WO2011097437A1

WO2011097437A1 - Double displacement probes for nucleic acid detection

Info

Publication number: WO2011097437A1
Application number: PCT/US2011/023686
Authority: WO
Inventors: Eric Todd Kool; Daniel J. Kleinbaum
Original assignee: The Board Of Trustees Of The Leland Stanford Junior University
Priority date: 2010-02-05
Filing date: 2011-02-04
Publication date: 2011-08-11

Abstract

A branched linker structure is provided, including two quenchers, two activating leaving groups adjacent to electrophilic centers on branched tethers, for attachment to the terminal of any polynucleotide probe. The two electrophilic centers of the branched linker can react in a templated reaction with a second modified polynucleotide probe carrying a nucleophilic group. A templated reaction occurs when the two probes are hybridized near one another on a target nucleic acid sequence, resulting in release of the two quenchers, and unmasking of an adjacent fluorophore. Double displacement of the quenchers is required before a fluorescent signal is generated. Probes including double displacement linkers give low background fluorescence and enhanced signal to background ratios as demonstrated in in vitro and in vivo experiments. The branched linkers of the present invention are simple and inexpensive to prepare, and can be appended to any polynucleotide in automated steps on a standard DNA synthesizer.

Description

DOUBLE DISPLACEMENT PROBES FOR NUCLEIC ACID DETECTION

BACKGROUND OF THE INVENTION

[01 ] This invention was made with Government support under Grant No: 1065480-3-

PAHCW awarded by the National Institutes of Health. The Government has certain rights in this invention.

[02] The detection of specific nucleic acid sequences is immensely useful in molecular medicine. The possibilities for useful detection and quantitation of specific genes and gene products are nearly endless. Genotyping methods are of interest for prenatal diagnosis; as well as detecting changes in genotype associated with disease, for example during oncogenesis. Genotyping methods also find use in pharmacogenomics, to determine an individual's profile for drug metabolism, including the likelihood of adverse reactions and responsiveness to treatment. Other important areas of research include analysis of mRNA for expression, alternative splicing and SNP variation. In addition to analysis of expression, and of sequence polymorphisms, there is significant interest in simply determining whether a target sequence is present in a sample, for example in the detection and identification of microbial species in clinical and environmental samples.

[03] Fast, simple and accurate methods of detecting and analyzing the presence or absence of nucleic acids, which may differ by as little as one nucleotide from others, are of great interest. In some cases, the nucleic acids may be present in minute quantities or concentrations, which underscore the need for high sensitivity as well. Many methods of detecting the presence of nucleic acid sequences are known in the art, including Northern and Southern blots, microarray hybridization, and the like. These methods have typically relied on hybridization kinetics between the target and probe species, coupled with varying temperature and ionicity to provide specificity. However, there are some significant drawbacks to these methods in terms of specificity and sensitivity.

[04] A number of laboratories have investigated the use of nonenzymatic fluorescence based approaches for RNA or DNA detection, relying on the formation of bonds, hybridization of fluorescent oligonucleotides, or changes in secondary structure to detect genetic sequences. For example, see Xu and Kool (1997) Tetrahedron Lett. 38:5595-5598; Paris et al. (1998) N.A.R. 26:3789-3793; Okamoto et al. (2003) JACS 125:9296-9297; Tyagi and Kramer (1996) Nat. Biotech. 14:303-308; Kuhn et al. (2001 ) Antisense Nucleic Acid Drug Dev. 1 1 :265-270; and International Patent application WO 2004/010101 . Also of interest are U.S. Patents no. 5,571 ,903 (Gryaznov); and 4,958,013 (Letsinger).

[05] Self-ligation reactions have been developed for sequence detection, where the chemistry for joining two short oligonucleotide probes is incorporated into the ends of the probe molecules themselves. Such self-ligation reactions can be highly selective for single nucleotide differences in the target molecule. See, for example, Xu and Kool (2000) JACS 122:9040-9041 ; Xu et al. (2001 ) Nat. Biotech. 19:148-152; Ficht et al. (2004) JACS 126:9970-9981 ; and Gryaznov and Letsinger (1993) JACS 1 15:3808-3809. In a recent advance, the chemistry for ligation was activated by a group that acted both as leaving group and as fluorescence quencher, thus enabling the probes to become fluorescent in the presence of a complementary target (Sando and Kool (2002) J. Am. Chem. Soc. 124 (10): 2096-2097).

[06] Previous designs of SN2 ligation probes required only one displacement for unquenching (Abe and Kool, J. Am. Chem. Soc. 2004, 126, 13980); they have been used to discriminate between different strains of bacteria based on small differences in ribosomal RNA sequences(Silverman et al. Chembiochem. 2006, 7, 1890; Silverman and Kool, Nucleic Acids Res 2005, 33, 4978; Sando and Kool, J. Am. Chem. Soc. 2002, 124, 9686; G. P. Miller, A. P. Silverman, E. T. Kool, Bioorg. Med. Chem. 2008, 16, 56] and to detect high- copy-number mRNAs in mammalian cells [H. Abe, E. T. Kool, Proc. Natl. Acad. Sci. U S A 2006, 103, 263]. Background signals from these probes arise largely from non-templated displacement of the quencher by cellular nucleophiles such as water and thiols.

[07] To address this issue, double displacement (DD) linkers are described herein which slow the rate at which background signal is produced by having two fluorescence quenchers as leaving groups on the probe. The displacement of one of these quenchers should not produce a large increase in fluorescence, increasing the amount of time before the non-specific fluorescence dominates the amount of signal produced. Structures of suitable probes which are useful in detecting target sequences present at low concentrations or in low numbers are described herein, that provide for low background fluorescence both in vitro and in bacterial cells and increased signal to background ratios. Other publications

[08] Strategies for using pairs of modified oligonucleotides to generate amplified products or signals have been described. Ma and Taylor, Proc. Natl. Acad. Sci. U S A 2000, 97, 1 1 159-1 1 163; Ma and Taylor, Bioorg. Med. Chem. 2001 , 9, 2501 -2510; and Brunner et al., J. Am. Chem. Soc. 2003, 125, 12410-1241 1 have described the combination of a hydrolysis catalyst on one oligonucleotide with a leaving group (in the form of an ester) on the other, resulting in the release of multiple leaving groups for each targeted complementary strand of DNA. Those approaches have generated ca 3-35 turnovers. The former has reported the generation of fluorescence signals, albeit without the demonstration of turnover (Ma and Taylor, Bioconjugate Chem. 2003, 14, 679-683). None of these approaches rely on ligation or nucleophilic displacement. Ligations of amino-conjugated oligonucleotides have been investigated by Luther et al., Nature 1998, 396, 245-248 and by Zhan and Lynn, J. Am. Chem. Soc. 1997, 1 19, 12420-12421 . The former approach requires denaturation cycles for turnover. The latter strategy isothermally generates as much as >50 turnovers in ligation, but it requires a separate reagent (borohydride), and it is not clear how the approach could generate easily detectable signals, such as those provided by the present invention. The approach described herein requires neither denaturation steps nor a separate reagent.

Ficht et al., supra, developed peptide nucleic acid (PNA) probes that ligate by native chemical ligation; such probes have not been demonstrated to undergo turnover, nor do they generate fluorescent signals. RNA-detecting ribozymes are well documented to undergo turnover (Wang and Sen, J. Mol. Biol. 2001 , 310, 723-734; Komatsu et al., Biochemistry 2002, 41, 9090-9098); however, a recent example of a DNAzyme designed to generate fluorescence signals in detection of an RNA documented only 4 signals per target (Sando et al., J. Am. Chem. Soc. 2003, 125, 15720-15721 ). These approaches did not utilize nucleophilic displacement.

SUMMARY OF THE INVENTION

Compositions of modified polynucleotide probes and branched linker reagents for their synthesis are provided. The polynucleotide probes are modified at the 5' terminus by two quenchers, which are both linked to the polynucleotide through activating leaving groups and branched tethers. Each activating leaving group activates an adjacent carbon atom to attack by a nucleophilic group, leading to release of the quencher. A modified polynucleotide that includes a fluorophore provides for a detectable change in fluorescence following the release of both quenchers from the modified polynucleotide. Branched linker reagents for synthesis of the modified polynucleotides are simple and inexpensive to prepare, and they can be appended to any polynucleotide in automated steps on a standard DNA synthesizer.

In some methods of interest, the modified polynucleotide is reacted with a second polynucleotide, where the sequences of the first and second polynucleotide usually hybridize to neighboring sites of a target sequence. The second polynucleotide usually comprises a nucleophile attached to the 3' hydroxyl, which can attack the electrophilic atom adjacent to the first activating leaving group in a ligation (nucleophilic displacement) reaction, thereby displacing the first quencher. Further nucleophilic reaction can occur at the electrophilic atom adjacent to the second activating leaving group, thereby displacing the second quencher.

In a ligation reaction between a pair of probes, the nucleophile displaces a first quencher on an adjacent tether, thereby linking the pair of probes through an electrophilic center of the tether. Further reaction with the nucleophile then occurs at a second electrophilic center displacing a second quencher. In some embodiments, one of the quenchers may be displaced by hydrolysis. The branched linker is optionally of a composition that destabilizes hybridization between the ligation product and its complementary target sequence without destabilizing the transition state for ligation. Under appropriate conditions, known to those of ordinary skill in the art, the ligation product can be released from the target sequence if desired, and therefore the target sequence can be made available for additional rounds of probe binding and ligation.

[14] The probes are useful in a variety of nucleophilic reactions triggered by specific hybridization to a target nucleic acid. Depending on the nature of the nucleophilic groups, leaving groups and adjacent electrophilic centers, reactions triggered by hybridization may result in release fluorescence quenchers; release of fluorescent tags; release of biologically active molecules, chemical ligation; and the like. The compositions and methods can be used in both in vitro and in vivo applications.

[15] In one embodiment of the invention, a branched universal linker reagent is provided, which can be reacted with any polynucleotide sequence to provide a modified polynucleotide of the invention, conveniently during synthesis. Such linker reagents may be provided with two or more suitable detectable moieties, or may be provided as a linker reagent to which the detectable moieties are added during a subsequent synthesis reaction. The linker can be added to any polynucleotide using conventional solid state oligonucleotide synthesis reactions, for example in an automated synthesizer using phosphoroamidite or H- phosphonate chemistry.

[16] In a related embodiment, a modified polynucleotide is provided, where the polynucleotide comprises two 5' attached quenchers, linked via a branched linker containing electrophiles, activating leaving groups and tethers. In addition to the quenchers, the polynucleotide and/or the nucleophilic polynucleotide may further comprise a fluorescent tag. The modified polynucleotides may be provided as a single species, in pairs as described above, or as a plurality of species and/or pairs. The modified polynucleotides or branched universal linker may be provided in solution; in a purified form, e.g. lyophilized; bound to a solid support, such as beads, arrays, and the like.

[17] In another embodiment, methods are provided for the specific detection of polynucleotides, including mRNA, genomic DNA, extrachromosomal DNA, rRNA, viral RNA, etc., in a variety of platforms. Samples suitable for analysis include isolated polynucleotides; cell lysates; whole cells and tissues, which may be live or fixed; and whole organisms. Kits for practice of the methods are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[18] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.

[19] Figure 1 . Structures of three branched dabsyl linkers, based on isobutyl, 1 ,3-pentyl, and 1 ,5-pentyl linkers.

[20] Figure 2. Initial Quenching Efficiency of Single vs. Double Displacement Probes.

Initial emission spectra of different quencher probes (butyl linker or 1 ,5-pentyl DD linker) are shown.

[21 ] Figure 3. Initial quenching of fluorescence in modified polynucleotides containing two quenchers attach via branched linkers (isobutyl, 1 ,3-pentyl and 1 ,5-pentyl). Comparison to a modified polynucleotide including a single quencher.

[22] Figure 4. Fluorescence time course of templated ligation reactions at 25^. Different quencher probes (butyl linker or 1 ,5-pentyl DD linker) and nucleophile probe were incubated with or without template. The fluorescence was measured every 120 s (494 nm excitation and 522 nm emission).

[23] Figure 5. Fluorescence time course of templated ligation reactions at 37 ^<C. Different quencher probes (butyl linker or 1 ,5-pentyl DD linker) and nucleophile probe were incubated with or without template. The fluorescence was measured every 120 s (494 nm excitation and 522 nm emission).

[24] Figure 6. Signal to background ratio for probes in vitro. Fluorescent signals observed over time for various probes (containing two dabsyl quenchers attached via isobutyl, 1 ,3-pentyl or 1 ,5-pentyl linkers) incubated with a target sequence; either with a nucleophile probe (shown with solid lines); or without a nucleophile probe (shown with broken lines). Comparison to a modified polynucleotide including a single quencher.

[25] Figure 7. Fluorescence endpoint of templated ligation reactions. Different quencher probes (butyl linker or 1 ,5-pentyl DD linker) and a phosphorodithioate probe were incubated with or without template. The fluorescence emission spectra of each sample is shown (494 nm excitation).

[26] Figure 8. Degradation of 3' terminal phosphorodithioate DNAs over time. The arrow indicates the peak corresponding to the intact phosphorodithioate DNA.

[27] Figure 9. Equivalents of displaced dabsylate over time. The amount of free dabsylate in solution was monitored by HPLC from reactions of 1 ,5-pentyl DD probe, template strand and phosphorodithioate probe (Blue) or phosphoromonothioate probe (Green). The 1 ,5-pentyl DD probe and template with no nucleophile probe present is also shown (Red). [28] Figure 10. Detection of 16S rRNA in intact Escherichia Coli, showing lower background signal. The top two panels show the 1 ,5-pentyl Double Displacement probe with (A) and without (B) phosphorodithioate probe. The bottom two panels show the single displacement (butyl linker) probe with (C) and without (D) phosphorodithioate probe.

[29] Figure 11. In vivo results for probes. Fluorescent signals observed over time for various probes (containing two dabsyl quenchers attached via isobutyl, 1 ,3-pentyl or 1 ,5- pentyl linkers) incubated with a target sequence; either with a phosphorothioate nucleophile probe (top images); or without a nucleophile probe (bottom images). Comparison to a modified polynucleotide including a single dabsyl quencher.

DETAILED DESCRIPTION OF THE INVENTION

[30] Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.

[31 ] In this specification and the appended claims, the singular forms "a," "an" and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

[32] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[33] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

[34] All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing those components that are described in the publications that might be used in connection with the presently described invention. [35] As used herein, compounds which are "commercially available" may be obtained from standard commercial sources including Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall U.K.), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA); Molecular Probes (Eugene, OR); Applied Biosystems, Inc. (Foster City, CA); and Glen Research (Sterling, VA).

[36] As used herein, "suitable conditions" for carrying out a synthetic step are explicitly provided herein or may be discerned by reference to publications directed to methods used in synthetic organic chemistry. The reference books and treatise set forth above that detail the synthesis of reactants useful in the preparation of compounds of the present invention, will also provide suitable conditions for carrying out a synthetic step according to the present invention.

[37] As used herein, "methods known to one of ordinary skill in the art" may be identified though various reference books and databases. Suitable reference books and treatise that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et at., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, "Heterocyclic Chemistry", 2nd Ed., John Wiley & Sons, New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure", 4th Ed., Wiley-lnterscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C., may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.

[38] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[39] "Optional" or "optionally" means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, "optionally substituted aryl" means that the aryl radical may or may not be substituted and that the description includes both substituted aryl radicals and aryl radicals having no substitution. The term lower alkyl will be used herein as known in the art to refer to an alkyl, straight, branched or cyclic, of from about 1 to 6 carbons.

[40] The compounds of the invention may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids. The present invention is meant to include all such possible isomers, as well as, their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

COMPOSITIONS

[41] Compositions are provided for the preparation of modified polynucleotide probes.

The polynucleotides are modified by reaction with a branched universal linker reagent of the invention. The modified polynucleotide comprises two or more detectable moieties linked through activating leaving groups and tethers, usually to the 5' or 3' terminus. Conveniently, the branched universal linker is used to introduce the modification to the polynucleotide during in vitro non-enzymatic oligonucleotide synthesis, e.g. synthesis via phosphoroamidite or H-phosphonate chemistry; etc., although for some purposes the modification may be introduced post-synthetically or during enzymatic synthesis, such as PCR, etc. [42] Modified polynucleotides for ligation reactions have the following general structure

where Q and Q² are detectable moieties, such as quenchers;

L and L² are activated leaving groups;

T , T² and T³ are tethers;

Z is carbon or nitrogen;

A is a polynucleotide; and

R is OR¹ , SR , O^", S^", or a lower alkyl, which may be linear or branched; and R is methyl or other lower alkyl, straight or branched; or β-cyanoethyl.

[43] In certain embodiments, a modified polynucleotide of the invention has the structure:

where Q and Q² are quenchers;

L and L² are activated leaving groups;

T , T² and T³ are tethers;

A is a polynucleotide; and

R is OR¹ , SR¹ , O^" or S^", wherein R is a linear or branched lower alkyl.

[44] In certain embodiments, in structure II, the activated leaving group L and the quencher Q may be part of the same group, i.e., the activated leaving group may be included in the structure of a quencher group. For example, when a dabsyl quencher is used, the activated leaving group is a sulfonate group that is part of the dabsyl group structure. Similarly, the activated leaving group L² and the quencher Q² may be part of the same group. The combined L -Q¹ group or L²-Q² group may also be referred to as quenchers.

[45] In certain embodiments, in structure II, A is a polynucleotide comprising at least one fluorophore quenched by Q and Q².

[46] In certain embodiments, in structure II, A is a polynucleotide comprising at least one fluorophore quenched by Q and Q² where the fluorophore is at least one of fluorescein, 5- carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2',4',1 ,4,-tetrachlorofluorescein (TET), 2',4', 5',7',1 ,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy-4',5'-dichloro-6- carboxyfluorescein (JOE); Cy3, CY5, Cy5.5, a dansyl derivative; 6- carboxytetramethylrhodamine (TAMRA), a BODIPY fluorophore, tetrapropano-6- carboxyrhodamine (ROX), ALEXA dye, and Oregon Green.

[47] In certain embodiments, in structure II, A is a polynucleotide of at least 6 bases in length and not more than about 100 bases in length.

[48] In certain embodiments, in structure II, L and L² are independently selected from a sulfonate, a carbonyl ester, a para nitrophenyl ester, a nitrophenyl ester, a trifluoroacetyl ester, a nosylate, a brosylate, a tosylate. In particular embodiments, in structure III, L and L² are sulfonate.

[49] In certain embodiments, the activated leaving group L or L² may be part of the quencher Q or Q², respectively, such that L -Q¹ and/or L²-Q² are combined in a single group. For example, when a dabsylate quencher is used, the quencher incorporates the leaving group (i.e. a sulfonate group) as part of the quencher. In certain embodiments, in structure II, Q and Q² are independently selected from DABSYL (dimethylamino- azobenzene-sulfonyl), DANSYL (5-dimethylaminonaphthalenesulfonyl), DIMAPDABSYL ((p-dimethylamino- phenylazo)-azobenzenesulfonyl); an azobenzene-sulfonyl group, a benzenesulfonyl group, or an arenesulfonyl optionally comprising a substituent selected from amino, dialkylamino, nitro, fluoro, cyano; anthraquinone, nitrothiazole, nitroimidazole, rhodamine, tetramethyl-6-carboxyrhodamine (TAMRA), ROX, cyanine, coumarin, BODIPY, fluorescein, and ALEXA dyes. In particular embodiments, in structure II, one or both of Q and Q² are dabsyl.

[50] In certain embodiments, in structure II, L and Q together comprise a group selected from dabsylate (dimethylamino-azobenzene-sulfonate), dansylate (5- dimethylaminonaphthalenesulfonate), dimapdabsylate ((p-dimethylamino- phenylazo)- azobenzenesulfonate); an azobenzene-sulfonyloxy group, a benzenesulfonyloxy group, or an arenesulfonyloxy optionally comprising a substituent selected from amino, dialkylamino, nitro, fluoro, cyano; anthraquinone, nitrothiazole, nitroimidazole, rhodamine, tetramethyl-6- carboxyrhodamine (TAMRA), ROX, cyanine, coumarin, BODIPY, fluorescein, and ALEXA dyes. [51 ] In certain embodiments, in structure II, L² and Q² together comprise a group selected from dabsylate (dimethylamino-azobenzene-sulfonate), dansylate (5- dimethylaminonaphthalenesulfonate), dimapdabsylate ((p-dimethylamino- phenylazo)- azobenzenesulfonate); an azobenzene-sulfonyloxy group, a benzenesulfonyloxy group, or an arenesulfonyloxy optionally comprising a substituent selected from amino, dialkylamino, nitro, fluoro, cyano; anthraquinone, nitrothiazole, nitroimidazole, rhodamine, tetramethyl-6- carboxyrhodamine (TAMRA), ROX, cyanine, coumarin, BODIPY, fluorescein, and ALEXA dyes.

[52] In certain embodiments, in structure II, T , T² and T³ are independently a single bond or a chain of from about 1 to about 20 methylene groups in length, where the methylene backbone is optionally substituted with between one to seven sulfur, nitrogen and/or oxygen heteroatoms; optionally comprising one, two, or three unsaturated bonds in the tether backbone, wherein each of the backbone atoms may be substituted or unsubstituted.

[53] In certain embodiments, in structure III, T , T² and T³ are independently a single bond, an oligo(ethylene glycol), an ether, a thioether, a tertiary amino, a straight, branched or cyclic alkyl or alkenyl group; optionally substituted with an alkyl, an aryl, an alkenyl, an aralkyl, an aralkenyl, a cycloalkyl, a cycloalkylalkyl or a cycloalkylalkenyl group.

[54] In certain embodiments, in structure II, T , T² and T³ are independently absent or present, and when present independently selected from the group consisting of:

wherein n is from 1 to 20; and and n₂ are independently selected to be from 1 to 20; Πι + n₂ are usually not more than about 20; and y is from 1 to 7;

the alkyl or alkenyl is optionally substituted with an alkyl, an aryl, an alkenyl, an aralkyl, an aralkenyl, a cycloalkyl, a cycloalkylalkyl or a cycloalkylalkenyl group;

R³ is selected from an alkyl, usually branched or linear lower alkyl; hydroxy, alkoxy, aryloxy, haloalkoxy, cyano, nitro, mercapto, alkylthio, -S(0)_pR⁶ (where p is 0 to 2), -S(0)_pN(R⁶)₂ (where p is 0 to 2); -OR⁶, -C(0)OR⁶, -C(0)N(R⁶)₂, -N(R⁶)₂, -N(R⁶)C(0)OR⁷, -N(R⁸)C(0)R⁸, and -R⁸-N=N-0-R⁷; where each R⁶ , R⁷ or R⁸ is independently selected from the group consisting of hydrogen, an alkyl, an alkenyl, a haloalkyl, a haloalkenyl, an aryl, an aralkyl, an aralkenyl, a cycloalkyl, a cycloalkylalkyl and a cycloalkylalkenyl. [55] In certain embodiments, in structure II, T , T² and T³ are independently selected from a single bond, -CH₂- and -(CH₂)₂-.

[56] In certain embodiments, in structure II, L and Q together are dabsylate; L² and Q² together are dabsylate; and T , T² and T³ are independently a single bond, -CH₂- or -

(CH₂)₂-.

[57] In certain embodiments, in structure II, A is a polynucleotide that is attached to a support.

[58] Nucleophilic probes of the invention may include a polynucleotide modified with a nucleophilic group, e.g., modified at the 3' or 5' terminus with a phosphorothioate, a phosphorodithioate, a phosphorotrithioate, a phosphoselenoate, a thiophenol, a thioaryl group, or other known nucleophilic groups. In certain embodiments, a nucleophilic probe of the invention has the structure:

where X¹ , X² and X³ are independently selected from oxygen and sulfur, where usually at least one of X¹ , X² and X³ is sulfur; and Y is a polynucleotide.

In certain embodiments, a composition of the invention comprises a modified polynucleotide of structure I, and a second modified polynucleotide comprising a

nucleophilic group. In particular embodiments the second modified polynucleotide has the structure III, as described above.

