10/641,677, filed August 15,2003 and a continuation-in-part of U. S. Application Serial No. 10/314,012, filed December 5,2002, all of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION Gene expression in diseased and healthy individuals is oftentimes different and characterizable. The ability to monitor gene expression in such cases provides medical professionals with a powerful diagnostic tool. This form of diagnosis is especially important in the area of oncology, where it is thought that the overexpression of an oncogene, or the underexpression of a tumor suppressor gene, results in tumorogenesis. See Mikkelson et al. R Cell. Biochem.

1991, 46, 3-8.

One can indirectly monitor gene expression, for example, by measuring nucleic acid (e. g., mRNA) that is the transcription product of a targeted gene.

The nucleic acid is chemically or biochemically labeled with a detectable moiety and allowed to hybridize with a localized nucleic acid of known sequence sometimes, know here as a probe. The detection of a labeled nucleic acid at the probe position indicates that the targeted gene has been expressed. See, e. g., International Application Publication Nos. WO 97/27317, WO 92/10588 and WO 97/10365.

The labeling of a nucleic acid is typically performed by covalently attaching a detectable group (label) to either an internal or terminal position.

Scientists have reported a number of detectable nucleotide analogues that have been enzymatically incorporated into an oligo-or polynucleotide. Langer et al., for example, disclosed analogues of dUTP and UTP that contain a covalently bound biotin moiety. Proc. Natl. Acad. Sci. USA 1981, 78, 6633-6637. The analogues, shown below, possess an allylamine linker arm that is attached to the C-5 position of the pyrimidine ring at one end and a biotin moiety at the other.

The dUTP and UTP analogues, wherein R is H or OH, were incorporated into a polynucleotide.

Petrie et al. disclosed a dATP analogue, 3- [5- [ (N-biotinyl-6- aminocaproyl)-amino] pentyl]-1- (2-deoxy-p-D-erythro-pentofuranosyl)-1 H- pyrazolo [3,4-d] pyrimidin-4-amine-5'-triphosphate. Bioconjugate Chem. 1991,2, 441-446. The analogue, shown below, is modified at the 3-position with a linker arm that is attached to a biotin moiety. Petrie et al. reported that the compound wherein R is biotin is incorporated into DNA by nick translation.

Prober et al. disclosed a set of four dideoxynucleotides, each containing a succinylfluoresceindye. Sciencel987, 238, 336-341. The dideoxynucleotides, one of which is shown below, were enzymatically incorporated into an oligonucleotide through a template directed extension of a primer. The compounds provided for a DNA sequencing method based on gel migration.

Herrlein et al. disclosed modified nucleoside trisphosphates of the four DNA bases. Helv. Chim. Acta 1994,77, 586-596. The compounds, one of which is shown below, contain a 3'-amino group containing radioactive or fluorescent moieties. Herrlein et al. further described the use of the nucleoside analogues as DNA chain terminators.

Cech et al. disclosed 3'-amino-functionalized nucleoside triphosphates.

Collect. Czech. Chem. Commun. 1996, 61, S297-S300. The compounds, one of which is shown below, contain a fluorescein attached to the 3'-position through an amino linker. Cech et al. proposed that the described functionalized nucleosides would be useful as terminators for DNA sequencing.

The development of novel nucleic acid labeling compounds that are effectively incorporated into a nucleic acid to provide a readily detectable composition would benefit genetic analysis technologies. It would aid, for example, in the monitoring of gene expression and the detection and screening of mutations and polymorphisms. Such a compound should be suitable for incorporation into a nucleic acid either by enzymatic or other means.

Furthermore, the nucleic acid to which the labeling compound is attached should maintain its ability to bind to a probe, such as a complementary nucleic acid.

Although nucleic acid labeling compounds for use as coupling agents for probes are available there is a continuing need for additional compounds that are more efficient labeling compounds. There also exists a need for compounds that have increased solubility. This will make the compounds more useful for monitoring gene expression.

SUMMARY OF THE INVENTION The present invention relates to nucleic acid labeling compounds. More specifically, the invention provides heterocyclic derivatives containing a detectable moiety. The invention also provides methods of making such heterocyclic derivatives. It further provides methods of attaching the heterocyclic derivatives to a nucleic acid.

The nucleic acid labeling compounds of the invention the general formula (I) :

wherein G is NH or OH (depicted herein as >C=O), A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; X is O, S, NR1 or CHR2, wherein Rl and R2 are, independently, H, alkyl or acid by either enzymatic or, e. g. , by chemical means; Y is H, N3, F, OR9, SR9 or NHR9, wherein Rg is H, alkyl or aryl; Z is H, N3, F or ORlo, wherein Rlo is H, alkyl or aryl; L is-CH=CH-Rll, or C=C-Rn wherein Rn is-O-. amino, thio, carbonyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, thio and amino alkyl ; Q is a detectable moiety, M is a connecting group, and wherein m is an integer ranging from 0 to about 3.

In addition, the nucleic acid labeling compounds of the present invention are capable of being enzymatically incorporated into a nucleic acid. The nucleic acids to which the compounds are attached substantially maintain their ability to bind to a complementary nucleic acid sequence.

The nucleic acid labeling compounds of the present invention are heterocyclic derivatives that have a detectable moiety. The invention also provides methods of making such heterocyclic derivatives. It further provides methods of attaching the heterocyclic derivatives to a nucleic acid.

The present invention provides nucleic acid labeling compounds that are capable of being enzymatically incorporated into a nucleic acid. The nucleic acids to which the compounds are attached substantially maintain their ability to bind to a complementary nucleic acid sequence.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a nonlimiting set of template moieties.

Figure 2 shows a nonlimiting set of heterocyclic groups: 4- aminopyrazolo [3,4-d] pyrimidine, pyrazolo [3, 4-d] pyrimidine, 1, 3-diazole (imidazole), 1, 2,4-triazine-3-one, 1, 2,4-triazine-3, 5-dione and 5-amino-1, 2,4- triazine-3-one.

Figure 3 shows a schematic for the preparation of N1-labeled 5- (p-D- ribofuranosyl)-2, 4 [lH, 3H] -pyrimidinedione 5'-triphosphate.

Figure 4 shows HIV array data for analog 42a (T7 labeling of RNA target).

Figure 5 shows HPLC incorporation efficiency of C-nucleotide 42a (T7 RNA pol, 1 kb transcript).

Figure 6 shows IVT incorporation of saturated versus unsaturated nucleic acid labeling compounds.

DETAILED DESCRIPTION OF THE INVENTION A general aspect of the instantly disclosed invention includes nucleic acid labeling compounds of formula (II) : A-O-CH2-T-Hc-L- (M) m-Q (II) wherein A is hydrogen or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; T is a template moiety; He is a heterocyclic group, including without limitation a base such as A, T, G, C, and U; or He is absent) L is a linker moiety; Q is a detectable moiety; and M is a connecting group, wherein m is an integer ranging from 0 to about 5. Thus, the connecting group may optionally not be present, depending on, inter alia, the nature of L and Q.

In one embodiment, the nucleic acid labeling compounds have formula (III) :

(Ill) Stereoisomers are also included within the scope of the invention, examples of two of which are shown below: (IIIB) wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid by either enzymatic or, e. g. , by chemical means; X is O, S, NR1 or CHR2, wherein Ri and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHR9, wherein Rg is H, alkyl or aryl; Z is H, N3, F or ORlo, wherein Rlo is H, alkyl or aryl; L is linker group; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.

In one embodiment, the nucleic acid labeling compounds have formula (IV) :

Stereoisomers are also included within the scope of the invention, examples of two of which are shown below: wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid by either enzymatic or, e. g. , by chemical means; X is O, S, NR1 or CHR2, wherein Ri and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHRg, wherein Rg is H, alkyl or aryl; Z is H, N3, F or ORIO, wherein Rio is H, alkyl or aryl; L is functionalized alkyl ; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.

In another embodiment, the nucleic acid labeling compounds used in the coupling have the following structures for example: or

wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; X is O, S, NRl or CHR2, wherein R and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHRg, wherein Rg is H, alkyl or aryl; Z is H, N3, F or ORIO, wherein Rlo is H, alkyl or aryl; L is linker group; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.

The hybridization product formed from this nucleic acid derivative comprises the nucleic acid derivative bound to a complementary probe. In one embodiment, the probe is attached to a glass chip.

In another embodiment, the nucleic acid labeling compounds used in the coupling have the following structures for example:

wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; X is O, S, NRl or CHR2, wherein R and R2 are, independently, H, alkyl or aryl; Y is H, N3, F, OR9, SR9 or NHRg, wherein Rg is H, alkyl or aryl; Z is H, N3, F or ORlo, wherein Rlo is H, alkyl or aryl; L is functionalized alkyl ; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.

The hybridization products formed from the nucleic acid derivatives of the invention comprise the nucleic acid derivative bound to a complementary probe. In one embodiment, the probe is attached to a glass chip.

The method of nucleic acid detection using the nucleic acid derivatives of the inventioin involves the incubation of the derivative with a probe. In one embodiment, the probe is attached to a glass chip.

In another embodiment, the methods of the invention include the steps of : (a) providing at least one nucleic acid coupled to a support; (b) providing a labeled moiety capable of being coupled with a terminal transferase to said nucleic acid; (c) providing said terminal transferase; and (d) coupling said labeled moiety to the nucleic acid using said terminal transferase.

In still another embodiment, the methods of the invention include the steps of : (a) providing at least two nucleic acids coupled to a support; (b) increasing the number of monomer units of said nucleic acids to form a common

nucleic acid tail on said at least two nucleic acids; (c) providing a labeled moiety capable of recognizing said common nucleic acid tails; and (d) contacting said common nucleic acid tails and said labeled moiety.

In still yet another embodiment, the methods of the invention include the steps of : (a) providing at least one nucleic acid coupled to a support; (b) providing a labeled moiety capable of being coupled with a ligase to said nucleic acid; (c) providing said ligase; and (d) coupling said labeled moiety to said nucleic acid using said ligase.

This invention also provides compounds of the formulas described herein.

Definitions "Alkyl"refers to a straight chain, branched or cyclic chemical group containing only carbon and hydrogen. Alkyl groups include, without limitation, ethyl, propyl, butyl, pentyl, cyclopentyl and 2-methylbutyl. Alkyl groups are unsubstituted or substituted with 1 or more substituents (e. g. , halogen, alkoxy, amino).

"Aryl"refers to a monovalent, unsaturated aromatic carbocyclic group.

Aryl groups include, without limitation, phenyl, naphthyl, anthryl and biphenyl.

Aryl groups are unsubstituted or substituted with 1 or more substituents (e. g. halogen, alkoxy, amino).

"Amido alkyl"refers to a chemical group having the structure -C (O) NR3R4-, wherein R3 is hydrogen, alkyl or aryl, and R4 is alkyl or aryl.

Preferably, the amido alkyl group is of the structure-C (O) NH (CH2) nR5-, wherein n is an integer ranging from about 2 to about 10, and Rs is 0, NR6, or C (O), and wherein R6 is hydrogen, alkyl or aryl. More preferably, the amido alkyl group is of the structure-C (O) NH (CH2) nN (H)-, wherein n is an integer ranging from about 2 to about 6. Most preferably, the amido alkyl group is of the structure-C (O) NH (CH2) 4N (H)-.

"Alkynyl alkyl"refers to a chemical group having the structure -C=-C-R4-, wherein R4 is alkyl or aryl. Preferably, the alkynyl alkyl group is of

the structure-C#C- (CH2) nR5-, wherein n is an integer ranging from 1 to about 10, and R5 is O, NR6 or C (O), wherein R6 is hydrogen, alkyl or aryl. More preferably, the alkynyl alkyl group is of the structure-C#C- (CH2),, N (H) -, wherein n is an integer ranging from 1 to about 4. Most preferably, the alkynyl alkyl group is of the structure-C=-C-CH2N (H) -.

"Alkenyl alkyl"refers to a chemical group having the structure -CH=CH-R4-wherein R4is alkyl or aryl. Preferably, the alkenyl alkyl group is of the structure-CH=CH- (CH2) nR5-, wherein n is an integer ranging from 1 to about 10, and Rs is 0, NR6 or C (O), wherein R6 is hydrogen, alkyl or aryl. More preferably, the alkenyl alkyl group is of the structure-CH=CH- (CH2) nN (H)-, wherein n is an integer ranging from 1 to about 4. Most preferably, the alkenyl alkyl group is of the structure-CH=CH-CH2N (H)-.

"Functionalized alkyl"refers to a chemical group of the structure - (CH2) nR7-, wherein n is an integer ranging from 1 to about 10, and R7 is 0, S, NH or C (O). Preferably, the functionalized alkyl group is of the structure - (CH2)"C (O)-, wherein n is an integer ranging from 1 to about 4. More preferably, the functionalized alkyl group is of the structure-CH2C (O)-.

"Alkoxy"refers to a chemical group of the structure-O (CH2) nR8-, wherein n is an integer ranging from 2 to about 10, and R8 is 0, S, NH or C (O).

Preferably, the alkoxy group is of the structure-O (CH2) nC (O)-> wherein n is an integer ranging from 2 to about 4. More preferably, the alkoxy group is of the structure-OCH2CH2C (O)-.

"Thio"refers to a chemical group of the structure-S (CH2) nR8-, wherein n is an integer ranging from 2 to about 10, and R8 is O, S, NH or C (O).

Preferably, the thio group is of the structure-S (CH2) nC (O)-, wherein n is an integer ranging from 2 to about 4. More preferably, the thio group is of the structure-SCH2CH2C (O)-.

"Amino alkyl"refers to a chemical group having an amino group attached to an alkyl group. Preferably an amino alkyl is of the structure-NH (CH2) nNH-, wherein n is an integer ranging from about 2 to about 10. More preferably it is of

the structure-NH (CH2) nNH-, wherein n is an integer ranging from about 2 to about 4. Most preferably, the amino alkyl group is of the structure - NH (CH2) 4NH-.

"Nucleic acid"refers to a polymer comprising 2 or more nucleotides and includes single-, double-and triple stranded polymers. "Nucleotide"refers to both naturally occurring and non-naturally occurring compounds and comprises a heterocyclic base, a sugar, and a linking group, preferably a phosphate ester. For example, structural groups may be added to the ribosyl or deoxyribosyl unit of the nucleotide, such as a methyl or allyl group at the 2'-O position or a fluoro group that substitutes for the 2'-O group. The linking group, such as a phosphodiester, of the nucleic acid may be substituted or modified, for example with methyl phosphonates or 0-methyl phosphates. Bases and sugars can also be modified, as is known in the art. "Nucleic acid, "for the purposes of this disclosure, also includes"peptide nucleic acids"in which native or modified nucleic acid bases are attached to a polyamide backbone.

The phrase"coupled to a support"means bound directly or indirectly thereto including attachment by covalent binding, hydrogen bonding, ionic interaction, hydrophobic interaction, or otherwise.

"Probe"refers to a nucleic acid that can be used to detect, by hybridization, a target nucleic acid. Preferably, the probe is complementary to the target nucleic acid along the entire length of the probe, but hybridization can occur in the presence of one or more base mismatches between probe and target.

"Perfect match probe"refers to a probe that has a sequence that is perfectly complementary to a particular target sequence. The test probe is typically perfectly complementary to a portion (subsequence) of the target sequence. The perfect match (PM) probe can be a"test probe", a"normalization control"probe, an expression level control probe and the like. A perfect match control or perfect match probe is, however, distinguished from a"mismatch control"or"mismatch probe. "In the case of expression monitoring arrays, perfect match probes are typically preselected (designed) to be complementary to particular sequences or subsequences of target nucleic acids (e. g. , particular

genes). In contrast, in generic difference screening arrays, the particular target sequences are typically unknown. In the latter case, prefect match probes cannot be preselected. The term perfect match probe in this context is to distinguish that probe from a corresponding"mismatch control"that differs from the perfect match in one or more particular preselected nucleotides as described below.

"Mismatch control"or"mismatch probe", in expression monitoring arrays, refers to probes whose sequence is deliberately selected not to be perfectly complementary to a particular target sequence. For each mismatch (MM) control in a high-density array there preferably exists a corresponding perfect match (PM) probe that is perfectly complementary to the same particular target sequence. In"generic" (e. g., random, arbitrary, haphazard, etc.) arrays, since the target nucleic acid (s) are unknown perfect match and mismatch probes cannot be a priori determined, designed, or selected. In this instance, the probes are preferably provided as pairs where each pair of probes differ in one or more preselected nucleotides. Thus, while it is not known a priori which of the probes in the pair is the perfect match, it is known that when one probe specifically hybridizes to a particular target sequence, the other probe of the pair will act as a mismatch control for that target sequence. It will be appreciated that the perfect match and mismatch probes need not be provided as pairs, but may be provided as larger collections (e. g., 3.4, 5, or more) of probes that differ from each other in particular preselected nucleotides. While the mismatch (s) may be located anywhere in the mismatch probe, terminal mismatches are less desirable as a terminal mismatch is less likely to prevent hybridization of the target sequence.

