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Definitions

• the invention relates to oligonucleotide-quencher- fluorescent-dye conjugates having improved characteristics, and to reagents suitable for incorporating novel quencher and fluorescent dye moieties into oligonucleotides.
• the invention also relates to the use of oligonucleotide- quencher- fluorescent-dye conjugates in detection methods for nucleic acid targets.
• Synthetic oligonucleotides have been used for years as sequence specific probes for complementary DNA and RNA targets. These methods have broad application in forensics, molecular biology and medical diagnostics since they allow the identification and quantitation of specific nucleic acid targets.
• FRET fluorescence resonance energy transfer
• the excited-state energy of the donor fluorophore is transferred to the neighboring acceptor by a resonance dipole-induced dipole interaction, which results in quenching of the donor fluorescence. If the acceptor molecule is a fluorophore, its fluorescence may sometimes be increased.
• the efficiency of the energy transfer between the donor and acceptor molecules is highly dependent on distance between the molecules. Equations describing this relationship are known.
• the Forster distance (RQ) is described as the distance between the donor and acceptor molecules where the energy transfer is 50% efficient.
• Other mechanisms of fluorescence quenching are also known, such as, collisional and charge transfer quenching.
• FRET-based detection assays have been developed in the fields of nucleic acid hybridization and enzymology. Several forms of the FRET hybridization assays are reviewed (Nonisotopic DNA Probe Techniques, Academic Press, Inc., San Diego 1992, pp. 31 1-352). Since its discovery, the polymerase chain reaction (PCR) has revolutionized molecular biology. This technique allows amplification of specific DNA sequences, thus allowing DNA probe assays to be executed from a single DNA target copy.
• PCR polymerase chain reaction
• PCR-based diagnostic assays have initially not been used routinely, in part due to problems with sample handling and possible contamination with non-source DNA. Recently, new homogeneous fluorescent-based DNA assays have been described which can detect the progress of PCR as it occurs (“real-time” PCR detection) using spectrofluorometric temperature cyclers. Two popular assay formats use DNA probes which become fluorescent as DNA amplification occurs (fluorogenic probes). The first format for "real-time” PCR uses DNA probes known as "molecular beacons" (Tyagi et al, Nat. Biotech., 16: 49-53 (1998)).
• Molecular beacons have a hairpin structure wherein the quencher dye and reporter dye are in intimate contact with each other at the end of the stem of the hairpin. Upon hybridization with a complementary sequence, the loop of the hairpin structure becomes double stranded and forces the quencher and reporter dye apart, thus generating a fluorescent signal.
• the second format for "real-time” PCR uses DNA probes which are referred to as "5'-nuclease probes" (Lee et al, Nucl. Acid Res., 21 : 3761- 3766 (1993)). These fluorogenic probes are typically prepared with the quencher at the 3 ' terminus of a single DNA strand and the fluorophore at the 5' terminus. During each PCR cycle, the 5'-nuclease activity of Taq DNA polymerase cleaves the DNA strand, thereby separating the fluorophore from the quencher and releasing the fluorescent signal.
• the 5'-nuclease assay requires that the probe be hybridized to the template strand during the primer extension step (60-65°C). They also disclose the simultaneous "real-time" detection of more than one polynucleotide sequence in the same assay, using more than one fluorophore/quencher pair.
• the 5'-nuclease PCR assay is depicted in Figure 1.
• 5'-nuclease probes had to be prepared with the quencher (usually tetramethylrhodamine (TAMRA)) positioned at an internal nucleotide in close proximity to the 5 '-fluorophore (usually fluorescein (FAM) or tetrachlorofluorescein (TET)) to get efficient FRET. Later it was found that this is not necessary, and the quencher and the fluorophore can be located at the 3' and 5' end of the ODN, respectively.
• TAMRA tetramethylrhodamine
• FAM fluorescein
• TET tetrachlorofluorescein
• dabcyl was used in the beacon- type probes but this is a different quenching mechanism wherein the dabcyl and fluorophore are in intimate contact (collisional quenching).
• Dabcyl was used in fluorogenic peptides as a quencher for the fluorophore EDANS (5-[(2- aminoethyl)amino]naphthalene-l-sulfonic acid) which emits at short (490 nm , blue) wavelength (Matayoshi et al. Science 247: 954-958 (1990)).
• EDANS also has a lower extinction coefficient than dabcyl so it is not surprising that fluorescent quenching was efficient.
• dabcyl can be used to quench fluorescein in a FRET type mechanism.
• FRET fluorescein
• other formats have been developed that use the FRET mechanism.
• single-stranded signal primers have been modified by linkage to two dyes to form a donor/acceptor dye pair in such a way that fluorescence of the first dye is quenched by the second dye.
• This signal primer contains a restriction site (United States Patent No. 5,846,726) that allows the appropriate restriction enzyme to nick the primer when hybridized to a target. This cleavage separates the two dyes and a change in fluorescence is observed due to a decrease in quenching.
• Non- nucleotide linking reagents to couple oligonucleotides to ligands have also been described (United States Patent No. 5,696,251). FRET systems also have applications in enzymology.
• Protease cleavable substrates have been developed where donor/acceptor dye pairs are designed into the substrate. