount of RNA with single nucleotide base discrimination. Representative experiment of a perfectly complementary duplex with varying amounts of RNA target nucleic acid (top gel). Mobility of the acylated product is retarded in a denaturing gel compared with free probe. Hybridization and reactivity of a target RNA containing a single base pair substitution is shown in the lower gel. In this Figure, the probe oligonucleotide is similar as that shown in FIG. 2B, but has been extended by 5 bp on either end and the target strand is a 30 nucleotide RNA containing a C-A mismatch as illustrated schematically in FIG. 1. The probe strand is reactive at all concentrations due to the presence of the mismatched base pair (bottom gel).

FIG. 11 illustrates quantification of the data shown in FIG. 10. Square- shaped data points represent hybridization of a perfect duplex and diamond-shaped data points represent the mismatched duplex.

FIGS. 12 and 12B illustrate use of 2'-amine acylation to detection different conformations in RNA. In FIG. 12A, acylation of 2'-amino guanosine nucleotides in tRNA^Asp were detected by primer extension. Extension reactions were resolved by denaturing gel electrophoresis for reactions performed under denaturing (50% formamide), native (10 mM MgCl₂,100 mM NaCl), or high salt (500 mM NaCl) conditions. Bands observed in the denaturing lanes established the maximum reactivity at each postion. Positions protected from modification under native or high salt conditions are indicated with arrowheads. In FIG. 12B local RNA flexibility was mapped in tRNA^Λsp by selective acylation of 2'-amine substituted nucleotides. Residues protected from modification (determined by primer extension) under conditions that stabilize native tRNA folding are boxed. Base pairs are shown as dots, tertiary interactions as dashed lines. Structure is drawn to suggest arrangement of base paired helices and non-canonical tertiary interactions in three dimensions. These experiments show that local conformation can be mapped using any 2'-substituted nucleotide.

FIG. 13 illustrates the design of an oligonucleotide probe for detection of codon 12 mutations in the human K-ras gene. The oligonucleotide is written in the 3'<— 5' direction, while 2'-NH₂ substitutions are shown as "X."

FIG. 14 illustrates application of the present invention to detect conformational changes in a nucleic acid that occur when a nucleic acid aptamer binds a ligand. In this Figure, a DNA aptamer undergoes a conformational change upon binding AMP (shown as "A"). In this case, the internal nucleotides in the AMP- binding aptamer are not paired and are reactive at their 2'-ribose position. Upon binding AMP, a pattern of stable, non- Watson-Crick base pairs form, yielding unreactive 2'-ribose positions. This figure is adapted from CH. Lin and D.J. Patel, Chem. Biol. 4, 817-832 (1997).

Detailed Description of the Invention

The present invention will now be described with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Unless otherwise defined, 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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Amino acid sequences disclosed herein are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence. Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC- IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one- letter code, or the three letter code, both in accordance with 37 CFR §1.822 and established usage. See, e.g., Patentln User Manual, 99-102 (Nov. 1990) (U.S. Patent and Trademark Office).

Except as otherwise indicated, standard methods may be used for the production of cloned genes, expression cassettes, vectors (e.g., plasmids), proteins and protein fragments according to the present invention. Such techniques are known to those skilled in the art. See e.g., J. Sambrook et al., Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989), and F. M. Ausubel et al., Current Protocols In Molecular Biology (Green Publishing Associates, Inc. and Wiley-Interscience, New York, 1991).

Methods of the present invention generally relate to detecting mutations in nucleic acid molecules; determining the extent and specificity of hybridization between nucleic acid molecules; and detecting conformational changes in nucleic acid molecules. As used herein, a nucleic acid molecule may be RNA (the term "RNA" encompassing all ribonucleic acids, including but not limited to pre-mRNA, mRNA, rRNA, hnRNA, snRNA and tRNA); DNA; peptide nucleic acid (PNA, as described in, e.g., U.S. Patent No.5,539,082 to Nielsen et al, and U.S. Patent No. 5,821,060 to Arlinghaus et al); and the analogs and modified forms thereof. Nucleic acid molecules of the present invention may be linear or circular, an entire gene or a fragment thereof, full-length or fragmented digested, "chimeric" in the sense of comprising more than one kind of nucleic acid, and may be single-stranded or double- stranded. Nucleic acid from any source may be used in the present invention; that is, nucleic acids of the present invention include but are not limited to genomic nucleic acid, synthetic nucleic acid, nucleic acid obtained from a plasmid, cDNA, recombinant nucleic acid, and nucleic acid that has been modified by known chemical methods, as further described herein. Nucleic acids may also be products of in vitro selection experiments (also called aptamers) and other nucleic acid molecules useful for their ability to bind or be bound by other ligands. See L. Gold, et al, Annu. Rev. Biochem. 64, 763-798 (1995); S.E. Osborne and A.D. Ellington, Chem. Rev. 97, 349- 370 (1997). Nucleic acids of the present invention may be obtained from any organism, including but not limited to bacteria, viruses, fungi, plants and animals, with animal nucleic acid being preferred, mammalian nucleic acid being more preferred, and human nucleic acid being most preferred. If desired, the nucleic acid may be amplified according to any of the known nucleic acid amplification methods that are well-known in the art (e.g., PCR, RT-PCR, QC-PCR, SDA, and the like). Nucleic acids of the present invention may be, and preferably are, purified according to methods known in the art. In general, nucleic acid molecules of the present invention are nucleic acid molecules that are suspected of containing at least one mutation, the determination of the presence of the mutation being desired by the practitioner; or for which the amount or concentration is useful to be determined; or that undergo a conformational change.

As used herein, the term "oligonucleotide" refers to a nucleic acid sequence of at least about five nucleotides to about 60 nucleotides, and more preferably from about 12 to about 40 nucleotides. The term "oligonucleotide" is herein interchangeably used with the term "probe," as commonly defined in the art. As with the target nucleic acid molecules described above, the oligonucleotides of the present invention may comprise RNA (the term "RNA" encompassing all ribonucleic acids, including but not limited to pre-mRNA, mRNA, hnRNA, snRNA, rRNA and tR A), DNA, PNA, and the analogs and modified forms thereof.

Although the skilled artisan will recognize that oligonucleotides comprise nucleic acids, for the purpose of clarity the terms "nucleic acid molecule" and "target nucleic acid molecule" are used interchangeably herein to refer to nucleic acid molecules that are suspected to contain at least one mutation (e.g., a deletion, insertion or point mutation), nucleic acid molecules that are suspected to contain at least one single nucleotide polymorphisms, nucleic acid molecules (and samples thereof) that are to be analyzed for absolute or relative concentrations or amounts, nucleic acid molecules that are to be analyzed for conformational changes, local flexibility or variability in conformation, nucleic acid molecules that are to be analyzed for the presence or absence of a genetic defect, or nucleic acid molecules that are otherwise meant or desired to be analyzed by the methods of the present invention.

In contrast, the terms "oligonucleotide" and "oligonucleotide probe" will be used interchangeably herein to refer to a nucleic acid sequence comprising at least one nucleotide modified at the 2'-ribose position, unless otherwise specified. Thus defined, oligonucleotides are used in the present invention to detect mutations in target nucleic acid molecules, or are used to determine absolute or relative amounts of target nucleic acid molecules, or to detect conformational changes in target nucleic acids, etc. Oligonucleotides of the present invention may be used because they are known to undergo conformational changes upon ligand binding.

The oligonucleotides of the invention are generally synthesized by any one of the known methodologies (i.e., by using solid phase synthesis or by enzymatic transcription), and are designed to be complementary to a selected target nucleic acid molecule, or to be able to bind a desired ligand as described herein. Oligonucleotide synthesizers are commercially available and their use is understood by persons of ordinary skill in the art as being effective in generating any desired oligonucleotide of reasonable length. In an embodiment of the invention, oligonucleotides are at least about 10 to about 12 nucleotides in length, but may be as short as about eight nucleotides, about five nucleotides, or even shorter. In another embodiment of the invention, oligonucleotides may be as long as about 30 or about 40 nucleotides in length, but may be about 50 nucleotides long, about 60 nucleotides long, or even longer.

In preferred embodiments of the present invention, the individual nucleotides of the nucleic acid molecules of the invention are connected through a sugar moiety via phosphorus linkages, as are the oligonucleotides of the present invention. The sugar moiety may be deoxyribose or ribose. Preferred phosphorus linkages include phosphodiester, phosphorothioate and phosphorodithioate linkages. In other embodiments of the invention, nucleotides can be joined via linkages that substitute for the internucleotide phosphate linkage. Other known modifications can optionally be made to the sugar, to the base, or to the phosphate group of the nucleotide. Representative modifications are disclosed in International Publication Numbers WO 91/10671, published Jul. 25, 1991; WO 92/02258, published Feb. 20, 1992; WO 92/03568, published Mar. 5, 1992; and U.S. Pat. No. 5,138,045, issued Aug. 11, 1992. The disclosures of each of the above referenced publications are herein incorporated by reference. Preferred modifications include PNA, 2'-O-methyl, and 2'-O-alkyl modifications.

Preferred nucleotide bases of the mvention may be any pyrimidine or purine base of DNA and RNA, or the derivatives and analogs thereof, including, but not limited to: guanine, adenine, cytosine, uracil, thymine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo-uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, other aza and deaza thymidines, other aza and deaza cytosines, other aza and deaza adenines, other aza and deaza guanines, 5- trifluoromethyl uracil and 5-trifluoro cytosine. The nucleotides of the invention may also comprise other modified nucleotides as are known in the art.

As set forth above, oligonucleotides of the present invention will comprise at least one nucleotide which comprises a substitution at the 2'-ribose position (normally, H in DNA or OH in RNA) of the nucleotide. The substitution is preferably an amino (NH ) group, but alternatively may be an aldehyde group, a ketone group, a thiol group, or an azido group. The oligonucleotide may also include 5' or 3' or internal groups including fluorescent groups, fluorescent quenchers (e.g., dabcyl), biotin or other groups.

In one basic embodiment of the present invention, an oligonucleotide is hybridized to a target nucleic acid molecule. The target nucleic acid molecule may be suspected of containing a mutation. The oligonucleotide comprises at least one nucleotide that is substituted at the 2'-ribose position. In an embodiment of the invention in which a suspected mutation is being detected, the oligonucleotide has a sequence complementary to the sequence the nucleic acid molecule would have ifthe mutation was not present. In other embodiments of the invention, the oligonucleotide has a sequence complementary to the known, deduced, or suspected sequence of the nucleic acid molecule. Hybridization may be carried out by methods known in the art. For example, the oligonucleotide may be hybridized to the nucleic acid molecule by heating the oligonucleotide and the nucleic acid molecule to 90 °C, and then slow- cooling to 22 °C over 20 minutes. The oligonucleotide is thus hybridized to the nucleic acid molecule, and is accordingly referred to as the "hybridized oligonucleotide."

Next, the hybridized oligonucleotide is contacted with a reactive compound comprising a reporter moiety, as defined herein. The reactive compound will be one that is known to react with the 2'-substituted hybridized oligonucleotide. For example, ifthe hybridized oligonucleotide comprises a nucleotide substituted at the 2'-ribose position with an amine, the hybridized oligonucleotide will be contacted with succinimidyl ester, because succinimidyl ester is known to acylate the amine group. The reactive compound will comprise a reporter or "visualization" moiety as defined herein, such as biotin, an enzyme conjugate or a fluorescent group. Optionally, after contacting the hybridized oligonucleotide with the reactive compound, unbound reactive compound (i.e., any reactive compound that has not reacted with the modified 2'-ribose position of the hybridized oligonucleotide) may be washed away or separated from the hybridized oligonucleotide that has bound the reporter moiety of the reactive compound. Techniques for carrying out such washing and/or separating steps are known to those skilled in the art. The skilled artisan may, in particular, choose to separate the unbound reactive compound from the hybridized oligonucleotide if detection of the reporter moiety is to be carried out by gel electrophoresis, as described below. Finally, the hybridized oligonucleotide is analyzed for the presence or absence of the reporter moiety bound thereto, wherein the presence of the reporter moiety bound to the hybridized oligonucleotide indicates that the reactive compound has reacted with (i.e., covalently modified) the 2'-substituted group of the hybridized oligonucleotide. Again using the example of the 2'-amine substituted oligonucleotide and the succinimidyl ester as the reactive group, ifthe reporter moiety is biotin then the presence of the biotin bound to the oligonucleotide indicates that the succinimidyl ester has acylated the 2'-amine group.

As used herein, "binding" of the reporter moiety to the hybridized oligonucleotide means that the reactive compound comprising the reported moiety has reacted with (i.e., covalently modified) the substituted 2'-ribose position of the hybridized oligonucleotide. In other words, the reporter moiety need not bind directly to the hybridized oligonucleotide; rather, the reporter moiety may be indirectly bound to the hybridized oligonucleotide via the reactive compound that comprises the reported moiety being chemically linked and bound to the hybridized oligonucleotide as a result of the reaction of the reactive compound with the hybridized oligonucleotide. Stated another way, the reactive compound will generally be bifunctional; one functionality will react directly with the substituted 2'-ribose position of the hybridized oligonucleotide, while the other functionality will be the reporter moiety. Ifthe substituted 2'-ribose position of the hybridized oligonucleotide does react with the reactive compound via the first functionality, the reporter moiety will be considered "bound" to the hybridized oligonucleotide. As still another illustration of the binding of the reporter moiety to the hybridized oligonucleotide, Scheme 1, Scheme 3 and Scheme 4, below, illustrate reactive compounds wherein the reporter moiety is indicated as an "R" group. Ifthe reaction as illustrated in either Scheme 1 or Scheme 3 occurs, the reporter moiety is considered "bound" to the nucleotide that is substituted at the 2'-position via the functionality of the reactive compound that interacts or reacts directly with the substituted 2'-group of the hybridized oligonucleotide. Ifthe oligonucleotide initially hybridized to the nucleic acid molecule had a sequence complementary to the sequence the nucleic acid molecule would have if a mutation were not present therein, then the indication that the 2'-substituted group has been modified by the reactive compound may indicate that a mutation is present in the nucleic acid molecule. A mismatch between the oligonucleotide and the nucleic acid molecule will make the substituted 2'-group more reactive with the reactive compound, whereas a perfect duplex will generally render the hybridized oligonucleotide unreactive or very slowly reactive with the reactive compound. Thus, presence of a mismatch between the oligonucleotide and the nucleic acid molecule may be an indication of a mutation in the nucleic acid molecule. Alternatively, ifthe oligonucleotide initially hybridized to the nucleic acid molecule had a sequence complementary to the sequence the nucleic acid molecule would have if a particular mutation were present therein, then the indication that the 2'-substituted group has been modified by the reactive compound may indicate that a mutation is not present in the nucleic acid molecule. The mutation may be a deletion, insertion or point mutation in the nucleic acid molecule. SCHEME 1

Scheme 1 is a schematic illustration of the 2'-NH₂ modification chemistry of the present invention. Reaction of a 2'-NH₂ substituted nucleotide with an activated ester yields the 2'-amide product. In Scheme 1 , "R" may be any reporter moiety.

The skilled artisan will recognize that the basic methods described herein may also be used to determine whether or not the hybridization between nucleic acid molecules is specific or not. In the context of this invention, "hybridization" shall mean hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed

Hoogsteen hydrogen bonding, between complementary nucleotides. For example, adenine and thymine are complementary nucleotide bases which pair through the formation of hydrogen bonds. "Complementary," as used herein, also refers to sequence complementarity between two nucleotides. The term "specifically hybridizable" is used to indicate that stable and specific binding occurs between the oligonucleotide and the nucleic acid molecule target. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid molecule sequence to be specifically hybridizable.

In one embodiment of the invention, either the nucleic acid molecule or the oligonucleotide is affixed to a solid support, according to techniques known in the art. In one embodiment, either the nucleic acid molecule or the oligonucleotide is affixed to a solid surface of a multiwell plate. In another embodiment, the nucleic acid molecule or the oligonucleotide is affixed to a solid support known as a "DNA chip," as exemplified by those described in U.S. Patent Nos. 5,874,219 and 5,871,928 to Rava et al. The disclosures of each of these patents are herein incorporated by reference in their entirety.

As set forth above, the present invention involves the hybridization of a 2'- modified oligonucleotides to a target nucleic acid molecule resulting in a hybridized oligonucleotide, followed by contacting the hybridized oligonucleotide with a reactive compound (sometimes referred to herein as an "activated compound"). Reactive compounds are able to selectively react with (i.e., selectively modify) the 2'-modified nucleotide of the hybridized oligonucleotide. Reactive compounds may be activated esters as this term is understood in the art (e.g., succinimidyl esters) or may be fluorogens, which are non- fluorescent compounds that react with primary amines to form fluorescent compounds. Preferred fluorogens include but are not limited to fluorescamine, o-phthaldialdehyde, 3-(4-carbxylbenzoyl)-quinoline-2-carboxaldehyde (CBQCA), and other compounds that are formed by the general reaction illustrated in Scheme 2, below. As described in Cohen and Cech, (1997), supra, 2'-NH₂ ribonucleotides react specifically (i.e., are acylated by) with reactive compounds (i.e., activated esters) to form a 2'-amide product. The acylation reaction of the 2'-NH₂ group with the reactive compound yields a 2'-amide product, which can be detected by detection methods described herein. A preferred reactive compound is an activated ester such as a succinimidyl ester. Those skilled in the art will recognize that the selection of the reactive compound will depend on the particular substitution at the 2'-position of the modified nucleotide or nucleotides of the oligonucleotide. For example, ifthe substitution is an NH group, then a succinimidyl ester may be used as the reactive compound. Other reactive compounds that are useful in the invention include isothiocyanates, dichlorotriazines, aldehydes, and sulfonyl halides, as shown in Scheme 3, below. Ifthe substituted group is an aldehyde group, then the reactive compound is preferably a hydrazine or an amine; ifthe substituted group is a thiol group then the reactive compound is preferably a thiol group, alkyl halide, haloacetamide or maleimide. The selection of the appropriate and compatible reactive compound based upon the 2'-substituted group that is utilized is within the skill of those that are knowledgeable in the art.

In one embodiment of the invention, the reactive compound of the present invention will comprise a reporter (or "visualization" or "detection") moiety. Referring to Scheme 1, the reporter moiety is represented by the group "R". As used herein, a reporter moiety may be a compound, a chemical group, a labeled (i.e., by radioactivity, luminescence, or fluorescence) element, molecule or compound, an enzyme conjugate, or any other molecule that is able to be detected by detection means that are known in the art and are described more fully herein. In a preferred embodiment of the invention, the reporter moiety is biotin. For example, in certain experiments described herein, the ester sulfosuccinimidyl-6-(biotinamido)hexanoate is used as an exemplary compound. In other embodiments, the reporter moiety is streptavidin, or is an intrinsically fluorescent group (e.g., fluoroscein or rhodamine). In yet another embodiment, the reporter moiety is an enzyme conjugate (e.g., horseradish or thermostable soybean peroxidase). In still other embodiments, the reporter moiety is a fluorescent compound as described above, or is a fluorescent quencher, or is an electrochemically active moiety or another group detectable by mass spectroscopy. An extensive list of commercially available fluorescent molecules useful in the practice of the present invention may be found in R. P. Haugland et al. , Handbook of Fluorescent Probes and Research Chemicals, Sixth Edition (Molecular Probes, Inc., Eugene, Oregon, (1996), HTML version located at www.probes.com), incorporated herein by reference in its entirety.

SCHEME 2

non-fluorescent reagents fluorescent products

Scheme 2 illustrates representative fluorogenic chemistries for mismatch-selective reaction of 2'-NH₂ nucleotides.

The method of detecting (i.e., visualizing, observing) the reporter moiety, and thus the presence of the mismatch will depend on the specific moiety used. For example, if streptavidin is used as the reporter moiety, the known method of streptavidin capture will be used to detect ifthe reaction between the reactive compound and the 2'-modified oligonucleotide has occurred. If a fluorophore is used as the reporter moiety, it may be detected using a fluorometric assay according to known methods (for example, by using capillary electrophoresis) or a microtiter plate reader. One preferred method of detecting the reaction between the reactive compound and the 2'-modified oligonucleotide utilizes a fluorescent or fluorogenic reagent as the reporter moiety. The detection of the reaction is then accomplished with fluorescence detection techniques known to those in the art. SCHEME 3

X =

(succinim idyl ester)

SCN-R (isothiocyanate)

CI0₂S-R (sulfonyl chloride)

(m aleim ide)

CIH₂-R (al yl halide]

RSS-R (disu Ifide )

H₂NHN-R

-CHO (hydrazine)

Scheme 3 illustrates representative chemistries compatible with reaction at the 2'-ribose position and useful with the methodology of the present invention for detecting mismatches, mutation, hybridization, and conformational changes in nucleic acids.

Another preferred detection method utilizes a peroxidase or another enzyme as the reporter moiety. Alternatively, moieties capable of being detected by electrochemistry techniques may also be used. Detection of mismatch-specific modification may also be accomplished in a multi-well colorimetric assay format, or by using high throughput liquid chromatographic methods, without resorting to gel electrophoresis. The probe oligonucleotide may also comprise a fluorescent group prior to reaction; the reactive group may then comprise an additional fluorescent group or fluorescent quencher. Detection would then be by fluorescence resonance energy transfer (FRET) (P.R. Selvin, Nature Struct. Biol. 7, 730-734 (2000)), fluorescence quenching (S. Tyagi and F.R. Kramer, Nature Biotech. 14, 303-308 (1996)) or eximer approaches.

The modification (i.e., an acylation) of the 2'-modified oligonucleotide of the present invention by the reactive compound to produce a detectable product indicates the presence of a mismatch between the hybridized 2'-modified oligonucleotide and the nucleic acid molecule. Ifthe 2'-modified oligonucleotide has a sequence complementary to the sequence the nucleic acid compound would have if a mutation were not present therein, then the mismatch is generally an indication of a mutation in the nucleic acid molecule. Ifthe 2'-modified oligonucleotide has a sequence complementary to the sequence the nucleic acid compound would have a particular mutation were present therein, then the mismatch generally indicates that no mutation has been found in the nucleic acid molecule. The mutation may be a deletion mutation, an addition (insertion) mutation, or a point mutation, as these terms are understood in the art. Mutations that can be identified by the methods of the present invention include, but are not limited to, those mutations in sequences that regulate transcription or translation of a gene, nonsense mutations, splice site alterations, and translocationxr

Scheme 4 illustrates that the 2'-ribose reactive reagents are bifunctional sensors for mutation and other detection as disclosed herein. The -R group may be any reporter moiety as described herein.

The methods of the present invention are useful for detecting the presence of or the susceptibility for the development of disorders related to genetic abnormality in a subject, including those disorders caused by germline or somatic mutation (e.g., cancer). As such, the present invention is suitable for both medical and veterinary uses. Suitable subjects include, but are not limited to, mammalian and avian subjects.

More preferred subjects are mammalian subjects such as humans, monkeys, pigs, cattle, dogs, horses, cats, sheep, and goats. The most preferred subjects are human subjects.

The present invention provides a method for diagnosing a subject with a disorder that is characterized, caused or related to genetic mutation, and for identifying subjects at risk for developing such a disorder. An at-risk subject is any individual who, by virtue of the presence of a genetic mutation, is believed to be at a higher risk than the general population for developing a disorder associated with the particular mutation. Using the method of the present invention, a sample of a nucleic acid molecule is obtained from the subject (i.e., by obtaining cells from the subject by biopsy, or from the blood or other bodily fluid of the subject and extracting nucleic acid according to known techniques). An oligonucleotide comprising at least one nucleotide comprising a 2'-ribose modification is hybridized to the nucleic acid molecule of the subject. Preferably, the oligonucleotide comprises a sequence complementary to a sequence of a nucleic acid molecule that is indicative of the subject's likelihood of having or developing a disorder that is characterized, caused or related to genetic mutation (i.e., the mutation, if present, is likely to be present in the targeted nucleic acid molecule). That is, the nucleic acid molecule may be such that a mutation contained therein indicates a likelihood of developing a disorder associated with the particular mutation being detected. Thus, if a mismatch is detected between the oligonucleotide and the nucleic acid molecule, the subject may be at higher risk of developing the disorder, or may in fact have the disorder.

In one embodiment of the invention, the method of the present invention may be used to diagnose the presence of or the susceptibility of developing inherited thrombophilia and thrombosis mortality, also called Factor V Leiden. This disorder is caused by the mutation in the gene encoding Factor V clotting factor. Other disorders that are associated with particular genetic mutations (and thus may be detected by the present invention) include, but are not limited to, sickle cell anemia, certain forms of hemophilia, fragile-X syndrome, spinal and bulbar muscular dystrophy, myotonic dystrophy, Huntington's disease, hereditary angioedema, Li-Fraumeni disease, cystic fibrosis, neurofibromatosis type 2, von Hippel-Lindau disease, as well as others. Genes that may be screened for mutations using methods of the present invention include genes that encode cell cycle control proteins (such as p21, p27 or pl6). Other genes that may be screened for mutations are those associated with certain disorders. These genes include but are not limited to genes that encode β-globin, phenylalanine hydroxylase, αi-antitrypsin, 21-hydroxylase, pyruvate dehydrogenase Elα-subunit, dihydropteridine reductase, rhodopsin, β-amyloid, nerve growth factor, superoxide dismutase, adenosine deaminase, β-thalassemia, ornithine transcarbamylase, collagen, β-hexosaminidase, topoisomerase II, hypoxanthine phosphoribosyltransferase, phenylalanine 4-monooxygenase, Factor VIII, Factor IX, nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, and phosphoribosyltransferase. In another embodiment, the method of the present invention is used to detect or diagnose the presence of cancer in a subject, or is used to determine ifthe subject is at risk of developing cancer. In a preferred embodiment, this method involves detecting mutations in an oncogene or tumor supressor gene. Exemplary mammalian oncogenes include but are not limited to abl, akt, crk, erb-A, erb-B, ets, fes/fps, fgr, fms, fos, jun, kit, mil/raf, mos, myb, myc, H-ras, K-ras, rel, ros, sea, sis, ski, src and yes. Exemplary mammalian tumor suppressor genes include but are not limited to any one of the p53, retinoblastoma (preferably RBI), adenomatous polyposis coli, NF-1, NF--2, MLH-1, MTS-1, MSH-2, and human non-polyposis genes. In a particularly preferred embodiment, the method of the present invention is used to detect a mutation in the K-ras proto-oncogene, and more specifically, a mutation in codon 12 of the K-ras oncogene. Alternatively, the method of the present invention is used to detect the presence of a mutation in the BRCA1 or BRCA2 gene of a human subject, with such a mutation indicating that the subject is at risk of developing breast cancer. As another alternative, the method of the present invention is used to detect the presence of mutations in genes associated with certain cancers, and/or the genes and/or regulatory regions that control the expression of the genes associated with certain cancers. Such genes include but are not limited to Her2, FRK, and Neu2, the over-expression of which genes are associated with the development of certain cancers.

The term "cancer" as used herein is intended to encompass cancers of any origin, including both tumor- forming and non-tumor forming cancers. The term "cancer" has its understood meaning in the art, for example, an uncontrolled growth of tissue or proliferation of cells that has the potential to spread to distant sites of the body (i.e., metastasize). As used herein, the term "cancer cell" is also intended to encompass those cells referred to as "pre-cancerous," i.e., cells that contain mutated or damaged DNA or other components that are likely to cause the cell to develop into a cancer cell. Exemplary cancers include osteosarcomas, angiosarcomas, fibrosarcomas and other sarcomas; leukemias; sinus tumors; ovarian, uretal, bladder, prostate and other genitourinary cancers; colon, esophageal and stomach cancers and other gastrointestinal cancers; lung cancers; lymphomas; myelomas; pancreatic cancers; liver cancers; breast cancers; kidney cancers; endocrine cancers; skin cancers; melanomas; angiomas; and brain or central nervous system (CNS) cancers. Tumors or cancers, as defined herein, may be any tumor or cancer, primary or secondary.

Preferred methods of the present invention are those which identify subjects at risk for tumor- forming cancers, and methods of preventing the same. The term "tumor" is also understood in the art, for example, as an abnormal mass of undifferentiated cells within a multi-cellular organism. Tumors can be malignant or benign. Preferably, the inventive methods disclosed herein are used to identify subjects at risk for developing malignant tumors.

Kits useful for detecting mutations in nucleic acids, and kits useful for diagnosing or determining a subject's risk of developing a disorder related to a genetic abnormality such as cancer, are also an aspect of the present invention. Such kits will comprise at least one container sized to house an oligonucleotide comprising at least one nucleotide with a 2'-ribose modification, preferably a 2'-NH₂ modification. The kit may also comprise a reactive compound (i.e., an activated ester) that reacts with the 2'-modified oligonucleotide wherein the reactive compound comprising a reporter moiety that is detectable in the presence of a mismatch between the oligonucleotide and a nucleic acid. The kit will also comprise printed instructions for assessing whether or not a nucleic acid molecule contains a mutation, and/or whether or not a subject is at risk for developing the disorder related to genetic abnormality, or whether a subject actually has the disorder. Buffers, labels and other reagents useful in carrying out the present invention may also be included in the kit.

Those skilled in the art will recognize that the mutation detection methodology of the present invention is also useful in methods of determining the specificity of hybridization between nucleic acid molecules, and in methods of quantitatively determining the extent of specific hybridization in a population of nucleic acid molecules. In such methods, the amount of single stranded nucleic acid molecules (i.e., non-hybridized nucleic acid) present as a fraction of a population of nucleic acid molecules may be calculated by measuring the hybridization of the nucleic acid population with a predetermined amount of 2'-ribose modified oligonucleotide probe. Hybridization may be measured by the amount of reporter moiety present in the population after hybridizing the nucleic acid molecule population with a predetermined amount of 2'-ribose modified oligonucleotide probe and then treating the hybridized probe with a reactive group. For example, in one embodiment, a constant concentration of probe oligonucleotide comprising a 2'-ribose substitution is used to determine the unknown concentration of target nucleic acid. The probe oligonucleotide is hybridized to a complementary target strand and is then contacted with a reactive compound (e.g., a succinimidyl ester). If stoichiometric binding is assumed, then the target strand concentration can be determined by the concentration at which the probe oligonucleotide becomes reactive to the reactive compound. In this way, the concentration nucleic acid molecule population is "titrated" against the constant concentration of the probe oligonucleotide, as follows: ifthe 2'-substituted probe strand is present at higher levels than the target strand, then some of the probe strands will be single stranded and reactive to the ester. Reaction of the oligonucleotide with the reactive compound comprising the probe moiety can be detected as described herein. However, when the probe strand is present at a concentration less than the target strand, all probe strands are perfectly hybridized and non-reactive towards the ester. Accordingly, no (or very slow) reaction of the reactive compound with the probe oligonucleotide will be detected. The concentration of the probe oligonucleotide and the nucleic acid molecule population will be equivalent when the reaction between the reaction compound and the probe oligonucleotide can just begin to be detected, as in a chemical titration. Carrying out such a method may occur as follows: a known concentration of an oligonucleotide is hybridized to nucleic acid molecules present in an unknown concentration in a sample to produce a population of oligonucleotides hybridized to the nucleic acid molecule. The oligonucleotide has a sequence complementary to a sequence present in the nucleic acid molecules of the sample, and the oligonucleotide comprises at least one nucleotide having a substitution at the 2'-ribose position. The sample containing the hybridized oligonucleotide is then contacted with a reactive compound comprising at least one reporter moiety under conditions that will allow the reporter moiety to be detected in the sample ifthe reactive compound reacts with the unhybridized oligonucleotides in the sample. The presence or absence of the reporter moiety in the sample is then detected, wherein the absence of the reporter moiety indicates that the concentration of the oligonucleotide is lower than the concentration of nucleic acid molecules in the sample, and the presence the reporter moiety indicates that the concentration of the oligonucleotide is higher than the concentration of nucleic acid molecules in the sample. The hybridization, contacting and detecting steps may be repeated with a different concentration of oligonucleotide until the detecting step indicates that the concentration of the oligonucleotide is approximately or exactly equal to the concentration of the nucleic acid molecules in the sample. The hybridization, contacting and detecting steps need not be repeated ifthe initial hybridizing, contacting and detecting steps indicate that the concentration of the nucleic acid molecules and the concentration of the probe oligonucleotide is equal. Alternatively, the sample of nucleic acid molecules may be contacted with different concentrations of probe oligonucleotide simultaneously or concurrently. This one embodiment of this alternative method may be carried out by placing aliquots of the nucleic acid molecule sample into, for example, separate wells of a multiwell plate, adding a different concentration of the probe oligonucleotide into each well under hybridizing conditions, contacting the sample in each well with a reactive compound comprising a reporter moiety, and then determining (either quantitatively or qualitatively) the presence or absence of the reported moiety bound to the hybridized oligonucleotide.

The above methods assume stoichiometric binding; that is, that all of the oligonucleotide added to the sample of nucleic acid molecules will hybridize to the nucleic acid molecule. If binding is not stoichiometric, then concentration of the target strand can be calculated using an equilibrium constant, which will be determinable to those skilled in the art. Alternatively, the concentration of nucleic acid molecules may be determined by hybridizing a known concentration of probe oligonucleotide substituted at a 2'-ribose position to a sample of nucleic acid molecules present in unknown concentration. A reactive compound comprising a reporter moiety is contacted with the sample comprising the hybridized oligonucleotides. The presence of absence of reporter moiety bound to the hybridized oligonucleotides is determined, and then compared to a reference or control, wherein the reference or control comprises the results of a series of reactions between a known concentrations of probe oligonucleotide and known concentrations of the target nucleic acid molecule. When the amount of reporter moiety detected in the sample of unknown nucleic acid molecule concentration is equivalent to the amount of reporter moiety detected in one of the control reactions, then the concentration of the nucleic acid molecules in the first sample may be considered equivalent to the concentration of nucleic acid molecule in the control reaction. The aforementioned methodologies may also be used, for example, to quantitatively determine the amount of cellular RNA and especially of a specific RNA present in a particular sample of cells, or the amount of cellular RNA present in a sample of total nucleic acid molecules obtained from selected cells as illustrated schematically in FIG. 9. Such a method finds particular use in detecting the overexpression or underexpression of mRNA in a cell or cells, for example, by genes associated with certain hereditary disorders or cancers, as set forth above.

The methodologies of the present invention are additionally useful in methods of detecting genetic variation in populations, and in methods of detecting genetic polymorphisms, including single nucleotide polymorphisms (SNPs). In these methods, an oligonucleotide is hybridized to a reference nucleic acid molecule to produce a hybridized oligonucleotide, wherein the oligonucleotide has a sequence complementary to the sequence that the nucleic acid molecule would have if a single nucleotide polymorphism was not present therein, and wherein the oligonucleotide comprises at least one nucleotide having a substitution at the 2'-ribose position. The hybridized oligonucleotide is contacted with a reactive compound comprising at least one reporter moiety. The presence of the reporter moiety bound to the hybridized oligonucleotide is detected. The detection of the reporter moiety bound to the hybridized oligonucleotide indicates that a single nucleotide polymorphism is present in the nucleic acid molecule. Alternatively, the oligonucleotide may have a sequence complementary to the sequence that the nucleic acid molecule would have if a single nucleotide polymorphism was present therein. The detection of the reporter moiety bound to the hybridized oligonucleotide indicates that a single nucleotide polymorphism is not present in the nucleic acid molecule. As used herein, a "reference" nucleic acid molecule is a nucleic acid molecule that may be known to be polymorphic between individuals, and/or may be known to contain one or more single nucleotide polymorphisms.

Those skilled in the art will recognize that selective modification of 2'-amine substituted nucleic acids is also useful for detecting conformational changes in RNA or DNA or other modified nucleic acid. In the application of the present invention, oligonucleotides that undergo conformational changes upon interacting with a large or small molecule ligand also experience a change in the local nucleotide stability. Conformational changes are detected by measuring the amount of reporter moiety present after the oligonucleotide has undergone a conformational change. The reactive group is added after the oligonucleotide has potentially bound the ligand.

Because oligonucleotide conformational changes can be linked to ligand binding, this same methodology can be further used to quantify the amount of ligand in a solution. For example, the methods of the present invention may be used to detect the binding of a ligand to an aptamer. In one embodiment, a first sample of an aptamer is contacted with a reactive compound comprising a reporter moiety, wherein the aptamer comprises at least one nucleotide having a substitution at the 2'-ribose position. The binding of the reporter moiety to the aptamer is detected in the first sample to determine ifthe aptamer binds the reporter moiety in the absence of a ligand. A second sample of the aptamer is contacted with a ligand, wherein the aptamer comprises at least one nucleotide having a substitution at the 2'-ribose position, and is then contacted with a reactive compound comprising a reporter moiety. The binding of the reporter moiety to the aptamer is detected in the second sample to determine ifthe aptamer binds the reporter moiety in the presence of the ligand. The binding of the reporter moiety to the aptamer in the absence of a ligand is compared with the binding of the reporter moiety to the aptamer in the presence of the ligand, wherein a difference in the binding of the reporter moiety between the aptamer in the first sample and the aptamer in the second sample indicates that the aptamer binds the ligand. Of course, a comparison of two samples of aptamers is not necessary; in some cases, the simple detection of non-reactivity of an aptamer comprising a nucleotide substituted at a 2'-ribose position with a reactive compound of the present invention will be an indication of ligand binding.

The following Examples are provided to illustrate the present invention, and should not be construed as limiting thereof. Abbreviations used in the Examples below are as follows: HEPES means 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EDTA means ethylenediaminetetraacetic acid; DMSO means dimethyl sulfoxide; DTT means dithiothreitol; TE means Tris-EDTA; bp means base pairs; mL means milliliters; M means molar; mM means millimolar; μM means micromolar; nM means nanomolar; K means 1000 rpm (revolutions per minute); cpm means counts per minute; min means minutes, T_m means melting temperature; unless otherwise specified, all temperatures are provided in degrees Celsius (°C)

EXAMPLE 1 RNA and DNA Preparation

The scheme for the design of a series of oligonucleotides used to detect mutations in nucleic acid molecules according to the present invention is shown in FIG. 1. As shown in the Figure, an oligonucleotide probe that is 20 nucleotides in length contains a ribose 2'-NH₂ substitution at position 10. Residue 10 is shown as 2'- deoxy-2'-amino-cytidine. The complement nucleic acid molecules hybridize to the oligonucleotide to form a perfect duplex, while the mismatched nucleic acid molecules hybridize to the oligonucleotide to yield a duplex containing a single mismatch. According to the present invention, nucleic acid molecules and oligonucleotides may be RNA or DNA. DNA is shown in the Figure; for RNA. uridine substitutes are substituted for thymidine.

In the experiments described herein, some RNA and DNA nucleic acid molecules and 2'-NH₂-containing DNA oligonucleotides were synthesized and partially purified at the Nucleic Acid Facility at North Carolina State University (Raleigh, North Carolina, USA), or at the nucleic acids facility of the Lineberger Comprehensive Cancer Center at the University of North Carolina (Chapel Hill, North Carolina, USA). The RNA nucleic acid molecules were synthesized according to standard protocols and cleaved from solid supports. Oligonucleotides were deprotected by methods known to those in the art. Phosphate protecting groups (such as benzoyl and dimethoxytrityl groups) were removed by incubation in 3 mL of ethanolic ammonium hydroxide for 4 hours at 55°C. Solutions were evaporated to dryness and resuspended in 0.75 mL of tetrabutyl ammonium fluoride (with overnight incubation in the dark and periodic vortexing) to remove silyl protecting groups at the 2' positions. Samples were diluted with TE Buffer (10 mM Tris, pH 7.5, 1 mM disodium EDTA) and desalted over NAP-10 columns containing Sephadex^®G50 resin (Pharmacia Biotech, Peapack, New Jersey, USA). All nucleic acid molecules were concentrated by ethanol precipitation and purified on 20% denaturing polyacrylamide gels (29:1 acrylamide:bisacrylamide, 7M Urea, and TBE (90 mM Tris-borate, 4 mM disodium EDTA)) for 2 hours at 30 watts. Full length oligomers were excised from the gel and electroeluted using an elutrap sysyem (Schleier and Schuell, Keene, New Hampshire, USA; using l/2x TBE, at 150 volts for 3 hours). Other oligonucleotides were synthesized and deprotected as recommended by Dharmacon Research (Boulder, Colorado, USA; see S.A. Scaringe, et al, J. Am. Chem. Soc. 120, 11820-11821 (1998)).

The 2'-NH₂ modified DNA oligonucleotide was purified as described in M. Sawadago et al, Nucleic Acids Research 19, 674 (1990). 1 mL of n-butanol was added for each 100 μL of DNA in concentrated ammonium hydroxide. The sample was centrifuged at 12K for 1 minute and the supernatant discarded. The sample was resuspended in TE buffer and purified on a 20% denaturing polyacrylamide gel. The band was excised and passively eluted overnight at 4°C in 500 mM potassium acetate (pH 6.0), lmM disodium EDTA. The remaining oligodeoxynucleotides were synthesized at the Lineberger Cancer Institute at the University of North Carolina at Chapel Hill (Chapel Hill, North Carolina, USA), purified on 20% denaturing polyacrylamide gels, excised from the gels, and passively eluted as described above. All oligomers were stored at -20°C in TE Buffer. 2'-NH₂ modified oligonucleotide (30 pmol) was 5'-[³²P] end-labeled according to the methods described in A. Krol and P. Carbon, Methods in Enzymology 180, 212-227 (1989), using T4 polynucleotide kinase (1000 units/mL, New England Biolabs) at 37°C in 70 mM Tris-HCl, lOmM MgCl and 5mM DTT. Radiolabeled oligonucleotides were purified on 20% denaturing gels as described above. The reactions (16 μL, typically in 100 mM HEPES at pH 8.0) contained 1000 cpms of the radiolabeled oligonucleotide probe (approximately 1 nM) and 0.72 μM of the nucleic acid molecule strand. The oligonucleotide probe and either a complementary nucleic acid molecule or mismatched nucleic acid molecule were annealed by incubation at 90°C for 3 minutes and cooled to room temperature 22°C over 20 minutes.