[59] In certain embodiments, a composition of the invention comprises a modified polynucleotide of structure II, and a second modified polynucleotide of structure III;

where in structure II:

L and Q together comprise a group selected from dabsylate (dimethylamino- azobenzene-sulfonate), dansylate (5-dimethylaminonaphthalenesulfonate), dimapdabsylate ((p-dimethylamino- phenylazo)-azobenzenesulfonate); an azobenzene-sulfonyloxy group, a benzenesulfonyloxy group, or an arenesulfonyloxy optionally comprising a substituent selected from amino, dialkylamino, nitro, fluoro, cyano; anthraquinone, nitrothiazole, nitroimidazole, rhodamine, tetramethyl-6-carboxyrhodamine (TAMRA), ROX, cyanine, coumarin, BODIPY, fluorescein, and ALEXA dyes;

L² and Q² together comprise a group selected from dabsylate (dimethylamino- azobenzene-sulfonate), dansylate (5-dimethylaminonaphthalenesulfonate), dimapdabsylate ((p-dimethylamino- phenylazo)-azobenzenesulfonate); an azobenzene-sulfonyloxy group, a benzenesulfonyloxy group, or an arenesulfonyloxy optionally comprising a substituent selected from amino, dialkylamino, nitro, fluoro, cyano; anthraquinone, nitrothiazole, nitroimidazole, rhodamine, tetramethyl-6-carboxyrhodamine (TAMRA), ROX, cyanine, coumarin, BODIPY, fluorescein, and ALEXA dyes;

T , T² and T³ are independently a single bond or a chain of from about 1 to about 20 methylene groups in length, where the methylene backbone is optionally substituted with between one to seven sulfur, nitrogen and/or oxygen heteroatoms; optionally comprising one, two, or three unsaturated bonds in the tether backbone, wherein each of the backbone atoms may be substituted or unsubstituted;

R is OR¹ , SR , O^", S^", or a linear or branched lower alkyl; where R is a linear or branched lower alkyl.

A is a polynucleotide comprising a fluorophore quenched by Q and Q²; and where in structure III:

X¹ , X² and X³ are independently selected from oxygen and sulfur, where usually at least one of X¹ , X² and X³ is sulfur; and

Y is a polynucleotide.

[60] In particular embodiments, a composition of the invention comprises a modified polynucleotide of structure II, and a second modified polynucleotide of structure III, as described above where T , T² and T³ are independently present or absent, and when present independently selected from the group consisting of:

wherein n is from 1 to 20; and η and n₂ are independently selected to be from 1 to 20; Πι + n₂ are usually not more than about 20; and y is from 1 to 7;

R³ is selected from an alkyl, usually branched or linear lower alkyl; a hydroxy, an alkoxy, an aryloxy, a haloalkoxy, a cyano, a nitro, a mercapto, an alkylthio, -S(0)_pR⁶ (where p is 0 to 2), -S(0)_pN(R⁶)₂ (where p is 0 to 2); -OR⁶, -C(0)OR⁶, -C(0)N(R⁶)₂, -N(R⁶)₂, -N(R⁶)C(0)OR⁷, -N(R⁸)C(0)R⁸, and -R⁸-N=N-0-R⁷; where each R⁶ , R⁷ or R⁸ is independently selected from the group consisting of hydrogen, an alkyl, an alkenyl, a haloalkyl, a haloalkenyl, an aryl, an aralkyl, an aralkenyl, a cycloalkyl, a cycloalkylalkyl and a cycloalkylalkenyl.

[61] In particular embodiments, a composition of the invention comprises a modified polynucleotide having structure II, and a second modified polynucleotide having structure III, where L and Q together are dabsylate; L² and Q² together are dabsylate; T , T² and T³ are independently selected from a single bond, -CH₂- and -(CH₂)₂-; R, A and Y are as described above; and the nucleophilic group is a phosphorothioate or a phosphorodithioate.

[62] Branched universal linker reagents for preparing polynucleotides useful in ligation reactions have the following general structure:

IV

where Q and Q² are detectable moieties, such as quenchers; L and L² are activated leaving groups; T , T² and T³ are tethers; Z is carbon or nitrogen; and X is a phosphoramidite or a H-phosphonate.

[63] In some embodiments, a branched linker may include two or more, such as three or more, detectable moieties.

[64] Where it is present, X has the general structure known in the art for such in vitro oligonucleotide synthesis reagents, e.g. H-phosphonate; phosphoroamidite; etc.

Descriptions of such reagents may be found, for example, in U.S. Patent no. 4,458,066;

Caruthers and Matteucci; Garegg et al. (1986) Tet. Lett. 27:4051 -4054; Froehler and

Matteucci (1986) Tet. Lett. 27:469-472; each herein specifically incorporated by reference.

[65] Where X is phosphoroamidite, it will have the structure:

where R is a protecting group as known in the art, usually methyl or other lower alkyl, or β- cyanoethyl; and R² is a substituted primary amine or a secondary amine; including, without limitation, a primary or secondary amine substituted with one or two lower alkyls such as methyl, propyl, isopropyl, butyl, etc., including N((CH(CH₃)₂)₂; N(CH₃)₂ N(CH₂CH₃)₂; tetrazole, imidazoles, cyclic amines, and the like. In one embodiment of the invention, X has the structure

Where X is an H-phosphonyl it will have the structure

[68] The tethers, T , T² and T³ are independently a single bond or a chain of from about 1 to about 20 methylene groups in length, for example from about 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18 or 20, where the methylene backbone is optionally substituted with a sulfur, nitrogen or oxygen heteroatom, which tether may comprise one, two, three, five, seven or more backbone heteroatoms. The bonds between methylenes may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a tether backbone. Each of the backbone atoms may be substituted or unsubstituted, for example with an alkyl, aryl or alkenyl group. T , T² and T³ may include, without limitations, oligo(ethylene glycol); ethers, thioethers, tertiary amines, alkyls, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1 -methylethyl (/so-propyl), n-butyl, n-pentyl, 1 ,1 - dimethylethyl (/-butyl), and the like.

[69] Some specific examples of T , T² and T³ include:

^■(CH₂)_n- (CH₂)_n1-0 (CH

where n is from 1 to 20; and η and n₂ are independently selected to be from 1 to 20; Πι + n₂ are usually not more than about 20; and y is from 1 to 7. The alkyl or alkenyl is optionally substituted, which substituent may include, without limitation, an alkyl, aryl, alkenyl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl or cycloalkylalkenyl group. It will be understood that substitution can occur on any carbon of the alkyl or alkenyl group; R³ is selected from an alkyl, usually branched or linear lower alkyl; hydroxy, alkoxy, aryloxy, haloalkoxy, cyano, nitro, mercapto, alkylthio, -S(0)_pR⁶ (where p is 0 to 2), -S(0)_pN(R⁶)₂ (where p is 0 to 2); -OR⁶, -C(0)OR⁶, -C(0)N(R⁶)₂, -N(R⁶)₂, -N(R⁶)C(0)OR⁷, -N(R⁸)C(0)R⁸, and -R⁸-N=N-0-R⁷; where each R⁶ , R⁷ or R⁸ is independently selected from the group consisting of hydrogen, alkyl, alkenyl, haloalkyl, haloalkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl and cycloalkylalkenyl.

[70] L and L², the activating leaving groups, activate an adjacent atom for attack by a nucleophile. Such groups are known in the art. Specific non-limiting examples include sulfonate, carbonyl esters, para nitrophenyl esters, nitrophenyl esters, trifluoroacetyl esters, halogens, i.e. CI, Br, I, F; nosylate, brosylate, tosylate, perchlorate, triflate, mesylate; and the like. In particular embodiments, leaving groups of interest are sulfonate.

[71 ] The detectable moieties, Q and Q², provide for a detectable or functional change in the modified polynucleotide upon displacement of the leaving group by ligation, and may include, without limitation, fluorophores, quenchers, radioisotopes, a tag, a metal nanoparticle or a phosphorescent group. Certain detectable moieties of interest provide both Q and L or both Q² and L², e.g. DABSYL.

[72] In some embodiments, a probe of the invention may contain one or more detectable moieties attached to the probe via any suitable means known in the art.

[73] In one embodiment of the invention, Q and Q² are independently a quenching group, or quencher, where "quenching group" refers to any fluorescence-modifying group that can alter at least partly the light emitted by a fluorescent group. A polynucleotide having quenchers as Q and Q² will frequently also comprise one or more donor fluorophores. A fluorophore-quencher pair comprises two molecules having overlapping spectra, where the fluorophore emission overlaps the acceptor absorption, so that there is energy transfer between the excited fluorophore and the other member of the pair. It is not essential that the excited molecule actually fluoresce, it being sufficient that energy transfer can occur between the two. In certain embodiments, two or more quenchers may be paired with a single fluorophore, leading to more efficient quenching of the fluorescence.

[74] Any fluorescence quencher can be used as Q and/or Q². A quencher can be a

DABSYL (dimethylamino-azobenzene-sulfonyl) group, DANSYL (5- dimethylaminonaphthalenesulfonyl); DIMAPDABSYL ((p-dimethylamino- phenylazo) azobenzenesulfonyl), other azobenzene-sulfonyl groups, benzenesulfonyl groups, or arenesulfonyl groups, any of which may comprise substituents such as amino, dialkylamino, nitro, fluoro, and cyano groups; anthraquinone, nitrothiazole, and nitroimidazole compounds; rhodamine dyes (e.g., tetramethyl-6-carboxyrhodamine (TAMRA); ROX; cyanine; coumarin; BODIPY dyes; fluorescein dyes; ALEXA dyes; and the like. [75] Where the detectable moieties Q and/or Q² are quenchers, the modified polynucleotide may further comprise one or more donor or acceptor fluorophore(s), which is quenched prior to the release of the quencher. Any known method of incorporating a fluorophore into a nucleic acid molecule can be used. It is preferred that a fluorophore be located close to the quenchers, but this is not required. The fluorophore can generally be located at any distance from the quenchers sufficient to permit detection of ligation by monitoring the change in fluorescent properties. For example, the fluorophore can be located 1 , 2, 3 or more, and usually will be not more than about 10, 15 or 20 nucleotides away from the quenchers.

[76] The covalent attachment of dyes to nucleic acids can be achieved by a variety of methods known to those of skill in the art. The covalent attachment of dyes to nucleic acids is reviewed in Davies et al. (2000) Chem. Soc. Rev. 29:97-107, which is incorporated herein by reference in its entirety. Examples include, but are not limited to: incorporation of the dyes during the synthesis of nucleic acids, typically solid phase synthesis, post-synthetic labeling of either synthetic nucleic acids or nucleic acids derived through enzymatic reactions, e.g. the PCR reaction, replacement of a nucleotide with a fluorescence modified nucleotide during in vitro synthesis; enzymatic methods of incorporation of dyes into nucleic acids, e.g. the use of dye conjugated deoxynucleotide triphosphates in primer elongation reactions such as a PCR reaction; and the like.

[77] The efficiency of quenching (i.e. the unquenched fluorescence with the fluorescence quenching groups absent divided by the quenched fluorescence with the fluorescence quenching groups present) may be at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, at least about 100 fold, at least about 200 fold, at least about 300 fold, at least about 400 fold, at least about 500 fold, at least about 600 fold, at least about 700 fold, at least about 800 fold, at least about 900 fold, at least about 1000 fold, at least about 2000 fold, at least about 3000 fold, at least about 4000 fold, or at least about 5000 fold.

[78] The efficiency of quenching of a fluorophore in the presence of two or more quenching groups compared to where only a single quenching group is present may be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%. Probes of the present invention, containing two or more quenchers compared to probes where only a single quencher is present, may have increased signal to background ratios of at least about 1 .1 fold, at least about 1 .2 fold, at least about 1 .3 fold, at least about 1 .4 fold, at least about 1 .5 fold, at least about 1 .6 fold, at least about 1 .7 fold, at least about 1 .8 fold, at least about 1 .9 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, at least about 100 fold, at least about 200 fold, at least about 300 fold, at least about 400 fold, at least about 500 fold, at least about 600 fold, at least about 700 fold, at least about 800 fold, at least about 900 fold, at least about 1000 fold, at least about 2000 fold, at least about 3000 fold, at least about 4000 fold, or at least about 5000 fold, or more.

[79] In some embodiments, probes of the present invention containing two or more quenchers have decreased background signals compared to probes where only a single quencher is present. A decreased background signal may result from an improved efficiency of quenching. A background signal may result from side reactions which give inadvertent release of quencher (e.g., a hydrolysis side reaction). In some embodiments, double displacement probes of the invention have background signals arising from non- templated displacement of quencher by cellular nucleophiles such as water and thiols that are decreased compared to single displacement probes. In some embodiments, double displacement probes of the invention have increased signal to background ratios in templated double displacement reactions with nucleophilic probes, compared to single displacement probes. In some embodiments, double displacement probes of the invention have increased signal to background ratios, compared to single displacement probes, even when the nucleophilic probe contains only a single nucleophile. In this case, the displacement of one of the quenchers occurs via non-templated displacement (e.g., via a hydrolysis side reaction). It is unexpected that such reaction of a double displacement probe of the invention with a nucleophilic probe containing a single nucleophilic group would still give a significant advantage over a single displacement probe.