In a particularly preferred embodiment, the mismatch is located at or near the center of the probe such that the mismatch is most likely to destabilize the duplex with the target sequence under the test hybridization conditions. In a particularly preferred embodiment, perfect matches differ from mismatch controls in a single centrally-located nucleotide.

"Labeled moiety"refers to a moiety capable of being detected by the various methods discussed herein or known in the art.

The group A is either hydrogen or a functional group that permits the attachment of a nucleic acid labeling compound to a nucleic acid. Nonlimiting

examples of such groups include the following : monophosphate; diphosphate; triphosphate (H409P) ; phosphoramidite ((R2N) (R'O) P), wherein R is linear, branched or cyclic alkyl, and R'is a protecting group such as 2-cyanoethyl; and H-phosphonate (HP (O) O-HNR3), wherein R is linear, branched or cyclic alkyl.

The heterocyclic group (Hc) is a cyclic moiety containing both carbon and a heteroatom. Nonlimiting examples of heterocyclic groups contemplated by the present invention are shown in FIG. 2.: 4-aminopyrazolo [3,4-d] pyrimidine; pyrazolo [3,4-d] pyrimidine; 1, 3-diazole (imidazole); 1, 2,4-triazine-3-one ; 1,2, 4- triazine-3,5-dione ; and, 5-amino-1, 2,4-triazine-3-one.

The linker moiety (L) of the nucleic acid labeling compound is covalently bound to the heterocycle (He) at one terminal position. It is attached to the detectable moiety (Q) at another terminal position, either directly or through a connecting group (M). It is of a structure that is sterically and electronically suitable for incorporation into a nucleic acid. Nonlimiting examples of linker moieties include amido alkyl groups, alkynyl alkyl groups, alkenyl alkyl groups, functionalized alkyl groups, alkoxyl groups, thio groups and amino alkyl groups.

The detectable moiety (Q) is a chemical group that provides an signal.

The signal is detectable by any suitable means, including spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. In certain cases, the signal is detectable by 2 or more means.

The detectable moiety provides the signal either directly or indirectly. A direct signal is produced where the labeling group spontaneously emits a signal, or generates a signal upon the introduction of a suitable stimulus. Radiolabels, such as 3H, l25I, 35S, I4C or 32p, and magnetic particles, such as DynabeadsTM, are nonlimiting examples of groups that directly and spontaneously provide a signal.

Labeling groups that directly provide a signal in the presence of a stimulus include the following nonlimiting examples: colloidal gold (40-80 nm diameter), which scatters green light with high efficiency; fluorescent labels, such as fluorescein, texas red, rhodamine, and green fluorescent protein (Molecular Probes, Eugene, Oregon), which absorb and subsequently emit light; chemiluminescent or bioluminescent labels, such as luminol, lophine, acridine

salts and luciferins, which are electronically excited as the result of a chemical or biological reaction and subsequently emit light; spin labels, such as vanadium, copper, iron, manganese and nitroxide free radicals, which are detected by electron spin resonance (ESR) spectroscopy; dyes, such as quinoline dyes, triarylmethane dyes and acridine dyes, which absorb specific wavelengths of light; and colored glass or plastic (e. g. , polystyrene, polypropylene, latex, etc.) beads. See U. S. Patent Nos. 3,817, 837; 3,850, 752; 3,939, 350; 3,996, 345; 4,277, 437; 4,275, 149 and 4,366, 241.

A detectable moiety provides an indirect signal where it interacts with a second compound that spontaneously emits a signal, or generates a signal upon the introduction of a suitable stimulus. Biotin, for example, produces a signal by forming a conjugate with streptavidin, which is then detected. See Hybridization With Nucleic Acid Probes. In Laboratory Techniques in Biochemistry and Molecular Biology ; Tijssen, P. , Ed.; Elsevier: New York, 1993; Vol. 24. An enzyme, such as horseradish peroxidase or alkaline phosphatase, that is attached to an antibody in a label-antibody-antibody as in an ELISA assay, also produces an indirect signal.

A preferred detectable moiety is a fluorescent group. Flourescent groups typically produce a high signal to noise ratio, thereby providing increased resolution and sensitivity in a detection procedure. Preferably, the fluorescent group absorbs light with a wavelength above about 300 nm, more preferably above about 350 nm, and most preferably above about 400 nm. The wavelength of the light emitted by the fluorescent group is preferably above about 310 nm, more preferably above about 360 nm, and most preferably above about 410 nm.

The fluorescent detectable moiety is selected from a variety of structural classes, including the following nonlimiting examples: 1-and 2- aminonaphthalene, p, p'diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9-aminoacridines, p, p'-diaminobenzophenone imines, anthracenes, oxacarbocyanine, marocyanine, 3-aminoequilenin, perylene, bisbenzoxazole, bis- p-oxazolyl benzene, 1,2-benzophenazin, retinol, bis-3-aminopridinium salts, hellebrigenin, tetracycline, sterophenol, benzimidazolyl phenylamine, 2-oxo-3- chromen, indole, xanthen, 7-hydroxycoumarin, phenoxazine, salicylate,

strophanthidin, porphyrins, triarylmethanes, flavin, xanthene dyes (e. g., fluorescein and rhodamine dyes); cyanine dyes; 4, 4-difluoro-4-bora-3a, 4a-diaza- s-indacene dyes and fluorescent proteins (e. g. , green fluorescent protein, phycobiliprotein).

A number of fluorescent compounds are suitable for incorporation into the present invention. Nonlimiting examples of such compounds include the following: dansyl chloride; fluoresceins, such as 3,6-dihydroxy-9- phenylxanthhydrol; rhodamineisothiocyanate; N-phenyl-l-amino-8- sulfonatonaphthalene ; N-phenyl-2-amino-6-sulfonatonaphthanlene ; 4-acetamido- 4-isothiocyanatostilbene-2, 2'-disulfonic acid; pyrene-3-sulfonic acid; 2- toluidinonapththalene-6-sulfonate ; N-phenyl, N-methyl 2-aminonaphthalene-6- sulfonate ; ethidium bromide; sterine ; auromine-0, 2- (9'-anthroyl) palmitate; dansyl phosphatidylethanolamin; N, N'-dioctadecyl oxacarbocycanine; N, N'- dihexyl oxacarbocyanine ; merocyanine, 4- (3'-pyrenyl) butryate; d-3- aminodesoxy-equilenin; 12- (9'-anthroyl) stearate; 2-methylanthracene; 9- vinylanthracene; 2, 2'- (vinylene-p-phenylene) bisbenzoxazole; p-bis [2- (4-methyl- 5-phenyl oxazolyl) ] benzene; 6-dimethylamino-1, 2-benzophenzin; retinol; bis (3'- aminopyridinium)-1, 10-decandiyl diiodide; sulfonaphthylhydrazone of hellibrienin; chlorotetracycline; N- (7-dimethylamino-4-methyl-2-oxo-3- chromenyl) maleimide; N- [p- (2-benzimidazolyl) phenyl] maleimide; N- (4- fluoranthyl) maleimide; bis (homovanillic acid); resazarin; 4-chloro-7-nitro-2, 1, 3- benzooxadizole; merocyanine 540; resorufin ; rose bengal and 2,4-diphenyl- 3 (2H) -furanone. Preferably, the fluorescent detectable moiety is a fluorescein or rhodamine dye.

Another preferred detectable moiety is colloidal gold. The colloidal gold particle is typically 40 to 80 nm in diameter. The colloidal gold may be attached to a labeling compound in a variety of ways. In one embodiment, the linker moiety of the nucleic acid labeling compound terminates in a thiol group (-SH), and the thiol group is directly bound to colloidal gold through a dative bond. See Mirkin et al. Nature 1996, 382, 607-609. In another embodiment, it is attached indirectly, for instance through the interaction between colloidal gold conjugates of antibiotin and a biotinylated labeling compound. The detection of the gold

labeled compound may be enhanced through the use of a silver enhancement method. See Danscher et al. J Histotech 1993, 16, 201-207.