Kinetic studies as described in Example 3 below were performed on the oligonucleotide probe, or the oligonucleotide probe annealed to the complement nucleic acid molecule to form the perfect duplex, or the oligonucleotide probe annealed to one of the mismatched nucleic acid molecules to form mismatched duplexes as shown in the scheme of FIG. 1. Reactions were initiated by addition of sulfosuccinimidyl-6- (biotinamido) hexanoate reagent to a final concentration of either 50 or 75 mM from a stock solution of 500 or 750 mM, respectively, in dimethyl sulfoxide (DMSO). Reactions were quenched at 0, 2, 5, 10, 30, 60 minutes with 1 μL of IM dithiothreitol (DTT) followed by the addition of 7 μL of stop solution (85% (v/v) formamide, 50 mM disodium EDTA, 0.5X TBE, xylene cyanol and bromophenol blue) and placed on ice. Control "no reagent " experiments were performed by adding 1 μL of DMSO to the samples in place of the reagent. Control reactions performed with DNA and RNA probes containing a 2'-OH or 2'-H in place of the 2'-NH₂ group showed that all oligomers lacking the 2'-NH₂ substitutions are unreactive. Quenched reactions were centrifuged at 14K at room temperature for 5 minutes to pellet any precipitate. Reactions were heated at 90°C for 3 minutes and 2 μL of each reaction was loaded onto a 20%) denaturing acrylamide gel as described above. Bands were quantitated using a Molecular Dynamics Storm^® 840 Phosphorimager (Sunnyvale, California, USA) and the following equation was used to determine the fraction of unreacted material: fraction _^ lunreacted unreacted — j _μ T

-^■■unreacted ^xreacted where I is the band intensity after subtracting the background. Data points were plotted and fit to the first-order kinetic equation, as described by John and Weeks (D.M. John and K.M. Weeks, Chem. Biol. 1, 405-410 (2000)). EXAMPLE 2 Selective Acylation Incorporation of 2'-NH₂ substituted nucleotides into RNA or DNA introduces a good nucleophile that can be site-specifically modified using succinimidyl esters as shown in Scheme 1, above, and in Cohen and Cech (1997), supra. Referring to Scheme 1, a number of moieties can be incorporated in the position indicated as 'R." A single point mutation, G 1691 A, in the gene encoding the Factor V clotting factor was analyzed in order to illustrate the sensitivity of the method described herein. The G1691 A mutation affects five percent (5 %) of all Caucasian pre-surgical patients. The mutation, also called the Factor V Leiden (FVL) mutation, causes inherited thrombophilia and thrombosis morbidity. The presence of the FVL mutation was detected using the oligonucleotide model system illustrated in FIG. 1, as described below.

Detection of the mutation employed a 20-nucleotide probe oligonucleotide as shown in FIG. 1, which was complementary to the FVL sense strand and contained a unique 2'-NH₂-cytidine nucleotide. The synthesis of the 2'-NH₂ oligonucleotide was accomplished by the methods described herein. The oligonucleotide probe was hybridized to an excess of either a nucleic acid molecule strand that forms a perfect 20 bp duplex (wild type Factor V), or a nucleic acid molecule strand containing a mismatch at the position of the 2'-NH₂ cytidine (corresponding to the FVL mutation) as shown in FIG. 1.

Using the biotin-containing derivative of the succinimidyl ester shown in FIG. 2 A it was found that the rate of modification of a 20 bp model substrate (FIG. 2B) containing a single 2'-NH₂ moiety is diagnostically sensitive to the base-paired state of the 2'-NH₂ nucleotide. When probe oligonucleotide RNA is hybridized to a large excess of a complementary DNA nucleic acid molecule, acylation of the 2'-NH₂ position is slow (FIG. 2C, first panel). Modification was monitored by denaturing acrylamide electrophoresis as described above. The mobility of the modified oligonucleotide is retarded as compared with the unmodified oligonucleotide probe (FIG.3A).

Reaction of the mismatched 2'-NH₂ cytidine residue is 30-fold faster than reaction of the perfect duplex (FIG. 3B), providing a strong signal to identify the DNA mutation. When the same reaction is performed by hybridization of the test 2'- amine-containing ohgonucleotide DNA to each of three nucleic acid molecules that yield a mismatched base across from the 2'-NH₂ nucleotide, the rate of modification is increased about 30-fold. Modification of the mismatched position is as rapid as for the single stranded test oligonucleotide alone.

In the present experiment, the modified oligonucleotide was resolved from the unreacted oligonucleotide probe by denaturing electrophoresis (FIG. 3A). However, detection may also be achieved by streptavidin capture, or by replacing biotin with a fluorescent probe and using a fluorometric assay according to known methods. Mismatch-specific modification may also be detected using multi-well colorimetric assays and by using high throughput liquid chromatographic methods without resorting to gel electrophoresis.

EXAMPLE 3 Optimization of Specificity The selectivity of 2'-amine acylation chemistry at two temperatures, 35°C and

50 °C, was tested. 2'-amine acylation experiments were performed with excess (0.7 μM) complementary strand and 1-10 nM ³²P-5'-end labeled probe oligonucleotide. Probe and complementary strand were hybridized by heating to 90°C for 1-3 minutes and subsequently cooled to 22°C over 20 minutes. Reactions (16 μL) were initiated by addition of a 1 Ox solution of the succinimidyl ester in DMSO and aliquots (2 μL) were quenched by addition of 8 μL stop solution (125 mM DTT in 85% formamide, l/2χ TBE, 50 mM EDTA). 2*-amine acylation at 35 and 50°C was performed with a final concentration of 75 or 50 mM ester, respectively. Reactions were resolved on 20% denaturing gels and quantified using a phosphorimager. The succinimidyl ester reacts by 2'-amine acylation and also by solvent hydrolysis (Chamberlin, S.I. &

Weeks, K.M. (2000) J. Am. Chem. Soc. 122, 216-224). Acylation rates were obtained by fitting the fraction acylated product to an equation that accounts for these parallel reactions,

fraction product = 1 -

where ^_ac_yι and -^hydrolysis ^are the pseudo-first-order rate constants for 2'-amine acylation and reagent hydrolysis, respectively. ^hydrolysis ^was f°^und to be equal to 0.025 min^"1.

At 35°C none of the duplexes showed appreciable melting or fraying, while 50 °C was at or above T_m for the mismatch-containing duplexes, but below T_m for the perfect duplex (FIG. 4). Experiments performed at 50°C therefore monitor the global stability of the perfect versus the mismatch-containing duplexes. In contrast, 35°C represents a non-stringent temperature at which hybridization alone by the 2'-amine containing probe cannot discriminate thermodynamically between the perfect duplex and mismatch-forming target sequences. Thus, 2'-amine acylation will detect the mismatched base pairs at 35°C only if acylation is sensitive to local nucleotide flexibility. Thus, modification of 2'-amine substituted nucleotides detects mismatches at both stringent (35°C) and non-stringent temperatures.

EXAMPLE 4

2'-Ribose Modification Detects Mutations In Any Context And For Any

Mismatch

The present invention is used with varying nucleic acid chemistries, and mismatches are detected in any known duplex context. Perfect base pairing and mismatches were detected across from a 2'-NH₂ uridine nucleotide (FIG. 5). This oligonucleotide was the same as shown in FIG. 2B except that the 2'-NH₂ cytidine nucleotide was replaced by a 2'-NH₂ uridine. The probe oligonucleotide was hybridized either with DNA complementary strands (FIG. 5A) or with RNA complementary strands (FIG. 5B). FIG. 5A illustrates the method of the present invention with DNA-DNA duplexes. In the experiment shown in this FIG. 5, a 2'- NH₂ uridine DNA oligonucleotide was hybridized to DNA nucleic acid molecules. For this series of reactions, the reaction solutions contained 100 mM HEPES (pH 8.0), IM NaCl and 75 mM succinimidyl ester. The reaction was performed at 35°C over a period of 60 minutes. FIG. 5B illustrates the method of the present invention with DNA-RNA duplexes. In FIG. 5B, the same 2'-NH₂ uridine DNA oligonucleotide as in FIG. 5A was hybridized to RNA nucleic acid molecules. The reaction was performed over a period of 60 minutes at 35°C. These reactions contained 100 mM HEPES (pH 8.0), 1.5 M NaCl and 75 mM succinimidyl ester. A 2'-NH₂ DNA strand is also used to detect mutations in RNA strands across from a 2'-NH₂ cytosine nucleotide and under different conditions (FIG. 6). Conditions in FIG. 6A were 100 mM HEPES (pH 8.0) 1.5 M NaCl, 75 mM succinimidyl ester at 35°C. Conditions in FIG. 6B were without NaCl at 50°C.

An RNA strand is also used to detect mutations across from an RNA (FIG. 7A) or DNA strand (FIG. 7B). Reaction conditions were 150 mM Na phosphate (pH 7.6), 500 mM NaCl, 50 mM succinimidyl ester at 50°C (FIG. 7A) and 150 mM Na phosphate (pH 7.6), 19 mM NaCl, 4 mM MgCl₂ 50 mM succinimidyl ester at 22°C (FIG. 7B).

EXAMPLE 5 Detection of Nucleotide Deletions The present invention is also useful in the detection of deletions in nucleic acid molecules. A schematic of the nucleic acid molecule containing the deletion is shown in FIG. 8A. Hybridization of a complementary oligonucleotide to the nucleic acid molecule and contacting of the hybridized oligonucleotide with a reactive compound are carried out as described in Examples 1 and 2 above. Kinetic analysis was performed as described in Example 3 at 35°C and 75 mM succinimidyl ester. The results of the kinetic analysis is shown in FIG. 8B. As illustrated in FIG. 8B, reaction of the probe oligonucleotide with the target nucleic acid containing a deletion is significantly faster than for the target that forms a perfect duplex.