[80] In another embodiment, the detectable moiety is a fluorescent group, where

"fluorescent group" or "fluorophore" refers to a molecule that, when excited with light having a selected wavelength, emits light of a different wavelength, which may emit light immediately or with a delay after excitation. Fluorophores, include, without limitation, fluorescein dyes, e.g., 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2',4',1 ,4,-tetrachlorofluorescein (TET), 2',4', 5',7',1 ,4-hexachlorofluorescein (HEX), and 2', 7'- dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE); cyanine dyes, e.g. Cy3, CY5, Cy5.5, etc.; dansyl derivatives; 6-carboxytetramethylrhodamine (TAMRA), BODIPY fluorophores, tetrapropano-6-carboxyrhodamine (ROX), ALEXA dyes, Oregon Green, and the like. [81 ] Combinations of fluorophores also find use, e.g. where release of a fluorophore leads to a color change. Various combinations of detectable moieties are of interest in the probes of the invention. A fluorophore may be included in combination with two or more quenchers. For example, an electrophile probe may comprise two or more quenchers as Q and Q² and a covalently attached fluorophore; and a nucleophile probe may comprise a second fluorophore that, when in proximity of the first fluorophore, results in a color change.

[82] In some methods of the invention, a pair or plurality of polynucleotide probes is used.

A first modified polynucleotide probe comprises the detectable moieties, activating groups and tethers; while a second or additional polynucleotide probe comprises one or more nucleophiles, usually a 3' nucleophile, which reacts with the electrophilic probe in a ligation reaction, and may be referred to as a nucleophilic probe. In general, nucleophilic groups of the invention may include, without limitation, phosphorothioate, phosphorodithioate, phosphorotrithioate, and phosphoroselenoate groups, thiol and thiolate groups, hydroxy and oxyanion groups, amines, hydroxylamines, hydrazines, hydrazides, phosphines, thioacids and their conjugate bases, selenols and selenoates. The nucleophile is readily added to an oligonucleotide, for example using a commercially available phosphorylation reagent; and sulfurizing agent can be used to provide a phosphorothioate group. The nucleophilic probe may further comprise fluorophore(s) and/or fluorescence quenchers.

[83] "Probe" refers to a molecule that is capable of binding specifically to a target analyte, e.g., a polynucleotide, a peptide, a protein, an antibody, antigen, or fragment or analog thereof. Probes often include a specific binding moiety and two or more detectable moieties, e.g. a fluorophore, quencher, radioisotope, a tag, a metal nanoparticle, etc. Fluorophores and quenchers are of particular interest.

[84] The term "specific binding moiety" as used herein refers to a member of a specific binding pair, i.e. two molecules where one of the molecules through chemical or physical means specifically binds to the other molecule. The complementary members of a specific binding pair are sometimes referred to as a ligand and receptor, although two complementary polynucleotide sequences (including nucleic acid sequences used as probes and capture agents in DNA hybridization assays) are also specific binding pairs, as are antibody and antigen, peptide-MHC antigen and T cell receptor pairs; etc. The specific binding pairs may include analogs, derivatives and fragments of the original specific binding member. For example, an antibody directed to a protein antigen may also recognize peptide fragments, chemically synthesized peptidomimetics, labeled protein, derivatized protein, etc. so long as an epitope is present.

[85] In some embodiments, the specific binding moiety is a single stranded polynucleotide. In other embodiments, the specific binding moiety is a peptide, a protein an antibody, or a fragment thereof. [86] When the nucleophilic probe and the electrophilic probe are brought into close proximity, e.g. by hybridizing to neighboring sequences on a target polynucleotide, the nucleophilic probe reacts with a first tether on the electrophilic probe to release the first detectable moiety. Further reaction with a second tether on the electrophilic probe releases the second detectable moiety, The term "proximity" refers to the relative positions of the electrophile and nucleophile, and occurs when the two groups are sufficiently close to react. In certain embodiments, a hydrolysis reaction can occur with the second tether to release the second detectable moiety. In certain embodiments, the order of the reactions can be reversed, for example, a hydrolysis reaction can occur with the first tether to release the first detectable moiety, followed by reaction of the nucleophilic probe with the second tether to release the second detectable moiety.

[87] Reference may be made herein to the hybridization, or potential for hybridization, of probe sequences to a target sequence. For example, the polynucleotide portion of a first and second probe may hybridize, respectively, to "neighboring" sites on the target through base complementarity. Two such sites, when aligned on the target polynucleotide sequence, are considered to be "neighboring" if the sites are contiguous on the target; are separated by one, two, three or more bases on the target; or overlap by one, two three or more bases on the target.

[88] Oligonucleotide, or polynucleotide means either DNA, RNA, single-stranded or double-stranded, and derivatives thereof, including, but are not limited to: 2'-position sugar modifications; propynyl additions, for example at the at the 5 position of pyrimidines; 5- position pyrimidine modifications, 7- or 8-position purine modifications, modifications at exocyclic amines, 5-methyl cytosine; 5 bromo-cytosine; alkynyl uridine and cytosine; substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, including peptide nucleic acids (PNA), locked nucleic acids (LNA), etc., methylations, morpholino derivatives; phosphoroamidate derivatives; unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Derivatives can also include 3' and 5' modifications such as capping.

[89] The polynucleotide can be derived from a completely chemical synthesis process, such as a solid phase mediated chemical synthesis, or from a biological origin, such as through isolation from almost any species that can provide DNA or RNA, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of those processes. Modifications to introduce a universal primer of the invention may be performed post-synthetically; or a modified polynucleotide may be used as a primer in a synthetic reaction, e.g. PCR; and the like. [90] As is used in the art, the term "oligonucleotide" usually refers to shorter molecules, usually of at least about 3 bases in length, more usually at least 4, 5, or 6 bases; for many embodiments of the invention, preferred oligonucleotides are at least 7 bases, at least 8 bases, at least 10 bases, at least 12 bases, and not more than about 100 bases in length, usually not more than about 50 bases in length, or any length range between any two of these lengths. The term "polynucleotide" may refer to any length of nucleic acid greater than a single base; although in many instances will be used to refer to molecules as present in living organisms, which range from about 50 bases in length to many megabases, in the case of genomic DNAs. It will be understood by those of skill in the art that the linkers described herein are readily attached to any polynucleotide. For many assays of interest, one probe, which may be the electrophilic probe or the nucleophilic probe, will be of a length that is sensitive to small differences in sequence.

[91 ] Modified oligonucleotides of the invention may be provided in solution, or bound to a substrate. One, a pair or a plurality of modified probes may be provided in any configuration, although on a solid support it will be more usual for one member of a pair to be present on the support, and a second member to be provided in solution.

[92] By "solid substrate" or "solid support" is meant any surface to which the probes of the invention are attached. A variety of solid supports or substrates are suitable for the purposes of the invention, including both flexible and rigid substrates. By flexible is meant that the support is capable of being bent, folded or similarly manipulated without breakage. Examples of flexible solid supports include nylon, nitrocellulose, polypropylene, polyester films, such as polyethylene terephthalate, etc. Rigid supports do not readily bend, and include glass, fused silica, quartz, acrylamide; plastics, e.g. polytetrafluoroethylene, polypropylene, polystyrene, polystyrene and sulfonated polystyrene-divinyl benzene, quaternized product of chloromethylated polystyrene-divinyl benzene, PEG-polystyrene, PEG, polycarbonate, and blends thereof, and the like; metals, e.g. gold, platinum, silver, and the like; etc. The substrates can take a variety of configurations, including planar surfaces, filters, fibers, membranes, beads, particles, dipsticks, sheets, rods, pins, lanterns, etc.

[93] In one embodiment, the substrate comprises a planar surface, and probes are attached to the surface. The probes may be attached in a uniform pattern or in an array in a plurality of probe spots. The density of labeled probes on the substrate will be such that a signal from the label can be detected. As such, the density will vary depending on the identity of the particular label. Where the probes are spotted on the array, the spots can be arranged in any convenient pattern across or over the surface of the support, such as in rows and columns so as to form a grid, in a circular pattern, and the like, where generally the pattern of spots will be present in the form of a grid across the surface of the solid support. The total number of probe spots on the substrate will vary depending on the concentration of binding member-complementing domain conjugates, as well as the number of control spots, calibrating spots and the like, as may be desired. In an alternative method, where the probes are not bound to a solid support, the assays of the invention may utilize such reaction vessels as 96 well plates, etc., as are known in the art.

[94] In one embodiment, the substrate comprises a non-planar surface, and probes are attached to the surface, e.g., when the substrate is a bead. In another embodiment, the substrate is a collection of physically discrete solid substrates, e.g. a collection of beads, individual strands of fiber optic cable, and the like. Each discrete substrate can have probes distributed across the surface or attached in one or more probe spots on the substrate. The collection of physically separable discrete substrates may be arranged in a predetermined pattern or may be separated in a series of physically discrete containers (e.g., wells of a multi-well plate).

[95] The substrates can be prepared using any convenient means. One means of preparing the supports is to synthesize the probes, and then deposit them on the support surface. The probes can be deposited on the support using any convenient methodology, including manual techniques, e.g. by micropipette, ink jet, pins, etc., and automated protocols. The probes may also be covalently attached to the substrate, using methods known in the art. Alternatively, the probes can be synthesized on the substrate using standard techniques known in the art.

The above reaction scheme shows one possible mechanism of ligation and nucleophilic displacement between a polynucleotide including a 3' phosphorothioate as a nucleophile, and a modified polynucleotide including a double displacement (DD) dabsyl branched linker, resulting in displacement of two dabsylate groups. In the above scheme, the second dabsylate group is displaced by a hydrolysis reaction.

[97] The above reaction scheme shows one further possible mechanism of ligation and nucleophilic displacement between a polynucleotide including a 3' phosphorodithioate as a nucleophile, and a modified polynucleotide including a double displacement (DD) dabsyl branched linker, resulting in displacement of two dabsylate groups. The modified polynucleotide corresponds to previously described structure I, where Q and Q² are DABSYL; L and L² (which are provided by the DABSYL groups) are sulfonate groups; T and T² are single bonds; T³ is an alkyl tether of length n = 1 , i.e.,— CH₂— ; and Z is carbon. The reaction proceeds under a variety of conditions (e.g., see examples), and also including physiological conditions, as are found in vivo.

[98] The DD dabsyl branched linker is linked to the 5' end of the probe via (in this example) phosphoramidite chemistry. In some embodiments, after ligation of the nucleophilic probe to the tether and release of the two dabsyl groups, the product contains a flexible tether interrupting the two complementary half-segments that are complementary to neighboring positions on the target. Because of the length and flexibility of the tether, the ligation product is less favorable entropically than a similar product having a direct phosphodiester linkage. If desired, the ligation product of the invention can dissociate, even under isothermal conditions, allowing a new pair of probes to bind, generating multiple signals per target.

SYNTHETIC METHODS

[99] A modified polynucleotide is generated by reacting a branched universal linker with a polynucleotide of a desired length and sequence. Conveniently, the modification is performed in solid phase following synthesis of an oligonucleotide, using readily available methods, reagents and equipment (see, for example, U.S. Patent no. 4,458,066; or a review of the art in "Perspectives in Nucleoside and Nucleic Acid Chemistry"; ISBN: 3-90639-021 - 7, herein incorporated by reference).

[100] Linkers of the invention may be attached at any suitable position of a probe using readily available methods, reagents and equipment, as are known in the art.

[101 ] In a phosphoramidite method, a series of deprotection, coupling, capping, and oxidation steps is repeated until the nucleotide chain of interest is formed. The strand is formed 3' to 5'. The first step requires a column that has a protected form of the terminal (3') monomer chemically bound to its matrix. The nascent oligonucleotide chain will stay attached to this support as each activated monomer is linked to its 3' neighbor. In DNA synthesis, the protected monomers are deoxyribonucleoside 3-phosphoramidites containing dimethoxytrityl (DMT) blocking groups on the 5'-oxygen atoms. These monomers are activated by treatment with a weak acid prior to chain elongation. Deprotection is the first step of each chain elongation cycle. An acid wash removes the DMT blocking group from the terminal monomer. After deprotection, the terminal monomer has a ready-to- react hydroxyl group (OH) at its 5' end. The second step is coupling. Because phosphoramidites react with water, coupling is carried out under anhydrous conditions. In this step, the next activated monomer in the sequence is added to the vessel. The 5'-OH ends of unreacted terminal monomers are blocked via acetylation. In the fourth step, the phosphite triester is oxidized to the more stable phosphotriester linkage. This cycle is repeated for each added monomer.