The connecting groups (M) m can serve to covalently attach the linker group (L) to the detectable moiety (Q). Each M group can be the same or different and can independently be any suitable structure that will not interfere with the function of the labeling compound. Nonlimiting examples of M groups include the following: amino alkyl,-CO (CH2) sNH-,-CO-,-CO (O)-, - CO (NH) -, -CO (CH2) sNHCO (CH2) 5NH-,-NH (CH2CH20) kNH-,- NH (CH2CH20) kCH2CH2NHand-CO (CH2) 5- ; wherein, k is an integer from 1 to about 5, preferably k is 1 or 2; m is an integer ranging from 0 to about 5, preferably 0 to about 3.

In another embodiment L comprises a vinyl group (-CH=CH-) attached at its 1 position directly to the N of the base. In accordance with this aspect of the present invention, where L is vinyl, further substituents are attached to the 2 position of the vinyl groups such as such as for example Rl l (-CH=CH-Rl l).

Preferably Rll is alkyl, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, alkoxy, thio and amino alkyl. Most preferablbly Rll is C (O) R12, where R12 is a bond, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl alkoxy, thio and amino alkyl.

Importantly, as disclosed in accordance with one aspect of the present invention, the linker group L is selected to provide a linking function, which either alone or in conjunction with appropirate connecting groups (M) appropriately spaces the Q group from the Hc group (nucleotide base) at such a length and in such a configuration as to allow an appropriate assay to be performed on the Q group, but at the same time substantially preserving the ability of the nucleic acid labeling compound to act as a substrate for the appropriate enzyme, e. g. , terminal transferase and/or RNA polymerase. Those of skill in the art will also appreciate that the chosen Hc-L- (M) m-Q groups must not adversely impact the ability of a nucleic acid strand incorporating such group to undergo Watson-Crick type base pairing with complementary sequences.

In accordance with one aspect of the present invention, R12 is preferably a bond, Y is OH; Z is OH; L is- (CH2) 2C (O)-, and Q is selected from the group consisting of a fluorescein and a biotin; and a first M is-NH (CH2) 2NH-, and a second M is-CO (CH2) 5NH-, wherein m is 2.

Specific A groups are H or H409P3-.

A specific X group is O.

A specific Y group is H or OR9, A specific Z group is H, N3, F or ORlo.

More specific Y groups are hydrogen or OH.

More specific Z groups are hydrogen or OH.

A more specific Y group is OH.

A more specific Z group is OH.

Specific Rg groups are H, alkyl or aryl.

Specific Rio groups are H, alkyl or aryl.

A more specific Rg group is hydrogen.

A more specific Rio group is hydrogen.

Specific L groups are-CH=CH-Rll, or C-=C-Rll.

A more specific L group is-CH=CH-Rl, A more specific L group is-CH=CH-C (O)-.

Specific Rll groups are-O-, amino, thio, carbonyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, and amino alkyl.

More specific Ru groups are alkyl, alkoxy, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, thio and amino alkyl.

More specific Rll group is C (O) R12.

Specific R12 groups are a bond, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, alkoxy, thio and amino alkyl.

A more specific R12 group is a bond.

Specific Q groups are a hapten, a fluorophore, a metal chelator, an intercalator, a luminescent moiety, a metal aggregate, and a protein.

A more specific Q group is biotin or a fluorescent dye A more specific Q group is a biotin.

A specific biotin has the structure:

A specific M group is-NH (CH2) nNH-, or-CO (CH2) pNH- ; wherein n is an integer from about 2 to about 10; and p is an interger from about 2 to about 10.

A more specific M group is-NH (CH2) 2NH-, or-CO (CH2) sNH-.

A specific m is 2.

A specific A group is a triphosphate group having appropriate counterions.

Specific counterions are H+, Na+, Li+, K+, or NH4+.

A specific compound of the invention has the structure: or

In another embodiment, wherein A is a functional group the permits the attachment of the nucleic acid labeling compound to a nucleic acid, preferably A is a triphosphate group with apporpriate counterions. The counterions are selected from the group consisting of are H+, Na+, Li+, K+, or NH4+ ; X is O ; Y is OH; Z is OH; L is selected from the group consisting of-CH=CH-C (O)-NH- CH2-CH2-NH-C (O)-and-CH2-CH2-C (O)-NH-CH2-CH2-NH-C (O) ; M is- (CH2) 5-NH- and Q is biotin having the structure: In another embodiment, A is H or H409P3- ; X is O ; Y is H or OR9, wherein Rg is H, alkyl or aryl; Z is H, N3, F or ORIO, wherein Rio is H, alkyl or aryl; L is selected from the group consisting of-CH=CH-C (O)- and-CH=CH- CH (NH2)- ; Q is biotin or a fluorescein; and, a first M is-NH (CH2)"NH-, wherein n is an integer from about 2 to about 10, and a second M is-CO (CH2) pNH-, wherein p is an interger from about two to about 10 and m is 2.

In another embodiment, Y is H or OH; Z is H or OH; L is-CH=CH- C (O)-, Q is biotin or a carboxyfluorescein; and a first M is-NH (CH2) 2NH-, and a second M is-CO (CH2) 5NH-, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is-CH=CH-C (O)-, Q is a biotin or a carboxyfluorescein; and, a first M is-NH (CH2) 2NH-, and a second M is-CO (CH2) SNH-, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is-CH=CH-C (O)-, Q is or biotin; and, a first M is-NH (CH2) 2NH-, and a second M is-CO (CH2) 5NH-, wherein m is 2.

In yet another embodiment L comprises a vinyl group (-CH=CH-) attached at its 1 position directly to the N of the base. In accordance with this aspect of the present invention, where L is vinyl, further substituents may be attached to the 2 position of the vinyl groups such as such as for example Rl i (-CH=CH-Rll). Preferably Ri I is alkyl, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, alkoxy, thio and amino alkyl. Most preferablbly Rll is C (O) RI2 where R12 is a bond, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl alkoxy, thio and amino alkyl.

Importantly, as disclosed in accordance with one aspect of the present invention, the linker group L is selected to provide a linking function, which either alone or in conjunction with appropriate connecting group (M), provide appropriate spacing of the Q group from the Hc or base group at such a length and in such a configuration as to allow an appropriate assay to be performed on the Q group, but at the same time substantially preserving the ability of the nucleic acid labeling compound to act as a substrate for the appropriate enzyme, e. g. , terminal transferase and/or RNA polymerase. Those of skill in the art will also appreciate that the Hc-L- (M) m-Q groups must be chosen, in accordance with the present invention, to avoid subtantially inhibiting the ability of a nucleic acid strand incorporating such group to undergo Watson-Crick type base pairing with complementary sequences. Thus,-L- (M) m-Q may be any arrangements or grouping of molecules or atoms which functions to allow nucleic acids to be labeled and detected.

In accordance with one aspect of the present invention, R12 is preferably a bond, Y is OH; Z is OH; L is- (CH2) 2C (O)-, and Q is selected from the group consisting of a fluorescein and a biotin; and a first M is-NH (CH2) 2NH-, and a second M is-CO (CH2) 5NH-, wherein m is 2.

In another embodiment, A is H or H409P3- ; X is O ; Y is H or OR9, wherein Rg is H, alkyl or aryl; Z is H, N3, F or ORIO, wherein Rio is H, alkyl or aryl; ; L is-(CH2) nC (O)-7 wherein n is an integer ranging from about 1 to about

10; Q is biotin or a fluorescein; and, a first M is-NH (CH2) nNH-7 wherein n is an integer from about 2 to about 10, and a second M is-CO (CH2) sNH-, wherein m is 2.

In another embodiment, Y is H or OH; Z is H or OH; L is-(CH2) 2C (O)-, Q is biotin or a carboxyfluorescein; and a first M is-NH (CH2) 2NH-, and a second M is-CO (CH2) sNH-, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is- (CH2) 2C (O)-, Q is a carboxyfluorescein; and, a first M is-NH (CH2) 2NH-, and a second M is - CO (CH2) 5NH-, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is- (CH2) 2C (O)-, Q is biotin; and, a first M is-NH (CH2) 2NH-, and a second M is-CO (CH2) 5NH-, wherein m is 2.

In another embodiment, wherein A is a functional group the permits the attachment of the nucleic acid labeling compound to a nucleic acid; preferably, A is a triphosphate group with appolpriate counterions, said counterions selected from the group consisting of H+, Na+, Li+, K+, and NH4+; X is O ; Y is OH; Z is OH; L is selected from the group consisting of-CH=CH-C (O)-NH-CH2-CH2- NH-C (O)- and-CH2-CH2-C (O)-NH-CH2-CH2-NH-C (O) ; M is- (CH2) 5-NH- and Q is biotin having the structure: In another embodiment, A is H or H4O9P3- ; X is O ; Y is H or OR9, wherein Rg is H, alkyl or aryl; Z is H, N3, F or ORzo, wherein Rio is H, alkyl or aryl; L is-CH=CH-C (O)- ; Q is biotin or a fluorescein; and, a first M is -NH(CH2)nNH-, wherein n is an integer from about 2 to about 10, and a second M is-CO (CH2) pNH-, wherein p is an interger from about 2 to about 10 and m is 2.

In another embodiment, Y is H or OH; Z is H or OH; L is-CH=CH- C (O)-, Q is biotin or a carboxyfluorescein; and a first M is-NH (CH2) 2NH-, and a second M is-CO (CH2) 5NH-, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is-CH=CH-C (O)-, Q is a biotin or a carboxyfluorescein ; and, a first M is-NH (CH2) 2NH-, and a second M is-CO (CH2) 5NH-, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is-CH=CH-C (O)-, Q is biotin; and, a first M is-NH (CH2) 2NH-, and a second M is-CO (CH2) 5NH-, wherein m is 2.

In another embodiment L comprises a vinyl group (-CH=CH-) attached at its 1 position directly to the N of the base. In accordance with this aspect of the present invention, where L is vinyl, further substituents are attached to the 2 position of the vinyl groups such as such as for example Rll (-CH=CH-Rll).

Preferably Rl l is alkyl, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl, alkoxy, thio and amino alkyl. Most preferablbly Rl l is C (O) Rl2, where R12 is a bond, aryl, functionalized alkyl, amido alkyl, alkenyl alkyl alkoxy, thio and amino alkyl.

Importantly, as disclosed in accordance with one aspect of the present invention, the linker group L is selected to provide a linker function, which either alone or in conjunction with appropriate connecting groups (M), appropriately spaces the Q group from the Hc or base group at such a length and in such a configuration as to allow an appropriate assay to be performed on the Q group, but at the same time substantially preservers the ability of the nucleic acid labeling compound to act as a substrate for the appropriate enzyme, e. g. , terminal transferase and/or RNA polymerase. Those of skill in the art will also appreciate that the chosen Hc-L- (M) m-Q groups must be chosen in accordance with the present invention, to avoid subtantially inhibiting the ability of a nucleic acid strand incorporating such group to undergo Watson-Crick type base pairing with complementary sequences. Thus,-L- (M) m-Q may be any arrangements or gourping of molecules or atoms which functions to allow nucleic acids to be labeled and detected.

In accordance with one aspect of the present invention, Rl2 is preferably a

bond, Y is OH; Z is OH; L is- (CH2) 2C (O)-, and Q is selected from the group consisting of a fluorescein and a biotin; and a first M is-NH (CH2) 2NH-, and a second M is-CO (CH2) sNH-, wherein m is 2.

Nucleic Acid Labeling Nucleic acids can be isolated from a biological sample or synthesized, on a solid support or in solution for example, according to methods known to those of skill in the art. As used herein, there is no theoretical limitation on the length or source of the nucleic acid used in the labeling process. Limitations on length may, however, be imposed or suggested by the hybridization process. Exemplary methods of nucleic acid isolation and purification are described in Theory and Nucleic Acid Preparation. In Laboratory Techniques in Biochemist1y and Molecular Biology : Hybridization With Nucleic Acid Probes ; P. Tijssen, Ed.; Part 1 ; Elsevier: N. Y. , 1993. A preferred method of isolation involves an acid guanidinium-phenol-chloroform extraction followed by oligo dT column chromotography or (dT) n magnetic bead use. Sambrook et al. Molecular Cloning : A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory, 1989; Vols. 1-3; and Current Protocols in Molecular Biology ; F. Ausubel et al. Eds.; Greene Publishing and Wiley Interscience: N. Y. , 1987.

In certain cases, the nucleic acids are increased in quantity through amplification. Suitable amplification methods include, but are not limited to, the following examples: polymerase chain reaction (PCR) (Innis, et al. PCR Protocols. A guide to Methods and Application ; Academic Press: San Diego, 1990); ligase chain reaction (LCR) (Wu and Wallace. Genomics 1989, 4, 560; Landgren, et al. Science 1988, 241, 1077; and Barringer, et al. Gene 1990, 89, 117); transcription amplification (Kwoh et al. Proc. Natl. Acad. Sci. USA 1989, 86, 1173); and self-sustained sequence replication (Guatelli, et al. Proc. Nat.

Acad. Sci. USA 1990, 87, 1874). Each of these references is hereby incorporated by reference.

The nucleic acid labeling compound can be incorporated into a nucleic acid using a number of methods. For example, it can be directly attached to an

original nucleic acid sample (e. g., mRNA, polyA mRNA, cDNA) or to an amplification product. Methods of attaching a labeling compound to a nucleic acid include, without limitation, nick translation, 3-end-labeling, ligation, in vitro transcription (IVT) or random priming. Where the nucleic acid is an RNA, a labeled riboligonucleotide is ligated, for example, using an RNA ligase such asT4 RNA Ligase. See The Enzymes ; Uhlenbeck and Greensport, Eds.; Vol. XV, Part B, pp. 31-58; and, Sambrook et al. , pp. 5.66-5. 69 (incorporated here by reference). Terminal transferase is used to add deoxy-, dideoxy-or ribonucleoside triphosphates (dNTPs, ddNTPs or NTPs), for example, where the nucleic acid is single stranded DNA.

The labeling compound can also be incorporated at an internal position of a nucleic acid. For example, PCR in the presence of a labeling compound provides an internally labeled amplification product. See, e. g. , Yu et al. Nucleic Acids Research 1994,22, 3226-3232 (incorporated by reference). Similarly, IVT in the presence of a labeling compound can provide an internally labeled nucleic acid.

Probe Hybridization The nucleic acid to which the labeling compound is attached can be detected after hybridization with a nucleic acid probe. Alternatively, the probe can be labeled, depending upon the experimental scheme preferred by the user.

The probe is a nucleic acid, or a modified nucleic acid, that is either attached to a solid support or is in solution. It is complementary in structure to the labeled nucleic acid with which it hybridizes. The solid support is of any suitable material, including polystyrene based beads and glass chips. In a preferred embodiment, the probe or target nucleic acid is attached to a glass chip, such as a GeneChip° product (Affymetrix, Inc. , Santa Clara, CA). See International Publication Nos. WO 97/10365, WO 97/29212, WO 97/27317, WO 95/11995, WO 90/15070, and U. S. Pat. Nos. 5,744, 305 and 5,445, 934 which are hereby incorporated by reference.

Because probe hybridization is often a step in the detection of a nucleic acid, the nucleic acid labeling compound must be of a structure that does not substantially interfere with that process. The steric and electronic nature of the labeling compound, therefore, is compatible with the binding of the attached nucleic acid to a complementary structure.

High-density DNA probe arrays are proving to be a valuable tool for hybridization-based genetic analysis. These assays require covalent labeling of nucleic acid molecules with fluorescent or otherwise detectable molecules in order to detect hybridization to the arrays. We have pursued a program to develop a set of novel nucleotide analogs for enzymatic labeling of nucleic acid targets for a variety of array-based assays. A primary goal of the invention is to provide new reagents for two particular labeling procedures: (i. ), 3'end labeling of fragmented, PCR-generated DNA targets with terminal deoxynucleotidyl transferase (TdT); and (ii. ), template-directed internal labeling of in vitro transcription-generated RNA targets with T7 RNA polymerase (T7).

EXAMPLES The following examples are offered to illustrate, but not to limit, the present invention.

General Experimental Details Reagents were purchased from Aldrich Chemical Company (Milwaukee, WI) in the highest available purity. All listed solvents were anhydrous.

Intermediates were characterized by 1H NMR and mass spectrometry.

The general approach taken was to screen various base-substituted nucleotide analogs, using a rapid and quantitative HPLC-based assay, to empirically determine which analogs were efficient substrates for the polymerase of interest. The analogs selected for this study were nucleotides in which the native heterocyclic base was substituted with the following: 1- (imidazole-4- carboxamide), 1- (1, 3,6-trazine-2, 4-dione), 5- (1, 3-pyrimidine-2,4-dione), 3- (pyrazalo- [4, 3-d] pyrimidine), 1- (pyrazalo- [3, 4-d] pyrimidine) and a simple carboxamide moiety. Labeled versions of promising candidate molecules were

then designed and synthesized for further testing of relative incoproation efficiency and functional performance in array-based assays.

It was determined that TdT was generally tolerant of base substitutions, and that ribonucleotides were about as efficiently incorporated as 2'-deoxy, and 2', 3'-dideoxynucleotides. In contrast, T7 was relatively intolerant of heterocyclic base substitutions with the exception of the 5- (1, 3-pyrimidine-2,4- dione), i. e. the pseudo-uridine analog. Two new reagents, a C4-labeled 1- (2', 3'- didexoy-P-D-ribofuranosyl) imidazole-4 carboxamide 5'-triphophate and an N1- labeled pseudo-uridine 5'-triphophate, were found to be excellent substrates for TdT andT7, respectively. These new analogs prove array assay performance equivalent to that obtained using conventional labeling reagents.

Using the guidance provided herein, the methods for the synthesis of reagents and methods (enzymatic or otherwise) of label incorporation useful in practicing the invention will be apparent to those skilled in the art. See, for example, Chemistry of Nucleosides and Nucleotides 3, Townsend, L. B. ed., Plenum Press, New York, at chpt. 4, Gordon, S. The Synthesis and Chemistry of Imidazole and Benzamidizole Nucleosides and Nucleotides (1994); Gen Chem.

Chemistry of Nucleosides and Nucleotides 3, Townsend, L. B. ed. , Plenum Press, New York (1994); can be made by methods simliar to those set forth in Chemistry of Nucleosides and Nucleotides 3, Townsend, L. B. ed., Plenum Press, New York, at chpt. 4, Gordon, S. "The Synthesis and Chemistry of Imidazole and Benzamidizole Nucleosides and Nucleotides (1994); Lopez-Canovas, L. Et al., Arch. Med. Res 25: 189-192 (1994) ; Li, X. , et al., Cytometry 20 : 172-180 (1995); Boultwood, J. Et al., J ; Pathol. 148: 61 ff. (1986); Traincard, et al., Ann.

Immunol. 1340: 399-405 (1983).

Example 1 Procedure for HPLC Analysis of Enzymatic Incorporation of Modified Nucleotides.

Reaction Conditions TdT 3 uM dT 16 template

15 (30) uM NTP 40 U TdT (Promega) 1X buffer, pH 7.5 (Promega) Procedure: Incubate lhr. at 37 °C, then for 10 min. at 70 C, followed by the addition of EDTA (2 mM final concentration) in a volume of 50 pL HPLC Analysis Materials and Reagents 4.6 mm X 250 mm Nucleopac PA-100 ion-exchange column (Dionex) buffer A: 20 mM NaOH (or 20 mM Tris pH 8, in the case of TdT incorporation of nucleotide triphoshates that are not dye-labeled) buffer B: 20mM NaOH, 1M NaCl (or 20 mM Tris pH 8,1M NaCl, in the case of TdT incorporation of nucleotide triphoshates that are not dye-labeled).

General Procedure Dilute the reaction with 50 pLL of buffer A. Inject 50 uL of this sample onto the HPLC column and fractionate using a gradient of 5 to 100% buffer B over 30 minutes at a flow rate of 1 mL/min.. Detect the peaks simultaneously at 260 nm absorbance and the absorbance maximum of the dye (or the fluorescence emission maximum of the dye).

The incorporation efficiency is expressed as the fraction of oligonucleotide that is labeled. This number is determined by dividing the peak area measured at 260 nm absorbance of the labeled oligonucleotide by the sum of the peak areas of the unlabeled and labeled oligonucleotide. (The retention time of fluorescein-labeled dTl6 is on the order of 2 to 3 min. longer than the unlabeled dTl6.) The error in this type of assay is about 10%.

Example 2 Synthesis of NI-labeled 5- (, (3 D-ibofuf°anosyl)-2, 4 (1H, 3H)-pyrimidinedione 5'- triphosphate 42a and 42b (Figure 3).

To 5-(ß-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 39 (100mg, 0.41 mmol, leq.) in acetonitrile (5ml) was added 1 M TEAB, pH 9 (5ml) followed by methyl acrylate (5. 5ml, 61 mmol, 150eq). The reaction was stirred at room temperature overnight. The solvents were evaporated, and the residue was coevaporated with water (3X, 5ml) yielding 135 mg of acrylate 40. The acrylate 40 was then treated with neat ethylenediamine (2ml, excess) and two drops of TEA and heated to 100 °C. After 1 hour the excess EDA was evaporated, yielding 146 mg of the free amine (quantitative). The crude residue was then co-evaporated with pyridine (3x, 5ml, insoluble), resuspended in a mixture of pyridine and DMF and was cooled to 0° C. To this mixture was added TFA-imidazole (73.8mg, 1. 1 eq. ). The reaction was then allowed to warm to room temperature and stirred overnight. An additional 1 eq. of TFA-imidazole was added at this time and the reaction was stirred an additional 15 minutes. The solvent was then evaporated, and the residue was co-evaporated with water (2x, 5ml) and dissolved in 5ml of water. The white precipitate that formed was removed by filtration. The mother liquor, which contained the TFA-protected nucleoside 3, was separated into two aliquots and purified by reverse phase HPLC. The fractions were then pooled and evaporated to yield 20% (35mg) of pure 41, which was verified by'H NMR. Using standard procedures (eg. , Prober, et al. , EP 0252683), compound 41 was converted to the triphosphate, which was then conjugated to biotin and fluorescein to afford 42a and 42b.

Synthesis of the Nl-labeled 2-amino-5-(ß-D-ribofuranosyl)-4 (1H)- pyrimidinone, 55, involved alkylation at N1 using conditions similar to those described by Muehlegger, et al. (WO 96/28640) for the N1-alkylation of pyrazalo- [4, 3-d] pyrimidines (Scheme 2).

The IVT incorporation efficiency (the number of labeled analogs incorporated per transcript) of the Nl-fluorescein-X-5- (P-D-ribofuranosyl)- 2,4 (1H, 3H) -pyrimidinedione 5'-triphosphate 42a was measured by HPLC (diode

array UV detection at 260 nm and 495 nm) in an IVT amplification of a 1.24 kb transcript. See US patent application SN 09/126,645 for additional details on test methods used. Chart 1 summarizes the data obtained using different ratios of UTP/5 At a ratio of 1: 5, the incorporation and relative yield (measured relative to the yield obtained with UTP only) of transcript are optimal. This transcript was compared in a hybridization assay to transcript labeled using fluorescein. The preliminary results indicated that the Nl-fluorescein-X-5-(ß-D-ribofuranosyl)- 2,4 (1H, 3H) -pyrimidinedione 5'-triphosphate (42a) performed equivalently in a hybridization assay in terms of number of correct calls and in hybridization intensity (Charts 2 and 3). The hybridization assay used for this purpose was the Affymetrix HIV-PRT GeneChip assay (see Kozal, et al. Nature Medicine 1996, 2: 753-9.).

Similarly, the efficiency of DNA 3'-end labeling of a polythymidylate oligonucleotide (Tie) using terminal deoxynucleotidyl transferase and N1- fluorescein and biotin-labeled 5-(ß-D-ribofuranosyl)-2, 4 (1H, 3H) - pyrimidinedione 5'-triphosphate, was determined by HPLC. In this analysis, the percent conversion of oligo-T16 to the 3'-end labeled T16-Fl, is determined by AX-HPLC (see US patent application SN 09/126,645 for detailed procedures).

The data is summarized in Chart 4. The incorporation of the biotin and fluorescein triphosphates was very efficient as determined by HPLC.