EXAMPLE 6

Quantification of Absolute RNA Amounts With Single Nucleotide Specificity

FIG. 9 demonstrates the use of chemical modifications of a 2'-ribose position for quantification of target nucleic acids. A constant concentration of probe oligonucleotide (e.g. 0.1 nM) is used to determine the unknown concentration of target nucleic acid. The probe oligonucleotide (thick line, FIG. 9) is hybridized to a complementary target strand (thin line, FIG. 9) and is then reacted with a succinimidyl ester. If stoichiometric binding is assumed, then the target strand concentration can be determined by the concentration at which the probe oligonucleotide becomes reactive to the succinimidyl ester. Ifthe 2'-NH₂ probe strand is present at higher levels than the target strand, then some of the probe strands will be single stranded and reactive to the ester. Accordingly, the binding of the reporter moiety will be detected. Reaction of the oligonucleotide as resolved on a denaturing gel is shown schematically in the lower panels (FIG. 9). However, when the probe strand is present at a concentration less than the target strand, all probe strands are perfectly hybridized and non-reactive towards the ester, and the binding of reporter moiety will not be detected (or will be detected as indicative of a very slow reaction, if at all). If binding is not stoichiometric, the concentration of the target strand can be calculated using the equilibrium constant. FIG. 10 shows quantification of the absolute amount of RNA with single nucleotide base discrimination. A representative experiment of a perfectly complementary duplex with varying amounts of RNA target nucleic acid is shown in the top gel. The probe concentration was constant at 0.04 nM. The free probe is separated from the 2'-acylated product by denaturing electrophoresis analogously as described in Examples 1 and 2 above. The probe oligonucleotide is less reactive in the presence of larger concentrations of the RNA target nucleic acid because all of the probe oligonucleotide is based-paired. Mobility of the acylated product is retarded in a denaturing gel as compared with free probe. Hybridization and reactivity of a target RNA containing a single base pair substitution is shown in the lower gel. In this case, the probe oligonucleotide is the same as shown in FIG. 2B but contains 5 bp extensions on both sides. The target strand is a 30 nt RNA containing a C-A mismatch as illustrated schematically in FIG. 1. The probe strand is reactive at all concentrations due to the presence of the mismatched base pair (bottom gel). Both experiments were performed in 100 mM HEPES, pH 8.0; 1.5 M NaCl, at 35°C, acylation was carried out by 75 mM succinimidyl ester for 30 minutes. Quantification of the data shown in FIG. 10 is represented in FIG. 11. The fraction of unreacted material after 30 minutes is calculated and fit with a simple binding equation. At the transition midpoint, 340 attomoles of target RNA are detected in this experiment, although lower detection limits are also possible. These data illustrate that nucleic acid target molecule quantification is very selective for the correct sequence.

EXAMPLE 7 Detection of Conformational Changes in Nucleic Acid The present inventors have found that acylation of 2'-amine substituted RNA is "gated" by the underlying RNA structure. Using tRNA^Asp as a model system, a single 2'-amine substitution was introduced at every position in the RNA. RNAs were generated by in vitro transcription using T7 RNA polymerase using a mixture of ribonucleotides and a 2'-NH₂-substituted ribonucleotide (H. Aurup, et al, Biochemistry 31, 9636-9641 (1992)). The resultant RNA pools were constructed such that each full-length tRNA contained a single 2'-NH₂ substitution. Incorporation of a 2'-amine moiety introduces a chemical handle within the RNA that can be selectively modified with a reactive compound such as an activated ester. Reaction with a succinimidyl ester as shown in FIG. 1 yields the 2'- amide product. In these tRNA-based experiments, the 2'-amide product was detected as a stop to primer extension by reverse transcriptase, as is illustrated for guanosine nucleotides in FIG. 12A (2'-NH₂ nucleotides function as templates for reverse transcriptase while the bulkier 2'-acylated causes reverse transcriptase to stop). Analogous experiments were performed independently for each of the other three ribonucleotides.

The relative reactivity for each 2'-amine substituted nucleotide in tRNA^Λsp was determined, and it was found that some positions were significantly protected from modification (compare denaturing with native lanes in FIG. 12A; see also S.I. Chamberlin and K.M. Weeks, J. Am. Chem. Soc. ll, 216-224 (2000)). 2'-amine positions protected from modification under native RNA folding conditions are superimposed on a secondary structure model for tRNA^Asp in FIG. 12B. All helices and most tertiary interactions are unreactive towards the activated ester. Relative reactivities do not correlate with the static solvent accessibility of the 2'-ribose positions. Instead, relative reactivities show good correlation to measures of local nucleotide flexibility including crystallographically derived temperature factors (cf. Chamberlin and Weeks (2000), supra; data not shown). Thus, 2'-amine reactivity is correlated with the local stability of the nucleic acid structure. In the case of RNA, 2'- NH₂ reactivity is higher at the sites of a wide variety of positions in the RNA structure that are not involved in stable base pairing or tertiary interactions (FIG. 12B). The foregoing example illustrates that the basic methodology of the present invention may be used in a general method for mapping local RNA stability that requires minimal optimization and can be used to monitor RNAs of any size at nucleotide resolution. In a general example of such a method, RNA pools are generated containing, on average, a single 2'-amine substitution per transcript. Incorporation of a 2'-amine moiety introduces a reactive functionality within the RNA that can be selectively modified using an activated ester. This reaction yields the 2'- amide RNA product, which is detected as a stop to primer extension by reverse transcriptase. Acylation of the 2'-amine position is gated by the underlying RNA structure. Thus, base paired positions and nucleotides involved in tertiary interactions are less reactive than nucleotides located in less constrained loops. This approach provides a means for evaluating flexibility at single nucleotide resolution on small quantities of large RNAs.

The skilled artisan will appreciate from the examples described herein that this method for evaluating flexibility at single nucleotide resolution is equally applicable to determining local flexibility of both DNA and RNA, and that the oligonucleotides used to determine such flexibility may be DNA or RNA. Additionally, the method may be carried out on nucleic acid populations that are not present in pools, but may be present simply as one or more nucleic acid molecules comprising a 2'-ribose substitution at one or more nucleotides therein. Finally, the present methods may be used to determine the local conformation of unhybridized or single stranded nucleic acid molecules (including unhybridized oligonucleotides), or the local conformation of oligonucleotides hybridized to a nucleic acid molecule.

EXAMPLE 8

Detection of Codon 12 K-ras Mutations

The most common mutations that activate the K-ras proto-oncogene are mutations in codon 12, normally a glycine. See J.L. Bos, Cancer Res. 49, 4682-4689 (1989); E. Santos, et al., Science 223, 661-664 (1984). The relevant portion of the K- ras proto-oncogene is shown in FIG. 13. Mutation of either of the first two positions in the Gly 12 codon results in a coding change. Mutations in the third position of codon 12 are silent because all GGN codons still code for glycine. Mutated proteins do not interact appropriately with auxiliary proteins and are stuck in a GTP -bound or active state, affecting normal regulation of cell growth. See D.R. Lowy and B.M. Willumsen, Ann. Rev. Biochem 62, 851-891 (1993).

To code for glycine, codon 12 must contain the sequence GGN, where N is any nucleotide. The nucleic acid molecules to be tested are hybridized to a probe oligonucleotide in which two 2'-NH₂ cytidine residues pair with the two consecutive G nucleotides in codon 12. Mutation of either G residue will result in a C-X mismatch. Either of these mismatches is simultaneously detected as an increased reactivity towards NH₂-selective reagents.

For codon 12 mutations, all possible mismatches including C-T, C-C and C-A are detected. Each mismatch is tested individually for its effect on probe reactivity. The ability to detect K-ras mutations in clinical samples, in which cells harboring mutant genes may constitute a small fraction of total gene sequences, is of profound importance for early detection of cancer. The key feature of the chemistry of the present invention suggests that 1 in 600 detection specificity is possible. That the chemistry is compatible with other methods may provide an additional proofreading function. Certain applications of allele-specific oligonucleotide hybridization permit scoring of a given mutation at a level of 1 in 20 (for Ras applications, see P.J. Browett and J.D. Norton, Oncogene 4, 1029-1036 (1989); R. Rosell, et al., Clin. Cancer Res. 2, 1083-1087 (1996)). Background signals arise from annealing of oligonucleotides to sequences that differ from the target nucleic acid molecule by as few as one position. Selective modification of 2'-NH₂ containing oligonucleotides are thus used to proofread signals obtained via allele-specific hybridization. An approximately 30- fold kinetic discrimination for modification of mismatched oligonucleotides was observed as compared with perfectly paired probes. Thus, following allele-specific hybridization (with a 1 :20 signal/noise ratio), selective modification of 2'-NH₂ groups (with 1 :30 kinetic selectivity) potentially affords a 1:600 selectivity.

Allele-specific hybridization methods follow standard approaches involving PCR amplification, immobilization on nylon membranes, selective probe hybridization, and stringent washing. Subsequently, selectively hybridized oligonucleotide probes are reacted with the chemistry described herein to detect codon 12 K-ras mutants. 2'-NH₂ modification is detected using either fluorescent or chemiluminescent methods. The input of total DNA from cells lines carrying mutant and wild type K-ras sequences is varied to determine the maximum achievable selectivity.

EXAMPLE 9 Fluorogens

An alternative chemistry for selective modification of 2'-NH containing oligonucleotide probes is also examined. Fluorogens are simple non-fluorescent molecules that react with primary amines to yield strongly fluorescent products. See S. Udenfriend, et al, Science 178, 871-872 (1972); W.Y. You, Anal. Biochem. 244, 277-282 (1997). Representative chemistries using O-phthaldialdehyde (OP A) and 3- (4-carboxylbenzoyl)-quinoline-2-carboxaldehyde (CBQCA) are shown in Scheme 2, above, and fluorescamine (not shown). Fluorogenic chemistry affords two advantages for practical detection of codon 12 K-ras mutations. First, since the unreacted oligonucleotide probes are non- fluorescent, any background signal is significantly reduced and increases specificity. Second, the fluorogenic chemistry, while well- established for reaction of primary amines, is sterically exacting. Modification fixes the ribose 2'-NH₂ oligonucleotide probe into a bulky aromatic ring system. Incorporation of such a bulky group significantly increases the selectivity for mismatch-containing positions.