[102] In an H-phosphonate method, the monomer that is able to be activated is usually a 5'-DMT-base-protected, nucleoside 3'-hydrogen phosphonate. The presence of the H- phosphonate moiety on these monomers renders phosphate protection unnecessary. The same base protecting groups are used in phosphite triester chemistry. There are four steps to the synthesis of an oligonucleotide using H-phosphonate chemistry. In a first step the 5' protecting group is removed by exposure to trichloroacetic acid and in dichloromethane. In coupling, the monomer is activated by a hindered acyl chloride, the resultant anhydride reacts with a free oligonucleotide 5'-OH end, forming an H-phosphonate analog of the internucleotidic linkage. Capping is achieved using the triethylamine (TEA) salt of isopropyl phosphite. After synthesis of the entire sequence is complete, all of the H-phosphonate bonds are simultaneously oxidized to phosphodiester linkages. Through alternate oxidation steps, H-phosphonate oligonucleotides can be converted to phosphorothioates, phosphorotriesters and various other analogs.

[103] Where it is desired, one or more fluorescent group(s) can be added as a modified phosphoramidite or H-phosphonate reagent, usually from between about 1 to 20 nucleotides before the 5' terminus.

[104] When the oligonucleotide has reached the desired length, the final cycle is used to add a branched universal linker, which optionally includes the releasing groups of interest. Linkers may be synthesized as described in the Experimental section. The linker is usually reacted using the same reagents as the oligonucleotide synthesis reactions, as known to those of skill in the art.

[105] The completed modified oligonucleotide is then cleaved from the support and deprotected by treatment, e.g. with concentrated ammonium hydroxide, usually with a milder deprotection treatment using methods known in the art, e.g. potassium carbonate/methanol. A subsequent heat treatment removes the remaining protecting groups. The final product may be purified by chromatography or electrophoresis, including ion exchange, HPLC, PAGE, etc. In some cases, crude oligonucleotides can be precipitated, or passed over a desalting column, and used without further purification.

[106] In an alternative method, the Q and Q² groups are added separately from the rest of the linker via a synthetic reaction. Following or during addition of the linker precursor to the oligonucleotide, the Q and Q² groups are added. For example, a linker precursor containing DMT-protected hydroxyl groups on tethers may be deprotected and the Q and Q² groups added using standard phosphoroamidite chemistry steps. In some cases, this alternative method may be used when the activating leaving groups L and L² are not stable during polynucleotide synthesis, e.g., undergo nucleophilic side-reactions leading to cleavage and loss of quenchers during synthesis. In some embodiments, the activating leaving groups and detectable moieties (e.g., quenchers) of the linker may be installed post polynucleotide synthesis.

DIAGNOSTIC METHODS

[107] The present invention includes methods for the detection or quantification of a nucleic acid target sequence, comprising the steps of: contacting a sample suspected of containing the target sequence with a first modified polynucleotide probe, where the first modified polynucleotide probe is modified with a fluorophore and a branched universal linker containing two or more quenchers, as described herein; and a second, nucleophilic polynucleotide probe, wherein the first and second probes may hybridize to neighboring sequences on a target nucleic acid under conditions permissive for the release of Q and Q² from the first probe; and measuring the change in fluorescence of the sample, where the level of change is proportional to the amount of target sequence present in the sample. In some embodiments, the presence of two or more quenchers provides for improved initial quenching of the fluorophore and increased signal to background ratios. In some embodiments, the presence of the tethers provides for improved reaction speed, and for amplification if desired of the signal, through repeated rounds of ligation.

[108] Where Q and Q² are quenchers, the intensity or wavelength of emission will change, for example by release or transfer of the quencher away from a fluorophore. Where Q and/or Q² are a fluorophore, the level of fluorescence can increase, decrease, or change color.

[109] In certain embodiments, a method of interest is a method of detecting a target nucleic acid sequence in a sample, the method comprising:

contacting the sample suspected of containing the target nucleic acid sequence with a composition including a modified polynucleotide of structure I or II, and a second modified polynucleotide including a nucleophilic group; where the modified polynucleotide of structure I and the second modified polynucleotide hybridize to neighboring sequences on the nucleic acid sequence;

exposing the sample to conditions sufficient to lead to ligation of the modified polynucleotides and release of quenchers Q and Q²; and

measuring the change in fluorescence of the sample, where the level of change is proportional to the amount of target nucleic acid sequence present in the sample,

[ o] In certain embodiments, a method of interest is a method of detecting a target nucleic acid sequence in a sample comprising: (a) contacting the sample with a probe set for each target nucleic acid sequence, the probe set comprising:

(i) a first probe comprising the modified polynucleotide of structure I or II, where A is a polynucleotide comprising a sequence complementary to a first region of the target nucleic acid sequence; and

(ii) a second probe comprising: a nucleophilic moiety; and a second polynucleotide comprising a sequence complementary to a second region of the target nucleic acid sequence, wherein the second region is adjacent to the first region in the target nucleic acid sequence;

(b) exposing the sample to conditions sufficient to lead to ligation of the probes and release of quenchers Q and Q²; and

(c) measuring the change in fluorescence of the sample, where the level of change is proportional to the amount of target nucleic acid sequence present.

[111] The sequence of the probe(s) is selected to be complementary, competitive, mismatched, etc. with respect to a target sequence, as dictated by the specific interests of the method. In some embodiments, the probe sequences are chosen to be sufficiently selective that there is a detectable difference between binding to a perfect match at the target, and to a single nucleotide mismatch at the target. A highly selective probe binds with high preference to the exact complementary sequence on a target strand as compared to a sequence that has one or more mismatched bases. Less selective probes are also of interest for some embodiments, where hybridization is sufficient for detectable reactions to occur in the presence of one, two three or more mismatches, where a mismatch may include substitutions, deletions, additions, etc.

[112] A "target sequence" refers to the particular nucleotide sequence of the target polynucleotide, which may be hybridized by a probe or probes. Exemplary targets include viral polynucleotides, bacterial polynucleotides (such as mRNA, rRNA), and eukaryotic rRNA, mRNA, genomic DNA, etc.

[113] As used herein, a "test sample" is a sample suspected of containing nucleic acids to be analyzed for the presence or amount of an analyte polynucleotide. Nucleic acids of the test sample may be of any biological origin, including any tissue or polynucleotide- containing material obtained from a human. For example, the nucleic acids of the test sample may be from a biological sample that may include one or more of: tissue or organ lavage, sputum, peripheral blood, plasma, serum, bone marrow, biopsy tissue including lymph nodes, respiratory tissue or exudates, gastrointestinal tissue, cervical swab samples, semen or other body fluids, tissues or materials. Biological samples may be treated to disrupt tissue or cell structure, thereby releasing intracellular components into a solution which may contain enzymes, buffers, salts, detergents and the like. Alternative sources of nucleic acids may include water or food samples that are to be tested for the presence of a particular analyte polynucleotide that would indicate the presence of a microorganism.

[114] Applications for such methods include in vitro diagnostics, including clinical diagnostics, research in the fields of molecular biology, high throughput drug screening, veterinary diagnostics, agricultural-genetics testing, environmental testing, food testing, industrial process monitoring, etc. In vitro diagnostics and clinical diagnostics relate to the analysis of nucleic acid samples drawn from the body to detect the existence of a disease or condition, its stage of development and/or severity, and the patient's response to treatment. In high throughput drug screening and development, nucleic acids are used to analyze the response of biological systems upon exposure to libraries of compounds in a high sample number setting to identify drug leads. Veterinary diagnostics and agricultural genetics testing provide a means of quality control for agricultural genetic products and processes. In environmental testing, organisms and their toxins that characterize an environmental medium, e.g. soil, water, air, etc., are analyzed. Food testing includes the qualitative identification and/or quantitation of organisms, e.g. bacteria, fungi, etc., as a means of quality control.

[115] In such assays, a change in fluorescent signal is generated upon the presence of a complementary nucleic acid sequence in the analyte. The fluorescent signal is monitored and quantified with fluorescence detectors, such as fluorescence spectrophotometers, microplate readers, UV lamps, PCR, commercial systems that allow the monitoring of fluorescence in real time reactions, or, in some instances, by the human eye.

[116] In one embodiment, a homogeneous assay is conducted. In this embodiment of the invention, the nucleic acid probes hybridize with a complementary nucleic acid sequence, if present in the target, to release the Q and Q² groups and effect a change in fluorescence. With appropriate target standards and concentration versus signal standard curves the method can easily be used to quantitate the target. In addition to single stranded target nucleic acids, double stranded target nucleic acids can also be detected by the nucleic acid probe following denaturation. Targets that can be specifically detected and/or quantified with this method include, but are not limited to, plasmid DNA, cloning inserts in plasmid DNA, mRNA transcripts, ribosomal RNA, PCR amplicons, restriction fragments, synthetic oligonucleotides, as well as any other nucleic acids and oligonucleotides.

[117] In another embodiment, a plurality of probes is employed in assays to detect or quantify one or more nucleic acid targets, which assays may be performed in solution; in cells; on a solid substrate; etc. At least one nucleophile probe and at least one electrophile probe will be present; the selection of which probe comprises an electrophile and which comprises a nucleophile will be dictated by the specific requirements of the assay. Various formats may be used in such assays. The composition of fluorophores and/or quenchers will be selected to provide the desired information, including the use of multiple fluorophores with distinguishable signals.

[118] For example, multiplex assays may be performed to simultaneously assay for a plurality of targets. A single probe species comprising an oligo-dT sequence can be used with a plurality of probe species in the simultaneous detection of multiple mRNA sequences. A plurality of nucleophile probes and a plurality of electrophile probes may be used to simultaneously assay for the presence of complementary target sequences.

[119] Competition assays may also be performed. For example, a single nucleophilic probe complementary to a sequence of interest may be used with a plurality of electrophilic linker probes complementary to potentially variable neighboring sequences, e.g. polymorphic sequences, alternatively spliced sequences, etc. The probe having greatest complementarity can win the competition, yielding a fluorescence signal specific to that probe.

[120] A fluorescent signal is generated, e.g. on a substrate comprising probes, upon the presence of a complementary nucleic acid sequence in the analyte; in solution; etc. The fluorescent signal that is generated in the assay can be monitored and quantified with fluorescence detectors, including fluorescence imagers, e.g. commercial instruments supplied by Hitachi Corp., San Bruno, Calif., fluorescence microscopes, confocal laser microscopes (confocal fluorescence scanners), e.g. commercial instruments from General Scanning, Inc., Watertown, Mass.

[121] Assays based on detection of sequences present in individual cells may utilize fixed or living cells. Cells in a sample may be fixed, e.g. with 3% paraformaldehyde, and are usually permeabilized, e.g. with ice cold methanol; HEPES-buffered PBS containing 0.1 % saponin, 3% BSA; covering for 2 min in acetone at -20°C; and the like as known in the art. Living cells may also be assayed using the probes of the invention. Probes can be introduced into live cells using any one of many well-known methods for bringing oligonucleotides into cells, including electroporation, calcium phosphate transfection, ionic shock, microinjection, pore-forming peptides, uptake reagents, fusion of vesicles, etc. Many such reagents are commercially available. Such methods may utilize carrier molecules, including calcium-phosphate, DEAE dextran and cationic lipids. Nucleic acids can be adsorbed to unilamellar liposome vesicles comprising cationic lipids mixed with neutral lipids, which vesicles may be modified by the inclusion of various commercially available components, e.g. FuGENE 6; X-tremeGENE Q2; etc. (Roche Applied Science). Cationic polymers, including dendrimeric polyamines or homopolymers of positively charged amino acids such as poly-L-lysines, poly-D-lysines and poly-L-ornithines, HIV tat, Pseudomonas exotoxin, Drosophila Antennapedia and HSV-1 VP22 protein may also be used as carriers. Agents that enhance uptake may be covalently conjugated to the probes. Examples include cationic peptides, cholesterol, arginine-rich peptides, etc.

[122] Flow cytometry is a convenient method to quantitate fluorescence signals from cells.

Flow cytometry methods are known in the art, and described in the following: Flow Cytometry and Cell Storing (Springer Lab Manual), Radbruch, Ed., Springer Verlag, 2000; Ormerod, Flow Cytometry, Springer Verlag, 1999; Flow Cytometry Protocols (Methods in Molecular Biology , No 91 ), Jaroszeski and Heller, Eds., Humana Press, 1998; Current Protocols in Cytometry, Robinson et al., eds, John Wiley & Sons, New York, NY, 2000. The readouts of selected fluorophores are capable of being read simultaneously, or in sequence during a single analysis, allowing of up to 5 or more fluorescent colors simultaneously. Readouts from such assays may be the mean fluorescence associated with individual fluorescent molecules, or the average fluorescence intensity, the median fluorescence intensity, the variance in fluorescence intensity, or some relationship among these.