Scheme 2 NH2 NH, NH2 N NH, 4 N N /I H O BTMSA TMSO/ O DMF TMSO 0 DMF 0 HOH OH H' TM80 OTMS 51 52 NH2 o , N NNOCH3 HO ho 2) H+/MeOH > I OH OH 53 NH2 0 NHz o 1) EDA, heat NEZ 2) TFA-Im, 0°C 0 H HO 0 H 4 OH 0 54 NH- n i H 1) phosphorylation N A N NHFI v _N 2) NH40H Ovls p H O 3) Fluorescein-X-NHS H4P309-0 A i O OH OH 0 55 Chart 1. Incorporation efficiency ofNI-fluorescein-labeled 5- (, B-D- ribofuranosyl)-2, 4 (1H, 3H)-pyrimidinedione 5'-triphosphate 42a, determined by HPLC

The labeling reaction conditions are the standard conditions used in the Affymetrix HIV-PRT GeneChip product assay (see Kozal, et al. Nature Medicine 1996,2 : 753-9.).

Chart 2. Call accuracy of N1-fluorescein-labeled 5-(ß-D-ribofuranosyl)- 2, 4 (IH, 3H)-pyrimidinedione 5'-triphosphate 42a.

Data was obtained from Affymetrix HIV-PRT GeneChip hybridization assay (see Kozal, et al. Nature Medicine 1996,2 : 753-9.).

Chart 3. Hybridization signal offluorescein labeled triphosphate 42a

Data obtained from hybridization of labeled transcript to the Affymetrix HTV-PRT GeneChip array (see Kozal, et al. Nature Medicine 1996,2 : 753-9.).

Chart 4. TdT labeling efficiency of Fluorescein and Biotin labeled 5-(ß-D- ribofuranosyl)-2, 4(1H,3H)-pyrimidinedione 5'-triphosphate 42a and 42b, determined by HPLC.

Reaction conditions: TdT (40 units), 20 uM U*TP and 3.2 uM T16 oligo in 50 ul of water. Heated at 37°C for 1 hour and 70°C for 10 min. , followed by 1 ul of 100 mM EDTA. HPLC analysis was performed on a Dionex DNAPac PA-100 column.

Example 3: Synthesis of Biotin-#isoCTP, propenamide-linked (RLR-3b) (Scheme 3) Scheme 3

tCI SHAc WHAc MsC) NHAc NHA& l N : , .-zfiN f NH N : (V hl , rl, sli r ! M N.,,.,,,., N COMe ,--\0 1 2 3 23C NH2 ° N rt osCO2H N aN vN ~NH2 Ni2 0 1 2 3 NsCf NHz 0 , 5 , 0==\/EDC. pHS 0\ H 0-EDC, pH5-5 0 H HO OH HO tH HO'"OH HO OH J o 2 0 H O HN tt tP t3A ; H tM H M S BMms n S H J HOm tOv . : H 6 1'4H 0 HN-' t 2 0 H NH H H : 182 0 H 0 HNa }) 3 rSi A ~</>IY j N H HO tH 7

Peracetylated pseudoisocvtidine 2 Pseudoisocytidine (1) (2. 5g, 9mmoles) was dissolved in 40 ml dry pyridine. Acetic anhydride (8. 5 ml, 90 mmoles) was added and the mixture was stirred under argon for at least 4 hours at room temperature. The reaction can be monitored by HPLC (C18 column, buffer A: 0. 1M TEAA, pH 7.5 ; buffer B: acetonitrile; gradient: 5-95% B over 20 minutes). The pyridine was removed under vacuum and the residual oil was dissolved in 500 ml of ethyl acetate. More ethyl acetate may be added to get a clear solution since the product has limited solubility in ethyl acetate. The organic phase was washed three times with brine and dried over anhydrous Na2S04, filtered and the solvent removed. The white solid was recrystallized from ethyl acetate/hexane yielding 3.2 g (85%) of 2.

Propenoic acid methyl ester 3 Compound 2 (2.0 g, 4.86mmoles) and dimethylaminopyridine (1.2g, 9.73mmoles) were co-evaporated with 50 ml dry acetonitrile two times and then re-dissolved in 45 ml dry acetonitrile under argon. Methyl propiolate (0.82g, 0. 86ml, 9.73 mmoles) was added and the solution was stirred at room temperature for 24 hours. The reaction turned from a colorless to amber colored solution. The reaction was followed by HPLC until no more product was produced. The solvent was removed by rotary-evaporation and the residue was dissolved in 400 ml of ethyl acetate and 200 ml of brine. The aqueous layer was back extracted with two 200 ml-portions of ethyl acetate. The combined organic layer was dried over anhydrous Na2S04, filtered and the solvent removed. The residue was purified by flash column chromatography on silica gel (200 ml wet gel) using ethyl acetate as the eluent affording 850mg (35%) of 3 as a white foam.

Propenoic acid 4 Compound 3 (0.85g, 1. 7mmoles) was dissolved in chloroform (5ml) and aqueous concentrated hydrochloric acid (conc., 10 ml) was added. The rosy red solution turned a lemon yellow instantly. The reaction was stirred at room temperature for an additional 48 hours or until the reaction was complete as determined by RP-HPLC (C18 column, buffer A: 0. 1M TEAA, pH 7.5 ; B,

acetonitrile; gradient: 0% B for 9 minutes, 0 to 90% B over 10 minutes). The solvent and water were removed by rotary-evaporation. The product was purified by precipitation from methanol/acetonitrile and dried under vacuum to afford 500mg (94%) of 4.

Aminopropenamide 5 Compound 4 (500mg, 1.6 mmoles) and a buffered solution of ethylenediamine in water (8 ml of 2.0 M ethylenediamine in MES buffer, pH 5.5, containing 16 mmoles of EDA) were mixed and then 1-ethyl-3- (3- dimethylaminopropyl) carbodiimide (2g, 16 mmoles) was added to the reaction with vigorous stirring. After 1 hour the reaction was analyzed by LC/MS and determined to be complete. The compound was purified by preparative HPLC: PRP-1,30 x 250mm column; flow rate 25 ml/min; buffers: A, 0. 1M TEAA, pH 7.5, B, acetonitrile; gradient: 0% B for 9 minutes, 0 to 90% B over 10 minutes.

Salts were removed with a retention time of about 4 min. and the compound eluted from 6 to 7.5 minutes. The collected fractions were pooled and the solvent removed under vacuum. The residue which contained triethylammonium acetate was co-evaporated with water several times and finally the product was precipitated from methanol/acetonitrile to afford 290mg (51 %) of 5.

Biotin-propenamide 6 Compound 5 (280 mg, 0.79mmoles) was dissolved in dry DMF (5 ml) followed by the addition of triethylamine (160mg, 220 pi, 1. 58 mmoles). The pH of the solution was adjusted to 7.5 with the addition of more triethylamine, if necessary. Biotin-X-NHS ester (358 mg, 0.79 mmoles,) was then added to the mixture with stirring. After 1.5 hours the solvent volume was reduced under vacuum to about 1 ml. Caution: do no vacuum to dryness because this compound tends to aggregate and it will be difficult to redissolve. The compound was purified by preparative HPLC: PRP-1,30 x 250mm column; flow rate 25 ml/min ; buffers: A, 0. 1M TEAA, pH 7.5, B, acetonitrile; gradient: 0% B for 8 minutes, then 0 to 95% B over 20 minutes. Fractions were collected across the peak from 16-17 min and the solution of pooled fractions was quantitated for the presence of product spectrophotometrically (X289, assuming s = 8000). The

solvent was removed under vacuum and the residue was co-evaporated with water (30 ml) three times and methanol (50 ml) two times. The product was precipitated from methanol/acetonitrile yielding 379mg (69%) of 6.

Triphosphate Compound 6 (110mg, 0. 1585mmoles) was dried over P205 under vacuum for two days and then dissolved in trimethyl phosphate (dried over molecular sieves, 20 ml) with gentle heating to about 60°C. Once the material dissolved, the solution was cooled to ambient temperature and a trap-pack (ABI Trap-pak, P#GEN 084034) was added and the mixture was allowed to gently stir overnight.

The trap-pack was removed and to the solution at 0°C under argon was added POC13 (73 mg, 45, ul, 0.48 mmoles). The reaction was monitored by AX-HPLC for the conversion to the monophosphate, and after 4 hours, an additional 2 equivalents of POC13 were added and the reaction was allowed to stir for 2 more hours (or until 90% conversion was achieved).