EXAMPLE 10 Detection of Ligand Binding to a Nucleic Acid Aptamer

Nucleic acid sequences can be created by in vitro selection to bind specifically and with high affinity to practically any large or small molecule ligand of interest; this selection is method is sometimes referred to as SELEX. See L. Gold, et al, Annu. Rev. Biochem. 64, 763-798 (1995); T. Hermann and D.J. Patel, Science 287, 820-825 (2000). Nucleic acid molecules thus produced are called aptamers and are able to bind ligands. It is known in the art that most nucleic acid aptamers undergo a conformational change upon binding of a ligand. See Hermann and Patel, supra. One such example is the adenosine monophosphate aptamer shown in FIG. 14, after D. E. Huizenga et al., Biochemistry 34, 656-665 (1995) and C. H. Lin et al, Chem. Biol. 4, 817-832 (1997). The free DNA contains an unstructured internal loop. A 2'-amine nucleotide in this loop is reactive towards a succinimidyl ester. Upon binding the adenosine monophosphate ligands, significant new structures form, as shown in FIG. 14. Constrained by this structure, the 2'-ribose position is now unreactive. In this case, the lack of reaction with the succinimidyl ester that contains a reporter moiety indicates binding by the ligand.

The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED:

1. A method of detecting a mutation in a nucleic acid molecule suspected of containing a mutation, comprising: hybridizing an oligonucleotide to the nucleic acid molecule to produce a hybridized oligonucleotide, wherein the oligonucleotide has a sequence complementary to the sequence that the nucleic acid molecule would have if a mutation was not present, and wherein the oligonucleotide comprises at least one nucleotide having a substitution at the 2'-ribose position; contacting the hybridized oligonucleotide with a reactive compound comprising at least one reporter moiety; and then detecting ifthe reporter moiety is bound to the hybridized oligonucleotide, wherein the detection of the reporter moiety bound to the hybridized oligonucleotide indicates that a mutation is present in the nucleic acid molecule.

2. The method according to Claim 1 wherein the substitution at the 2'- ribose position is an amine substitution.

3. The method according to Claim 1 , wherein the reporter moiety is biotin.

4. The method according to Claim 1 , wherein the reactive compound is an activated ester.

5. The method according to Claim 4, wherein the activated ester is succinimidyl ester.

6. The method according to Claim 1, wherein the reactive compound is a fluorogen.

7. The method according to Claim 6, wherein the fluorogen is selected from the group consisting of O-phthaldialdehyde (OPA), 3-(4-carboxylbenzoyl)- quinoline-2-carboxaldehyde (CBQCA) and fluorescamine.

8. The method according to Claim 1, wherein the nucleic acid molecule comprises DNA.

9. The method according to Claim 1 , wherein the nucleic acid molecule comprises

RNA.

10. The method according to Claim 1, wherein the oligonucleotide comprises DNA.

11. The method according to Claim 1 , wherein the oligonucleotide comprises RNA.

12. The method according to Claim 1, wherein the nucleic acid molecule comprises

RNA and the oligonucleotide comprises DNA.

13. The method according to Claim 1, wherein the nucleic acid molecule comprises

DNA and the oligonucleotide comprises RNA.

14. The method according to Claim 1, wherein both the nucleic acid molecule and the oligonucleotide comprise DNA.

15. The method according to Claim 1, wherein both the nucleic acid molecule and the oligonucleotide comprise RNA.

16. The method according to Claim 15, wherein the nucleic acid molecule comprises codon 12 of the human K-ras gene.

17. A method of detecting a deletion in a nucleic acid molecule suspected of containing a deletion, comprising: hybridizing an oligonucleotide to the nucleic acid molecule to produce a hybridized oligonucleotide, wherein the oligonucleotide has a sequence complementary to the sequence that the nucleic acid molecule would have if a deletion was not present, and wherein the oligonucleotide comprises at least one nucleotide having a substitution at the 2'-ribose position; contacting the hybridized oligonucleotide with a reactive compound comprising at least one reporter moiety; and then detecting ifthe reporter moiety is bound to the hybridized oligonucleotide, wherein the detection of the reporter moiety bound to the hybridized oligonucleotide indicates that a deletion is present in the nucleic acid molecule.

18. The method according to Claim 17 wherein the substitution at the 2'- ribose position is an amine substitution.

19. The method according to Claim 17, wherein the reporter moiety is biotin.

20. The method according to Claim 17, wherein the reactive compound is an activated ester.

21. The method according to Claim 20 wherein the activated ester is succinimidyl ester.

22. The method according to Claim 17, wherein the reactive compound is a fluorogen.

23. The method according to Claim 22, wherein the fluorogen is selected from the group consisting of O-phthaldialdehyde (OPA), 3-(4-carboxylbenzoyl)- quinoline-2-carboxaldehyde (CBQCA) and fluorescamine.

24. The method according to Claim 17, wherein the nucleic acid molecule comprises DNA.

25. The method according to Claim 17, wherein the nucleic acid molecule comprises

RNA.

26. The method according to Claim 17, wherein the oligonucleotide comprises DNA.

27. The method according to Claim 17, wherein the oligonucleotide comprises RNA.

28. The method according to Claim 17, wherein the nucleic acid molecule comprises

RNA and the oligonucleotide comprises DNA.

29. The method according to Claim 17, wherein the nucleic acid molecule comprises

DNA and the oligonucleotide comprises RNA.

30. The method according to Claim 17, wherein both the nucleic acid molecule and the oligonucleotide comprise DNA.

31. The method according to Claim 17, wherein both the nucleic acid molecule and the oligonucleotide comprise RNA.

32. A method of detecting a mismatch in a nucleic acid molecule suspected of containing a mismatch, comprising: hybridizing an oligonucleotide to the nucleic acid molecule to produce a hybridized oligonucleotide, wherein the oligonucleotide has a sequence complementary to the sequence that the nucleic acid molecule would have if a mismatch was not present, and wherein the oligonucleotide comprises at least one nucleotide having a substitution at the 2'-ribose position; contacting the hybridized oligonucleotide with a reactive compound comprising at least one reporter moiety; and then detecting ifthe reporter moiety is bound to the hybridized oligonucleotide, wherein the detection of the reporter moiety bound to the hybridized oligonucleotide indicates that a mismatch is present in the nucleic acid molecule.

33. The method according to Claim 32 wherein the substitution at the 2'- ribose position is an amine substitution.

34. The method according to Claim 32, wherein the reporter moiety is biotin.

35. The method according to Claim 34, wherein the reactive compound is an activated ester.

36. The method according to Claim 35, wherein the activated ester is succinimidyl ester.

37. The method according to Claim 32, wherein the reactive compound is a fluorogen.

38. The method according to Claim 37, wherein the fluorogen is selected from the group consisting of O-phthaldialdehyde (OPA), 3-(4-carboxylbenzoyl)- quinoline-2-carboxaldehyde (CBQCA) and fluorescamine.

39. The method according to Claim 32, wherem the nucleic acid molecule comprises DNA.

40. The method according to Claim 32, wherein the nucleic acid molecule comprises

RNA.

41. The method according to Claim 32, wherein the oligonucleotide comprises DNA.

42. The method according to Claim 32, wherein the oligonucleotide comprises RNA.

43. The method according to Claim 32, wherein the nucleic acid molecule comprises RNA and the oligonucleotide comprises DNA.

44. The method according to Claim 32, wherein the nucleic acid molecule comprises

DNA and the oligonucleotide comprises RNA.

45. The method according to Claim 32, wherein both the nucleic acid molecule and the oligonucleotide comprise RNA.

46. The method according to Claim 32, wherein both the nucleic acid molecule and the oligonucleotide comprise RNA.

47. A method of detecting a single nucleotide polymorphism in a nucleic acid molecule, comprising: hybridizing an oligonucleotide to a reference nucleic acid molecule to produce a hybridized oligonucleotide, wherein the oligonucleotide has a sequence complementary to the sequence that the nucleic acid molecule would have if a single nucleotide polymoφhism was not present, and wherein the oligonucleotide comprises at least one nucleotide having a substitution at the 2'-ribose position; contacting the hybridized oligonucleotide with a reactive compound comprising at least one reporter moiety; and then detecting ifthe reporter moiety is bound to the hybridized oligonucleotide, wherein the detection of the reporter moiety bound to the hybridized oligonucleotide indicates that a single nucleotide polymoφhism is present in the nucleic acid molecule.

48. The method according to Claim 47 wherein the substitution at the 2'- ribose position is an amine substitution.

49. The method according to Claim 47, wherein the reporter moiety is biotin.

50. The method according to Claim 47, wherein the reactive compound is an activated ester.

51. The method according to Claim 50, wherein the activated ester is succinimidyl ester.

52. The method according to Claim 47, wherein the reactive compound is a fluorogen.

53. The method according to Claim 52, wherein the fluorogen is selected from the group consisting of O-phthaldialdehyde (OPA), 3-(4-carboxylbenzoyl)- quinoline-2-carboxaldehyde (CBQCA) and fluorescamine.

54. The method according to Claim 47, wherein the nucleic acid molecule comprises DNA.

55. The method according to Claim 47, wherein the nucleic acid molecule comprises

RNA.

56. The method according to Claim 47, wherein the oligonucleotide comprises DNA.

57. The method according to Claim 47, wherein the oligonucleotide comprises RNA.

58. The method according to Claim 47, wherein the nucleic acid molecule comprises

RNA and the oligonucleotide comprises DNA.

59. The method according to Claim 47, wherein the nucleic acid molecule comprises

DNA and the oligonucleotide comprises RNA.

60. The method according to Claim 47, wherein both the nucleic acid molecule and the oligonucleotide comprise DNA.

61. The method according to Claim 47, wherein both the nucleic acid molecule and the oligonucleotide comprise RNA.

62. A method of determining if a subject is at risk for developing a disorder characterized by a genetic mutation, comprising: hybridizing an oligonucleotide to a nucleic acid molecule obtained from the subject to produce a hybridized oligonucleotide, wherein the nucleic acid molecule comprises a sequence that is known to contain a mutation characterizing the genetic disorder ifthe subject is at risk of developing the genetic disorder; wherein the oligonucleotide has a sequence complementary to the sequence that the nucleic acid molecule will have ifthe mutation characterizing the disorder is not present; and wherein the oligonucleotide comprises at least one nucleotide having a substitution at the 2'-ribose position; contacting the hybridized oligonucleotide with a reactive compound comprising at least one reporter moiety; and then detecting ifthe reporter moiety is bound to the hybridized oligonucleotide, wherein the detection of the reporter moiety bound to the hybridized oligonucleotide indicates that a mutation is present in the nucleic acid molecule, and that the subject is at risk for developing the disorder.