[123] Microscopic analysis of single cell multiparameter and multicell multiparameter multiplex assays are used in the art, see Confocal Microscopy Methods and Protocols (Methods in Molecular Biology Vol. 122.) Paddock, Ed., Humana Press, 1998. These methods are described in U.S. Patent no. 5,989,833 issued Nov. 23, 1999.

[124] In a particular embodiment of the invention, RNA molecules from a biological source are detected and/or quantified. The RNA may be directly obtained from cells of interest; may be present in living or fixed cells; or may be converted to cDNA molecules and/or further amplified by PCR.

[125] In many instances, such assays are conducted with mRNA samples obtained from a biological system under different environmental conditions, such as exposures to varying concentration of a drug candidate or mixtures of drug candidates, which can provide data on the efficacy, the safety profile, the mechanism of action and other properties of the drug candidates that are required in drug development. Alternatively, tissue samples may be probed for the presence of clinical conditions, e.g. the presence of pathogens; expression of tumor associated sequences; and the like.

[126] In another embodiment of the invention, the probes are used to detect or quantify nucleic acid targets from genomic DNA, in order to analyze for the presence or absence of polymorphisms in the genomic DNA. The polymorphisms can be deletions, insertions, or base substitutions or other polymorphisms of the genomic DNA. Typically the polymorphisms are single nucleotide polymorphisms (SNPs), gene rearrangements, allelic variants; and the like. KITS

[127] Also provided are kits for practicing the subject methods. The kits according to the present invention may comprise at least: one modified polynucleotide; and (b) instructions for using the provided modified oligonucleotide(s). Such modified polynucleotides may be provided lyophilized, in solution, or bound to a substrate. Kits may further include a second polynucleotide to form a pair that may hybridize to neighboring regions of a target sequence.

[128] Kits may also be provided for use in the synthesis of oligonucleotides, comprising a branched universal linker; which is optionally loaded with a functional group; which may be provided with reagents for modifying a second polynucleotide probe, e.g. phosphorylating agents, etc. Such kits may also comprise modified H-phosphonate or phosphoroamidite derivatives, e.g. to introduce functional groups of interest into a modified polynucleotide.

[129] The subject kits may further comprise additional reagents which are required for or convenient and/or desirable to include in the reaction mixture prepared during the subject methods, where such reagents include phosphoroamidite reagents and buffers for DNA synthesis; columns.

[130] The various reagent components of the kits may be present in separate containers, or may all be precombined into a reagent mixture for combination with samples. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.

EXPERIMENTAL

[131 ] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the scope of the invention. Materials and Methods

[132] Synthesis of double displacement (DD) dabsyl linker phosphoramidite derivatives.

[133] Synthesis of DD 1 ,5-Pentyl Linker Phosphoramidite.

[134] Scheme, (i) 0₃ (g), -78^<€; NaBH₄, -78 ^«C to RT, 12 h (94%); (ii) Dabsyl-CI, Me₂N(CH₂)₃NMe_2! ACN, 3h, RT (20%); (iii) 2-cyanoethyl N,N- diisopropylchlorophosphoramidite, DIPEA, CH₂CI₂, RT, 2h (65%).

[135] 1 ,3,5-pentanetriol (1 ) [Wender et al., J. Am. Chem. Soc. 2002, 124, 13648].

[136] 1 ,6-heptadien-4-ol (5.0 g, 42 mmol) was dissolved in 200 mL anhydrous methanol in a flask. The reaction flask was cooled to -78 'Ό and ozone was bubbled through the solution until a deep blue color persisted. N₂ was then bubbled through the solution for 25 minutes to purge excess ozone. Sodium borohydride (12.7 g, 336 mmol) was added to the solution and it was gradually warmed from -78 'Ό to room temperature overnight. The reaction was acidified to pH ~3 with concentrated HCI and filtered to remove the white precipitate. The filtrate was concentrated under vacuum, redissolved in 100 mL methanol, and concentrated under vacuum. This was process was repeated two more times. The resulting crude mixture was purified by silica gel column chromatography (15% methanol in chloroform) to give the product 1 (4.74 g, 39.5 mmol) in 94% yield.

H-NMR (CD₃OD, 500 MHz) δ = 3.88 (septet, 1 H, C3-H, J=4.2 Hz), 3.70 (t, 4H, C1 ,5-H, J=6.4), 1 .73-1 .61 (m, 4H, C2.4-H); ³C-NMR (CD₃OD, 500 MHz) δ = 67.43, 60.12, 41 .04. HRMS [+Scan]; calculated m/z for C₅H₁₃0₃ 121 .0859; observed mass 121 .0857

[137] Bi-dabsylate Compound 2.

[138] 1 ,3,5-pentanetriol (1 ) (0.50 g, 4.2 mmol) was dissolved in 20 mL dry acetonitrile at room temperature. N,N,N',N'-Tetramethyl-1 ,3-propanediamine [Yoshida et al., Synthesis- Stuttgart 1999, 1633] (1 .53 ml_, 9.2 mmol) and Dabsyl-CI (2.96 g, 9.2 mmol) were sequentially added to the reaction mixture at room temperature and stirred for three hours. The solvent was removed under vacuum and the crude mixture was purified by silica gel column chromatography (0 to 10% ethyl acetate in dichloromethane) to give 2 (0.56 g, 0.81 mmol) in 19% yield.

H-NMR (CDCI₃, 400 MHz) δ = 7.99 (d, 2H, ArH, J=8.6), 7.95 (d, 2H, ArH, J=8.4), 7.91 (d, 2H, ArH, J=9.3), 6.75 (d, 2H, ArH, J=9.2), 4.29 (m, 2H, C1 ,5-H_a), 4.15 (m, 2H, C1 ,5-H_b), 3.99-3.91 (m, 1 H, C3-H), 3.12 (s, 12H, C6-H), 1 .89-1 .80 (m, 2H, C2,4-H_a), 1 .73-1 .65 (m, 2H, C2,4-H_b);

3C-NMR (CDCI₃, 500 MHz) δ = 156.53, 153.31 , 143.66, 134.93, 129.02, 126.00, 122.83, 1 1 1 .52, 67.52, 63.93, 40.39, 36.31 .

HRMS [+Scan]; calculated m/z for

695.2316; observed mass 695.2310. [139] Phosphoramidite compound 3.

[140] Compound 2 (0.17 g, 0.25 mmol) was dissolved in 2.5 ml_ dry dichloromethane under argon. Diisopropylethylamine (0.150 ml_, 0.83 mmol) and 2-cyanoethyl N,N- bisphosphorochloroamidite (0.060 ml_, 0.28 mmol) were sequentially added to the reaction mixture and stirred under argon for two hours at room temperature. The solvent was removed under vacuum and the crude mixture was purified by silica gel column chromatography (6:4:2% to 1 :1 :2%, hexanes:ethyl acetate:triethylamine) to give 3 (0.15 g, 0.16 mmol) in 65% yield.

H-NMR (CDCI₃, 400 MHz) δ = 8.00-7.86 (m, 12H, ArH), 6.75 (d, 4H, ArH, J=9.3), 4.29-3.98

(m, 4H), 3.78-3.70 (m, 1 H), 3.62-3.54 (m, 1 H), 3.50-3.40 (m, 2H), 3.10 (s, 12H), 2.60-2.55

(m, 2H), 1 .90-1 .84 (m, 2H), 1 .32-1 .20 (m, 4H), 1 .08 (d, 6H, CH₃(isopropyl), J=7.0), 1 .04 (d,

6H, CH₃(isopropyl), J=7.0).

³ P-NMR (CDCI₃, 400 MHz) δ = 149.67.

ESI-MS calculated for C₄₂H₅₅N₈0₈PS₂894.3322, found 894.98.

[141] Isobutyl and 1 ,3-Pentyl DD dabsyl linker synthesis

isobutyl phosphoramidite 1 ,3-pentyl phosphoramidite

[142] The isobutyl and 1 ,3-pentyl DD dabsyl linker phosphoramidites were prepared by similar methods to those described above.

[143] Double Displacement Linker Oligonucleotide Synthesis

[144] Pac-dA, iPr-dG, and Ac-dC phosphoramidites for UltraMild synthesis (Glen Research) were employed in synthesizing oligonucleotides containing the double displacement linker. The fluorescein label was introduced with fluorescein-dT phosphoramidite (Glen Research). Deprotection and cleavage from the CPG support was carried out by incubation in 0.05 M Potassium Carbonate in methanol (Glen Research) for four hours at room temperature. The oligonucleotides were purified by reverse-phase HPLC (Prosphere C18 30θΑ 10u 250 mm, eluting with 0.1 M triethylammonium acetate, pH 7.0 and acetonitrile). Probe structure was confirmed by MALDI-TOF mass spectrometry. D D AGTf CG AC ATCGTTTACG A : calculated mass C₂39H₂₈4N₇₄0₁₂₂P₁₈S₂ 6764.926; observed mass 6760.423.

[145] Butyl Linker Oligonucleotide Synthesis

[146] Single-dabsyl (butyl linker) probes were prepared and purified as previously described [H. Abe, E. T. Kool, J. Am. Chem. Soc. 2004, 126, 13980]. [147] Preparation of 3'-Phosphorothioate Oligonucleotide Strand

[148] 3'-Phosphorothioate probes were prepared and purified as previously described [ S.

Sando, E. T. Kool, J. Am. Chem. Soc. 2002, 124, 2096; S. Sando, E. T. Kool, J. Am. Chem. Soc. 2002, 124, 9686].

[149] Preparation of 3 '-Phosphorodithioate Oligonucleotide

[150] 3'-Phosphorodithioate DNAs were synthesized using 3'-Phosphate-ON CPG columns and dA-thiophosphoramidite reagent (both from Glen Research). The thiophosphoramidite was coupled to the solid support and sulfurized according to the procedure from Glen Research [Glen Research, Glen Report 2008 20(1 ), 4-6]. The remaining strand was synthesized using normal conditions and phosphoramidites for 3' to 5' DNA Synthesis. After synthesis, the column was washed twice with 1 mL of anhydrous diethylamine and then once with 5 mL anhydrous acetonitrile. Deprotection and cleavage from the CPG support was carried out by incubation in concentrated aqueous ammonium hydroxide with 20 mM DTT for three hours at 45 The mixture was then filtered to remove the glass beads and diluted with 2 ml_ of 50 mM Tris buffer, pH 9.0. The solution was then evaporated to dryness in the speedvac and redissolved in 1 ml_ dH₂0.

] Phosphorodithioate-substituted DNA strands were purified by anion-exchange HPLC on a Dionex DNAPac PA200 column using a linear gradient of 0 to 0.5 M NaCI in 20 mM Tris (pH 9.0) over fifty minutes. The presence of the phosophorodithiate was confirmed by mass spectrometry: MS ES^" calculated m/z for Ci₄₄Hi8i N₆o085Pi5S₂ ^2": 4641 .1 ; observed mass 4642.8.

This approach to phosphorodithioate-modified DNA oligonucleotides is different from previously reported methods. The previous method relied on the coupling of 2-(N,N- diisopropylamino)-1 ,3,2-dithiaphospholane to the 3' end of a strand synthesized with reverse phosphoramidites and has the advantage of providing access to both phosphorodithioate and phosphorotrithioate modifications [G. P. Miller, A. P. Silverman, E. T. Kool, Bioorg. Med. Chem. 2008, 16, 56]. However, it also requires prior synthesis of the phospholane reagent. The method described herein uses only commercially-available reagents and has the advantage of allowing us to perform all of the chemistry on solid support.

] Double Displacement Probe Synthesis

] During oligonucleotide synthesis one or more fluorescent group(s) are added to a growing oligonucleotide via addition of a modified phosphoramidite or H-phosphonate reagent, via standard methods, at a position of between 1 to 20 nucleotides before the 5' terminus. During the final cycle of oligonucleotide synthesis a branched universal linker is added, which includes the releasing groups of interest. Linkers are synthesized as described herein. The linker is reacted using the standard reagents and methods as the oligonucleotide synthesis reactions. The completed modified oligonucleotide is then cleaved from the support and deprotected by treatment with concentrated ammonium hydroxide or potassium carbonate/methanol. The modified oligonucleotide is subsequently treated with heat to remove the remaining protecting groups. Crude product oligonucleotide is precipitated, or passed over a desalting column, and may be used in some cases without further purification. The final product is purified by chromatography or electrophoresis, using standard methods.

] In an alternative method, the Q and Q² groups are added separately from the rest of the linker via a synthetic reaction. The Q and Q² groups are added either following or during addition of the linker precursor to the oligonucleotide. For example, a linker precursor containing DMT-protected hydroxyl groups on tethers is deprotected to give free hydroxyl groups, to which are added the L -Q¹ and L²-Q² groups using standard methods.