While monitoring the reaction, a solution of dry tetra (tri-n- butylammonium) pyrophosphate (2.35 mmoles) in 5 ml of dry DMF was prepared as follows: n-butylammonium pyrophosphate (Aldrich, P-8533, l. lg, 2.35mmoles) was dissolved in 5 ml dry DMF. To the solution was added tri-n- butylamine (218 mg, 280 u, l, 1.2 mmoles). The solvent was removed under vacuum and the residue was co-evaporated three times with 5ml of dry DMF. To the ammonium salt in 5ml of dry DMF was added additional tri-n-butylamine (1.12 ml, 2. 35mmoles)]. Then the reaction was added drop wise to the pyrophosphate solution with vigorous stirring. After 5 minutes, triethylammonium bicarbonate (1.0 M, pH 7.5, 20 ml) was added to quench the reaction and the mixture was then analyzed by HPLC (70% triphosphate). The solution was then diluted 100 times with water and loaded directly on to a DEAE ion-exchange column and purified using standard procedures.

Example 4 Synthesis of Biotin-iBUTP, propenamide-linked (RLR-2B) Scheme 4 o O HN NH H"O'D O HN NOCH3 </OMe °59 IN HC1, 60°C, 3 days HO dOV > HOwOi _ DMF, o/n )- (DMF, o/n Y HO OH HO OH la 2a (55% yield after silical Flash column purification) 0 0 0 0 \ IO/o O HN-'N-v'OH HNIN NHz HO 0 EDC, NH, (CHI) 2NH2 0 H I eg Biotin-X-NH" pH 5. 5-HO 0 (Et) 3N, DMF HO OH 3a (70% yield after recrystallization from H20) 4a (75% yield after RP-HPLC desalt) 0 H tN4NH~Nv NH-biotin 1) POCI3 O 0 1) POCI3 HO O 2) P2074 [(nBu) 3NH+] 4 HO OH 3) (Et) 3NH+HC03 Ho OH 5a (64% yield after RP-HPLC purification) o IN4N>NH~N) NH-biotin 0 0 L14P309-0 0 HO OH 6a (24% yield after IE-chromatography, RP-HPLC desalt and lyophilization)

Propenoic acid methyl ester 2a Compound la (2.5 g, 10.2 mmoles) and dimethylaminopyridine (1.25g, 10.2 mmoles) were dissolved in 125 ml dry DMF under argon. Methyl propiolate (0. 943g, 1.0 ml, 11.2 mmoles) was added and the solution was stirred at room temperature for 24 hours. The reaction turned from a colorless to amber colored solution. The reaction was followed by HPLC until no more product was produced. The solvent was removed by roto-evaporation and the residue was dissolved in 10 ml methanol-acetonitrile (1: 1 volume). It was purified by preparative PRP-1, 30 x 250mm column using water as buffer A and acetonitrile as buffer B with a flow rate 25 ml/min. Eluting from 5 to 95 % B in 15 minutes.

Collect the fraction from 9 to 10 minute. Remove solvent to afford 1. 1 (33%) as a white solid.

Propenoic acid 3a Compound 2 (l. lg, 3.35 mmoles) was dissolved in 80 ml 1.0 N HC1 and heated to 60 °C for 88 hours when LC-MS indicated the starting material is completed converted. The reaction mixture was evaporated to an oily residual by rotary-evaporation and redissolve in minimum amount of methanol. Add the methanol solution slowly to acetonitrile (at least 200 ml) to precipitate the free acid. Collect the solid and dried under vacuum to afford 1.0 g (94%) of white solid.

Aminopropenamide 4a Compound 3a (1.0 g, 3.18 mmoles) and a buffered solution of ethylenediamine in water (16 ml of 2.0 M ethylenediamine in 0.1 M MES buffer, pH 5.5, containing 32 mmoles of EDA) were mixed and then 1-ethyl-3- (3- dimethylaminopropyl) carbodiimide (4g, 32 mmoles) was added to the reaction with vigorous stirring. After 1 hour the reaction was analyzed by LC/MS and determined to be complete. Note: desalt a sample for LC-MS. The compound was purified by preparative HPLC: PRP-1,30 x 250mm column; flow rate 25 ml/min ; buffers: A, 0. 1M TEAA, pH 7.5, B, acetonitrile; gradient: 0% B for 9 minutes, 0 to 90% B over 10 minutes. Salts were removed with a retention time of about 4 min. and the compound eluted from 6 to 7.5 minutes. The fractions were pooled and the solvent removed under vacuum. The residue which contained triethylammonium acetate was co-evaporated with water several times and finally the product was precipitated from methanol/acetonitrile to afford 700 mg (62%) of 4a.

Biotin-propenamide 5a Compound 4a (102 mg, 0.286 mmoles) was co-evaporated with dry DMF twice (5 ml each) and then dissolved in dry DMF (1.5 ml) followed by the addition of triethylamine (29 mg, 40 u, l, 0.286 mmoles). The pH of the solution was adjusted to 7.5 with the addition of more triethylamine, if necessary. Biotin- X-NHS ester (0.286 mmoles, 130 mg) was then added to the mixture with stirring. After 1.0 hour, the reaction was monitored by HPLC for completion. The solvent volume was reduced under vacuum to about 1 ml. Caution: do not

vacuum to dryness because this compound tends to aggregate and it will be difficult to redissolve. The residual was redissolved in 5 ml water and 1 ml methanol.

The compound was purified by preparative HPLC: PRP-1, 30 x 250mm column; flow rate 25 ml/min; buffers: A, 0. 1M TEAA, pH 7.5, B, acetonitrile; gradient: 0% B for 11 minutes, then 0 to 95% B over 16 minutes. Fractions were collected across the peak from 19-21 min. The solvent was removed under vacuum and the residue was co-evaporated with water (30 ml) three times and methanol (50 ml) two times. The product was recrystallized from acetonitrile yielding 130mg (67%) of 5a.

Triphosphate 6a Compound 5a (130mg, 0.187 mmoles) was dried over P205 under vacuum for 24 hours and then dissolved in trimethyl phosphate (dried over molecular sieves, 20 ml) with gentle heating to about 60°C. Once the material dissolved the solution was cooled to ambient temperature and a trap-pack (ABI Trap-pak, P#GEN 084034) was added and allowed to gently stir overnight. The solution turned into a little cloudy when chilled on ice. The trap-pack was removed and to the solution at 0°C under argon was added POC13 (115 mg, 70 il, 0.748 mmoles). The reaction was monitored by AX-HPLC for the conversion to the monophosphate, and after 4 hours, an additional one equivalent of POC13 were added and the reaction was allowed to stir for 2 more hours (90% conversion). While monitoring the reaction, a solution of dry tetra (tri-N- butylammonium) pyrophosphate (0. 187x5x3. 3 = 3. 1 mmoles) in 6 ml dry DMF was prepared. Then the reaction was added drop wise to the pyrophosphate solution with vigorous stirring. After 5 minutes, triethylammonium bicarbonate (1.0 M, pH 7.5, 23 ml) was added to quench the reaction. The mixture was stirred on ice for 30 minutes and placed in a fridge overnight. The mixture was then analyzed by HPLC (70% triphosphate) and purified using standard TriLink procedures on DEAE.

The final reaction mixture may be diluted with mili Q water by a factor of 100, and then loaded on DEAE column. It is not recommended to rotovap off TEAB because the compound may be unstable under basic condition.

To prepare tetra (tri-N-butylammonium) pyrophosphate, TBA-PPi (Aldrich, P-8533,1. 5 TBA per PPi, 1.4g, 3. 1mmoles) was dissolved in 5 ml dry DMF. Add TBA 287 mg, 364 RI, 1.55 mmoles). Co-evaporate with 5 ml dry DMF at least three times. Redissolve in 5 ml anhydrous DMF. Add TBA (1.46 ml, 3.1 mmoles). Handle the materials in a glove box filled with Ar.

Example 5 IVT incorporation was determined spectrophotometrically using 260nm absorbance for the quantitation of RNA and a HABA-based colorimetric assay for quantitation of biotin for RLR-3b (Biotin-TisoCTP, vinyl-linked), RLR-2b (Biotin-TUTP, vinyl-linked) and RLR-2a (Biotin-TUTP, ethane (or saturated)- link). The vinyl linked analogs were both incorporated more efficiently than the saturated ethane analog as shown in Figure 7. Purified and fragmented RNA was quantitated by UV absorbance at 260 nm, and the amount of biotin incorporated was determined using a spectrophotometric-based assay for biotin (see, e. g., Swaisgood, H. E. et al 1996 Applied Biochemistry and Biotechnology, 56, 1. ) All patents, patent applications, and literature cited in the specification are hereby incorporated by reference in their entirety. In the case of any inconsistencies, the present disclosure, including any definitions therein will prevail.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.