63. The method according to Claim 62, wherein the disorder is selected from the group consisting of sickle cell anemia, certain forms of hemophilia, fragile-X syndrome, spinal and bulbar muscular dystrophy, myotonic dystrophy, Huntington's disease, hereditary angioedema, Li-Fraumeni disease, cystic fibrosis, neurofibromatosis type 2, and von Hippel-Lindau disease.

64. The method according to Claim 62, wherein the disorder is characterized by a mutation in the Factor V Leiden (FVL) clotting factor.

65. The method according to Claim 64, wherein the mutation is the G 1691 A mutation.

66. The method according to Claim 62, wherein the disorder is a form of cancer.

67. The method according to Claim 66, wherein the cancer is selected from the group consisting of pancreatic, colon and lung cancers.

68. The method according to Claim 62, wherein the genetic disorder is characterized by a mutation in the human K-ras gene.

69. The method according to Claim 62, wherein the nucleic acid molecule comprises the human K-ras gene

70. The method according to Claim 69, wherein the nucleic acid molecule a fragment of the human K-ras gene, wherein the fragment comprises a sequence of DNA known to contain a mutation correlated with the development of cancer.

71. The method according to Claim 69, wherein the nucleic acid molecule comprises codon 12 of the human K-ras gene.

72. The method according to Claim 62 wherein the substitution at the 2'- ribose position is an amine substitution.

73. The method according to Claim 62, wherein the reactive compound is succinimidyl ester.

74. The method according to Claim 62, wherein the reactive compound is a fluorogen.

75. A method of determining the concentration of nucleic acid in a sample, comprising: hybridizing a known concentration of an oligonucleotide to nucleic acid molecules present in an unknown concentration in a sample to produce a population of oligonucleotides hybridized to the nucleic acid molecule, wherein the oligonucleotide has a sequence complementary to a sequence present in the nucleic acid molecules of the sample, and wherein the oligonucleotide comprises at least one nucleotide having a substitution at the 2'-ribose position; contacting the sample with a reactive compound comprising at least one reporter moiety under conditions that will allow the reporter moiety to be detected in the sample ifthe reactive compound reacts with the unhybridized oligonucleotides in the sample; detecting the presence or absence of the reporter moiety in the sample, wherein the absence of the reporter moiety indicates that the concentration of the oligonucleotide is lower than the concentration of nucleic acid molecules in the sample; wherein the presence the reporter moiety indicates that the concentration of the oligonucleotide is higher than the concentration of nucleic acid molecules in the sample; and then repeating the hybridization, contacting and detecting steps with a different concentration of oligonucleotide until the detecting step indicates that the concentration of the oligonucleotide is approximately equal to the concentration of the nucleic acid molecules in the sample.

76. The method according to Claim 75 wherein the substitution at the 2'- ribose position is an amine substitution.

77. The method according to Claim 75, wherein the reporter moiety is biotin.

78. The method according to Claim 75, wherein the reactive compound is an activated ester.

79. The method according to Claim 78, wherein the activated ester is succinimidyl ester.

80. The method according to Claim 75, wherein the reactive compound is a fluorogen.

81. The method according to Claim 80, wherein the fluorogen is selected from the group consisting of O-phthaldialdehyde (OPA), 3-(4-carboxylbenzoyl)- quinoline-2-carboxaldehyde (CBQCA) and fluorescamine.

82. The method according to Claim 75, wherein the nucleic acid molecules comprise DNA.

83. The method according to Claim 75, wherein the nucleic acid molecules comprise

RNA.

84. The method according to Claim 75, wherein the oligonucleotide comprises DNA.

85. The method according to Claim 75, wherein the oligonucleotide comprises RNA.

86. The method according to Claim 75, wherein the nucleic acid molecules comprise

RNA and the oligonucleotide comprises DNA.

87. The method according to Claim 75, wherein the nucleic acid molecules comprise

DNA and the oligonucleotide comprises RNA.

88. The method according to Claim 75, wherein both the nucleic acid molecules and the oligonucleotide comprise DNA.

89. The method according to Claim 75, wherein both the nucleic acid molecules and the oligonucleotide comprise RNA.

90. A method of determining the amount of cellular RNA in a sample, comprising: hybridizing a known concentration of an oligonucleotide to cellular RNA present in an unknown concentration in a sample to produce a population of oligonucleotides hybridized to the cellular RNA, wherein the oligonucleotide has a sequence complementary to a sequence present in the cellular RNA of the sample, and wherein the oligonucleotide comprises at least one nucleotide having a substitution at the 2'-ribose position; contacting the sample with a reactive compound comprising at least one reporter moiety under conditions that will allow the reporter moiety to be detected in the sample ifthe reactive compound reacts the with the unhybridized oligonucleotides in the sample; detecting the presence or absence of the reporter moiety in the sample, wherein the absence of the reporter moiety indicates that the concentration of the oligonucleotide is lower than the concentration of cellular

RNA in the sample; wherein the presence of an excess of the reporter moiety indicates that the concentration of the oligonucleotide is higher than the concentration of cellular RNA in the sample; and then repeating the hybridization, contacting and detecting steps with a different concentration of oligonucleotide until the detecting step indicates that the concentration of the oligonucleotide is approximately equal to the concentration of the cellular RNA in the sample.

91. The method according to Claim 90 wherein the substitution at the 2'- ribose position is an amine substitution.

92. The method according to Claim 90, wherein the reporter moiety is biotin.

93. The method according to Claim 90, wherein the reactive compound is an activated ester.

94. The method according to Claim 90, wherein the activated ester is succinimidyl ester.

95. The method according to Claim 90, wherein the reactive compound is a fluorogen.

96. The method according to Claim 95, wherein the fluorogen is selected from the group consisting of O-phthaldialdehyde (OPA), 3-(4-carboxylbenzoyl)- quinoline-2-carboxaldehyde (CBQCA) and fluorescamine.

97. The method according to Claim 90, wherein the oligonucleotide comprises DNA.

98. The method according to Claim 90, wherein the oligonucleotide comprises RNA.

99. A method of mapping the local conformation in a RNA molecule, comprising: generating a population of RNA pools, wherein each RNA molecule in an individual pool comprises a substitution at the 2'-ribose position of one nucleotide of the RNA being mapped, such that each RNA pool in the population of RNA pools represents one nucleotide of the RNA molecule having a substitution at the 2'-ribose position of that nucleotide; contacting each RNA pool with a reactive compound comprising at least one reporter moiety under conditions that will allow the reporter moiety to be detected in the pool ifthe reactive compound reacts with the 2'- substituted nucleotide; detecting the presence or absence of the reporter moiety in each RNA pool, wherein the absence of the reporter moiety indicates that the nucleotide represented by the RNA pool is stably base-paired or involved in a tertiary RNA structure, and the presence of the reporter moiety indicates that the nucleotide represented by the RNA pool is not stably base-paired or involved in a tertiary RNA structure; and then mapping each nucleotide of the RNA molecule by indicating its involvement in base-pairing or tertiary structure, or lack thereof, thereby producing a map of the RNA molecule indicative of the local conformation of the RNA molecule.

100. The method according to Claim 99 wherein the substitution at the 2'- ribose position is an amine substitution.

101. The method according to Claim 99, wherein the reporter moiety is biotin.

102. The method according to Claim 99, wherein the reactive compound is an activated ester.

103. The method according to Claim 102, wherein the activated ester is succinimidyl ester.

104. The method according to Claim 99, wherein the reactive compound is a fluorogen.

105. The method according to Claim 104, wherein the fluorogen is selected from the group consisting of O-phthaldialdehyde (OPA), 3-(4-carboxylbenzoyl)- quinoline-2-carboxaldehyde (CBQCA) and fluorescamine.

106. A method of detecting the binding of a ligand to an aptamer, comprising contacting a first sample of an aptamer with a reactive compound comprising a reporter moiety, wherein the aptamer comprises at least one nucleotide having a substitution at the 2'-ribose position; detecting the binding of the reporter moiety to the aptamer in the first sample to determine ifthe aptamer binds the reporter moiety in the absence of a ligand; contacting a second sample of the aptamer with a ligand, wherein the aptamer comprises at least one nucleotide having a substitution at the 2'-ribose position; contacting the second sample of the aptamer with a reactive compound comprising a reporter moiety; detecting the binding of the reporter moiety to the aptamer in the second sample to determine ifthe aptamer binds the reporter moiety in the presence of the ligand; and then comparing the binding of the reporter moiety to the aptamer in the absence of a ligand with the binding of the reporter moiety to the aptamer in the presence of the ligand, wherein a difference in the binding of the reporter moiety between the aptamer in the first sample and the aptamer in the second sample indicates that the aptamer binds the ligand.

107. The method according to Claim 106 wherein the substitution at the 2'- ribose position is an amine substitution.

108. The method according to Claim 106, wherein the reporter moiety is biotin.

109. The method according to Claim 106, wherein the reactive compound is an activated ester.

110. The method according to Claim 109, wherein the activated ester is succinimidyl ester.

111. The method according to Claim 106, wherein the reactive compound is a fluorogen.

112. The method according to Claim 111, wherein the fluorogen is selected from the group consisting of O-phthaldialdehyde (OPA), 3-(4-carboxylbenzoyl)- quinoline-2-carboxaldehyde (CBQCA) and fluorescamine.

113. A kit for detecting a mutation in a nucleic acid molecule, comprising at least one container comprising: at least one oligonucleotide complementary to the nucleic acid molecule, wherein the oligonucleotide comprises a nucleotide with a substitution at the 2'-ribose position; at least one reactive compound that can modify the oligonucleotide at the substituted 2'-ribose position, wherein the reactive compound comprises a reporter moiety; and printed instructions for determining whether or not a mutation is present in the nucleic acid molecule.

114. The kit according to Claim 113, wherein the reactive compound is selected from the group consisting of activating esters and fluorogens.

115. The kit according to Claim 114, wherein the reactive compound is a succinimidyl ester.

116. The kit according to Claim 114, wherein the reactive compound is a fluorogen selected from the group consisting of O-phthaldialdehyde (OPA), 3-(4- carboxylbenzoyl)-quinoline-2-carboxaldehyde (CBQCA) and fluorescamine.

117. The kit according to Claim 113, wherein the substitution at the 2'- ribose position is an amine substitution.

118. The kit according to Claim 113, wherein the reporter moiety is biotin.

119. The kit according to Claim 113, wherein the oligonucleotide comprises DNA.

120. The kit according to Claim 113, wherein the oligonucleotide comprises RNA.