[156] When the activating leaving groups L and L² are not stable during polynucleotide synthesis, (i.e., they undergo nucleophilic side-reactions leading to cleavage and loss of quenchers during synthesis), then the L -Q¹ and L²-Q² groups of the linker are installed post polynucleotide synthesis, using standard methods.

[157] Templated Double Displacement Reactions

[158] Reactions were performed in 70 mM PIPES Buffer (pH 7.0) containing 10 mM MgCI₂ and 50 μΜ dithiothreitol on a Flexstation II 384 microplate reader. Reaction mixtures contained 100 nM each of quencher probe and template and 120 nM phosphorodithioate strand. Reactions were initiated by addition of phosphorodithioate strand to the wells containing the quencher probe and template strand. The emission was measured in two- minute intervals at 522 nm with excitation at 494 nm for eight hours. The temperature was maintained at 25^ for all reactions unless otherwise noted. The control reactions were performed with the DNA template omitted from the reaction mixture.

[159] Testing intracellular application in RNA Detection

[160] E.coli K12 cells were grown to mid-log phase (OD600 = 0.4-0.6) in Luria-Bertani (LB) media at 37 ^<Ό with rapid shaking. Aliquots of media were centrifuged for five minutes at 5000 rpm. The supernatant was removed and pellets were resuspended in 0.1 ml_ phosphate-buffered saline solution (pH 7.4). The cells were centrifuged again for five minutes and the supernatant was removed. The pellets were resuspended in 0.1 ml_ hybridization buffer (6x SSC and 0.05% SDS). Aliquots of bacteria suspended in hybridization buffer were treated with double displacement probe (200 nM), nucleophile probe (2 uM), and helper probes (3 uM each). The reaction mixtures were incubated in the dark at 37^<Ό and then monitored by fluorescence microscopy without any washing steps.

[161] Structures of Single and Double Displacement Linkers

[162] (A) Single displacement (butyl) linker described previously; (B) Double displacement 1 ,5-pentyl linker; (C) Double displacement isobutyl linker; (D) Double displacement 1 ,3- pentyl linker.

[163] Initial Quenching Efficiency of Single vs. Double Displacement Probes (see Figure 2)

[164] Initial emission spectra of different quencher probes containing the butyl linker or the 1 ,5-pentyl DD linker are shown. Conditions: 100 nM probe in pH 7.0 buffer containing 70 mM PIPES, 10 mM MgCI₂, 50 uM DTT at 25^<C. A seven-point running average was used to smooth the data, which was noisy due to very low fluorescence. Figure 3 shows the initial emission spectra of different quencher probes containing the butyl linker, the 1 ,5-pentyl DD linker, the isobutyl DD linker, or the 1 ,3-pentyl DD linker.

[165] Fluorescence timecourse of templated ligation reactions at 25 °C (Figure 4).

[166] 100 nM different quencher probes containing the butyl linker or the 1 ,5-pentyl DD linker and 120 nM nucleophile probe were incubated with or without template at 25^ in pH 7.0 buffer containing 70 mM PIPES, 10 mM MgCI₂, 50 uM dithiothreitol. The fluorescence was measured every 120 s with 494 nm excitation and 522 nm emission. Reactions were repeated in triplicate; representative traces are shown. [167] Fluorescence timecourse of templated ligation reactions at 37°C (Figure 5).

[168] 100 nM different quencher probes containing the butyl linker or the 1 ,5-pentyl DD linker and 120 nM nucleophile were incubated with or without template at 37 ^<C in pH 7.0 buffer containing 70 mM PIPES, 10 mM MgCI₂, 50 uM DTT. The fluorescence was measured every 120 s using 494 nm excitation and 522 nm emission. Reactions were repeated in triplicate; representative traces are shown.

[169] Figure 6 shows signal and background signals for probes in vitro. Fluorescent signals observed over time for various probes (containing two dabsyl quenchers attached via isobutyl, 1 ,3-pentyl or 1 ,5-pentyl DD linkers) incubated with a target sequence; either with a nucleophile probe (shown with solid lines); or without a nucleophile probe (shown with broken lines). Comparison is shown to a modified polynucleotide including a single dabsyl quencher.

[170] Fluorescence endpoint of templated ligation reactions (Figure 7).

[171] 100 nM different quencher probes containing the butyl linker or the 1 ,5-pentyl DD linker and 120 nM phosphorodithioate were incubated with or without template at 25^ for twenty-four hours in pH 7.0 buffer containing 70 mM PIPES, 10 mM MgCI₂, 50 uM DTT. The emission of each sample was then measured from 500 to 600 nm with a 494 nm excitation. A seven-point running average was used to smooth the data.

[172] Degradation of 3' terminal phosphorodithioate DNAs at 37°C (Figure 8).

[173] Conditions: 4 μΜ phosphorodithioate strand was suspended in pH 7.0 buffer containing 70 mM PIPES, 10 mM MgCI₂, 50 uM DTT and incubated at 37^<C for the indicated length of time. 100 μΙ_ aliquots of the sample were analyzed by anion-exchange HPLC on a Dionex DNAPac PA200 column. The arrow indicates the peak corresponding to the intact phosphorodithioate DNA.

[174] Equivalents of Displaced Dabsylate Over Time (Figure 9).

[175] The amount of free dabsylate in solution was monitored by HPLC from reactions containing 1 uM of 1 ,5-pentyl DD probe, 1 uM template strand and 1 .2 uM phosphorodithioate probe (Blue) or 1 .2 uM phosphoromonothioate probe (Green). The 1 ,5- pentyl DD probe and template with no nucleophile probe present is also shown (Red). The reactions were run at 25^. [176] Detection of 16S rRNA in intact Escherichia Coli, showing lower background signal (Figure 10).

[177] Phosphorodithioate probes (2 uM), unlabeled helper DNAs (3 uM), and quencher probes containing the butyl linker or the 1 ,5-pentyl DD linker (200 nM) were mixed with cells in 6x SSC buffer and 0.05% SDS and incubated for one hour at 37 ^<C. The cells were imaged by epifluorescence microscopy. The top two panels show the 1 ,5-pentyl Double Displacement probe with (A) and without (B) phosphorodithioate probe. The bottom two panels show the single displacement (butyl linker) probe with (C) and without (D) phosphorodithioate probe.

[178] Figure 1 1 shows in vivo results for various probes. Fluorescent signals observed over time for various probes (containing two dabsyl quenchers attached via isobutyl, 1 ,3- pentyl or 1 ,5-pentyl linkers) incubated with a target sequence; either with a phosphorothioate nucleophile probe (top images); or without a nucleophile probe (bottom images). Comparison to a modified polynucleotide including a single dabsyl quencher is shown.

[179] Synthesis of unmodified oligonucleotides. All oligonucleotides were synthesized on 1 μηιοΐβ scale on an ABI model 392 synthesizer using standard β- cyanoethylphosphoroamidite coupling chemistry. Deprotection and cleavage from CPG support was carried out by incubation in concentrated ammonia for 14 h at 55^°C. Following deprotection, oligonucleotides were purified by PAGE, and quantitated by UV absorbance using the nearest neighbor approximation to calculate molar absorptivities.

[180] Preparation of dabsyl- and fluorescein-labeled oligonucleotides. Pac-protected dA, iPr-Pac-preotected dG, and acetyl-protected dC phosphoroamidites for ULTRA MILD SYNTHESIS (Glen Research) were employed in synthesizing oligonucleotides containing a dabsyl group. The fluorescein label was introduced with fluorescein-dT phosphoroamidite (Glen Research). Deprotection and cleavage from the CPG support was carried out by incubation in 0.05 M potassium carbonate in methanol for 6 h at room temperature. After concentration, oligonucleotides were purified by reverse-phase HPLC (Allotec BSD-C18 column 250 mm, eluting with 0.1 M triethylammonium acetate pH7.0/acetonitrile). For 3'- Phosphorothioate sequences, the first nucleotide added after the phosphorylation reagent was sulfurized by the sulfurizing reagent (Glen Research). 5'-³²P labeling was carried out using T4 polynucleotide kinase (NEB) and γ-³²Ρ-ΑΤΡ (Amersham).

Results and Discussion

[181] Design of Double Displacement Probes

[182] A number of criteria were taken into account in designing a linker that would enable double displacement. The linker needed to include suitable SN2 electrophilic centers at two positions and be compatible with DNA synthesis. One possible linker precursor is 1 ,3,5- pentanetriol which has functional groups suitable for both quencher coupling and phosphoramidite forming reactions, making the target linker synthetically accessible. The quenchers are coupled to relatively unhindered positions, making it a good SN2 substrate. The synthesis of phosphoramidite linker reagents is described herein.

[183] The activating leaving group (e.g., dabsylate) serves both to activate the linker substrate for electrophilic attack and to quench a nearby fluorophore. The linker includes two such quencher/leaving groups; these two groups provide advantages over earlier approaches. First is superior quenching of the nearby fluorophore, yielding lower initial fluorescence of the unreacted probe. Such an effect has been demonstrated with molecular beacons, where adding two and three quenchers improved the quenching efficiency [C. J. Yang, H. Lin, W. Tan, J. Am. Chem. Soc. 2005, 127, 12772]. Second is nonspecific reaction of the DD probe with water or other adventitious nucleophiles might displace one quencher, but this background can be mitigated by having a second quencher present still suppressing the emission of the fluorophore.

[184] For templated reactions with the double displacement probes, a nucleophile- conjugated DNA probes may be used: e.g., a 3'-phosphorothioate that is capable of performing a nucleophilic attack to displace a quencher. Alternatively, a 3' phosphorodithioate conjugated probe may be used that is capable of performing two nucleophilic attacks to displace two quenchers.

[185] Initial Quenching Efficiency

[186] The initial quenching properties for the double displacement and butyl linker strands were compared to determine whether the new probe was a better quencher of the fluorophore. The double displacement probe was found to have initial quenching that was more than four-fold greater than the butyl linker probe (Figure 2), thus confirming substantially lower initial background fluorescence of the DD probe.

[187] Monitoring Displacement Reaction by Fluorescence

[188] The double displacement reaction was monitored by observing the change in fluorescence emission at 522 nm over time at 25^ (Figure 4) or 37 ^<Ό (Figure 5). At both temperatures, the kinetics for the double displacement reaction were slower than for the single-dabsyl substrate, but the background rate was also markedly lower. At 25 °C, the phosphorothioate and phosphorodithioate nucleophiles produced similar fluorescence enhancements, suggesting that they employ the same mechanism of action. Additionally, after eight hours the reaction was still not complete, as evidenced by the fact that it did not reach the same fluorescence level as the single-dabsyl reaction. However, the signal-to- background was still considerably higher than the single-dabsyl reaction and when the double displacement reaction was allowed to run overnight, it eventually reached the same level of fluorescence as the single-displacement reaction (Figure 7).

[189] At 37^<C, the level of fluorescence produced by the phosphorodithioate was significantly lower and quickly reached a plateau, below the level expected for the completed reaction. Later experiments carried out to measure the thermal stability of the phosphorodithioate group on DNA showed that it degraded over a few hours. Therefore, in this case, the fluorescence intensity may reach a premature plateau due to a dearth of nucleophile with which to react. The phosphorothioate DNA is more stable than the phosphorodithioate case and therefore is not affected by the increase in temperature.

[190] Characterizing the Reaction Mechanism

[191] In order to gain insight into the mechanism of the reaction, the displacement of the quencher group was monitored quantitatively by following the increase in dabsylate leaving group concentration over time by HPLC. The rates of dabsylate appearance for reactions with and without a nucleophile probe primarily differ in the first eight hours of the reaction (Figure 9). At eight hours, the reaction with the nucleophile had lost 1 .5 equivalents of dabsylate and the reaction without the nucleophile had lost 0.5 equivalents. From then on, the rates of dabsylate appearance are very similar. By the time the reactions with the nucleophile displace two equivalents of dabsylate, the reaction with no nucleophile present had displaced one equivalent. This supports the notion that while background (water) displacement of one of the quenchers is occurring, it does not generate a signal until a templated nucleophile can react to displace the second one.

[192] Detection of rRNA in E. Coli

[193] The ability of the double displacement probes to detect ribosomal RNA in Escherichia Coli was tested. The probes were designed to bind to a sequence in the 16S rRNA that had been previously described [A. P. Silverman, E. T. Kool, Nucleic Acids Res. 2005, 33, 4978]. Bacterial cells were treated with quencher, nucleophile, and unlabeled helper probes [B. M. Fuchs, F. O. Glockner, J. Wulf, R. Amann, Appl. Environ. Microbiol. 2000, 66, 3603] in the presence of 0.05% SDS and 6x SSC buffer and incubated for one hour at 37^<C with no washing steps. The phosphorothioate strand was used as the nucleophile because it is significantly more stable at 37 ^<C than the phosphorodithioate. In control experiments to determine the background level for these experiments, the nucleophile probes were omitted.

[194] Inspection of the bacterial cells under the epifluorescence microscope revealed a strongly positive signal for the 16S RNA and a very low background from nonspecific reaction (Figures 10 and 1 1 ). The signal-to-background ratio for the double displacement probes was clearly better than for the single displacement probes. While the double displacement probes did not produce as much signal as the butyl linker probes, presumably because the reaction rate is slower, the background is substantially lower for the DD probes, as fluorescence in the bacteria cells was present in very low levels when the nucleophile probe was omitted. This result is consistent with the in vitro data, showing that the double displacement probes are less susceptible than the butyl linker probes to non- templated unquenching in cells, making this scaffold more bio-orthogonal than single displacement probes.

[195] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved.

ABBREVIATIONS

[196] ACN is acetonitrile; CPG is controlled pore glass; DD is double displacement; DNA is deoxyribonucleic acid; DMT is dimethoxytrityl; RNA is ribonucleic acid; RT is room temperature; TEA is triethylamine.

Claims

WHAT IS CLAIMED IS :

1 . A modified polynucleotide having the structure:

wherein Q and Q² are quenchers;

L and L² are activated leaving groups;

T\ T² and T³ are tethers;

A is a polynucleotide; and

R is OR¹ , SR , O^" or S^~, wherein R is a linear or branched lower alkyl.

2. The modified polynucleotide of Claim 1 , wherein:

A comprises at least one fluorophore quenched by Q and Q².

3. The modified polynucleotide of Claim 2, wherein said fluorophore is at least one of fluorescein, 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2',4',1 ,4,- tetrachlorofluorescein (TET), 2', 4', 5',7',1 ,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy- 4',5'-dichloro-6-carboxyfluorescein (JOE); Cy3, CY5, Cy5.5, a dansyl derivative; 6- carboxytetramethylrhodamine (TAMRA), a BODIPY fluorophore, tetrapropano-6- carboxyrhodamine (ROX), ALEXA dye, and Oregon Green.

4. The modified polynucleotide of Claim 1 , wherein:

said polynucleotide is at least 6 bases in length and not more than about 100 bases in length.

5. The modified polynucleotide of Claim 1 , wherein:

L and L² are independently selected from a sulfonate, a carbonyl ester, a para nitrophenyl ester, a nitrophenyl ester, a trifluoroacetyl ester, a nosylate, a brosylate, a tosylate, a perchlorate, a triflate, and a mesylate.

6. The modified polynucleotide of Claim 1 , wherein: L and L² are sulfonate.

7. The modified polynucleotide of Claim 1 , wherein:

L and Q together comprise a group selected from dabsylate (dimethylamino- azobenzene-sulfonate), dansylate (5-dimethylaminonaphthalenesulfonate), dimapdabsylate ((p-dimethylamino- phenylazo)-azobenzenesulfonate); an azobenzene-sulfonyloxy group, a benzenesulfonyloxy group, or an arenesulfonyloxy optionally comprising a substituent selected from amino, dialkylamino, nitro, fluoro, cyano; anthraquinone, nitrothiazole, nitroimidazole, rhodamine, tetramethyl-6-carboxyrhodamine (TAMRA), ROX, cyanine, coumarin, BODIPY, fluorescein, and ALEXA dyes; and

L² and Q² together comprise a group selected from dabsylate (dimethylamino- azobenzene-sulfonate), dansylate (5-dimethylaminonaphthalenesulfonate), dimapdabsylate ((p-dimethylamino- phenylazo)-azobenzenesulfonate); an azobenzene-sulfonyloxy group, a benzenesulfonyloxy group, or an arenesulfonyloxy optionally comprising a substituent selected from amino, dialkylamino, nitro, fluoro, cyano; anthraquinone, nitrothiazole, nitroimidazole, rhodamine, tetramethyl-6-carboxyrhodamine (TAMRA), ROX, cyanine, coumarin, BODIPY, fluorescein, and ALEXA dyes.

8. The modified polynucleotide of Claim 1 , wherein:

T , T² and T³ are independently a single bond or a chain of from about 1 to about 20 methylene groups in length, where the methylene backbone is optionally substituted with between one to seven sulfur, nitrogen and/or oxygen heteroatoms; optionally comprising one, two, or three unsaturated bonds in the tether backbone, wherein each of the backbone atoms may be substituted or unsubstituted.

9. The modified polynucleotide of Claim 1 , wherein:

T , T² and T³ are independently a single bond, an oligo(ethylene glycol), an ether, a thioether, a tertiary amino, a straight, branched or cyclic alkyl or alkenyl group; optionally substituted with an alkyl, an aryl, an alkenyl, an aralkyl, an aralkenyl, a cycloalkyl, a cycloalkyl alkyl or a cycloalkylalkenyl group.

10. The modified polynucleotide of Claim 1 , wherein:

T , T² and T³ are independently absent or present, and when present independently selected from the group consisting of:

the alkyi or alkenyl is optionally substituted with an alkyi, an aryl, an alkenyl, an aralkyl, an aralkenyl, a cycloalkyl, a cycloalkylalkyl or a cycloalkylalkenyl group;

R³ is selected from an alkyi, usually branched or linear lower alkyi; hydroxy, alkoxy, aryloxy, haloalkoxy, cyano, nitro, mercapto, alkylthio, -S(0)_pR⁶ (where p is 0 to 2), -S(0)pN(R⁶)₂ (where p is 0 to 2); -OR⁶, -C(0)OR⁶, -C(0)N(R⁶)₂, -N(R⁶)₂, -N(R⁶)C(0)OR⁷, -N(R⁸)C(0)R⁸, and -R⁸-N=N-0-R⁷; where each R⁶ , R⁷ or R⁸ is independently selected from the group consisting of hydrogen, an alkyi, an alkenyl, a haloalkyl, a haloalkenyl, an aryl, an aralkyl, an aralkenyl, a cycloalkyl, a cycloalkylalkyl and a cycloalkylalkenyl.

1 1 . The modified polynucleotide of Claim 1 , wherein:

T , T² and T³ are independently selected from a single bond, -CH₂- and -(CH₂)₂-.

12. The modified polynucleotide of Claim 1 , wherein:

Q and Q² are dabsyl;

L and L² are sulfonate groups; and

T , T² and T³ are independently a single bond, -CH₂- or -(CH₂)₂-.

13. The modified polynucleotide of Claim 1 , wherein:

said polynucleotide is attached to a support.

14. A composition comprising:

the modified polynucleotide according to Claim 1 ; and

a second modified polynucleotide comprising a nucleophilic group.

15. The composition of Claim 14, wherein: said second modified polynucleotide has the structure:

Λ x²- — O Y

X³

wherein X¹ , X² and X³ are independently selected from oxygen and sulfur; and Y is a polynucleotide.

16. The composition of Claim 15, wherein:

A is a polynucleotide comprising a fluorophore quenched by Q and Q².

17. The composition of Claim 16, wherein:

T , T² and T³ are independently present or absent, and when present independently selected from the group consisting of:

R³ is selected from an alkyi, usually branched or linear lower alkyi; a hydroxy, an alkoxy, an aryloxy, a haloalkoxy, a cyano, a nitro, a mercapto, an alkylthio, -S(0)_pR⁶ (where p is 0 to 2), -S(0)_pN(R⁶)₂ (where p is 0 to 2); -OR⁶, -C(0)OR⁶, -C(0)N(R⁶)₂, -N(R⁶)₂, -N(R⁶)C(0)OR⁷, -N(R⁸)C(0)R⁸, and -R⁸-N=N-0-R⁷; where each R⁶ , R⁷ or R⁸ is independently selected from the group consisting of hydrogen, an alkyi, an alkenyl, a haloalkyl, a haloalkenyl, an aryl, an aralkyl, an aralkenyl, a cycloalkyl, a cycloalkylalkyl and a cycloalkylalkenyl.

18. The composition of Claim 17, wherein:

L and Q together comprise a dabsylate;

L² and Q² together comprise a dabsylate;

T and T² are independently selected from a single bond, -CH₂- and -(CH₂)₂-; and two of X¹ , X² and X³ are sulfur; and one of X¹ , X² and X³ is oxygen.

19. A composition comprising a linker of the structure:

wherein Q and Q² are quenchers;

L and L² are activated leaving groups;

T , T² and T³ are tethers; Z is carbon or nitrogen; and

X is a phosphoramidite or a H-phosphonate.

20. The composition of Claim 19, wherein:

T , T² and T³ are each independently a single bond or a chain of from about 2 to about 20 methylene groups in length, where the methylene backbone is optionally substituted with between one to seven sulfur, nitrogen and/or oxygen heteroatoms; optionally comprising one, two, or three unsaturated bonds in the tether backbone, wherein each of the backbone atoms may be substituted or unsubstituted.

21 . The composition of Claim 19, wherein:

T , T² and T³ are independently a single bond, an oligo(ethylene glycol), an ether, a thioether, a tertiary amino; a straight, branched or cyclic alkyi or alkenyl; optionally substituted with an alkyi, an aryl, an alkenyl, an aralkyl, an aralkenyl, a cycloalkyl, a cycloalkyl alkyi or a cycloalkylalkenyl group.

22. The composition of Claim 19, wherein:

T , T² and T³ are independently present or absent; and when present, independently selected from the group consisting of:

wherein n is from 2 to 20; and and n₂ are independently selected to be from 1 to 20; Πι + n₂ are usually not more than about 20; and y is from 1 to 7;

R³ is selected from an alkyi, usually branched or linear lower alkyi; a hydroxy, an alkoxy, an aryloxy, a haloalkoxy, a cyano, a nitro, a mercapto, an alkylthio, -S(0)_pR⁶ (where p is 0 to 2), -S(0)_pN(R⁶)₂ (where p is 0 to 2); -OR⁶, -C(0)OR⁶, -C(0)N(R⁶)₂, -N(R⁶)₂, -N(R⁶)C(0)OR⁷, -N(R⁸)C(0)R⁸, and -R⁸-N=N-0-R⁷; where each R⁶ , R⁷ or R⁸ is independently selected from the group consisting of hydrogen, an alkyl, an alkenyl, a haloalkyl, a haloalkenyl, an aryl, an aralkyl, an aralkenyl, a cycloalkyl, a cycloalkylalkyl and a cycloalkylalkenyl.

23. The composition of Claim 19, wherein X is of the structure:

wherein R is a protecting group; and

R² is a primary amino or secondary amino group.

24. The composition of Claim 19, wherein:

25. The composition of Claim 19, wherein:

26. The composition of Claim 22, wherein:

L and Q together comprise a dabsylate;

L² and Q² together comprise a dabsylate;

Z is carbon; and T , T² and T³ are independently selected from a single bond, -CH₂- and -(CH₂)₂--

27. A method of detecting a target nucleic acid sequence in a sample, said method comprising:

contacting said sample suspected of containing said target nucleic acid sequence with the composition of Claim 14, wherein said modified polynucleotide of Claim 1 and said second modified polynuclotide hybridize to neighboring sequences on said nucleic acid sequence;

exposing said sample to conditions sufficient to lead to ligation of the modified polynucleotides and release of quenchers Q and Q²; and

measuring the change in fluorescence of the sample, wherein the level of change is proportional to the amount of target nucleic acid sequence present in the sample.

28. A method of detecting a target nucleic acid sequence in a sample comprising:

(a) contacting the sample with a probe set for each target nucleic acid sequence, said probe set comprising:

(i) a first probe comprising the modified polynucleotide of Claim 1 , wherein A is a polynucleotide comprising a sequence complementary to a first region of said target nucleic acid sequence; and

(ii) a second probe comprising:

a nucleophilic moiety; and

a second polynucleotide comprising a sequence complementary to a second region of said target nucleic acid sequence, wherein said second region is adjacent to said first region in said target nucleic acid sequence;

(c) measuring the change in fluorescence of said sample, wherein the level of change is proportional to the amount of target nucleic acid sequence present.

29. A method of modifying a polynucleotide, the method comprising:

reacting a 3' or 5' hydroxyl of a polynucleotide with the composition of Claim 22.

30. The method of Claim 29, wherein said reacting is performed following in vitro synthesis of said polynucleotide.

31 . A kit for the detection of a nucleic acid sequence of interest, the kit comprising a modified polynucleotide according to Claim 1 , and instructions for use.

32. A kit for modification of a polynucleotide, the kit comprising a universal linker according to Claim 1 , and instructions for use.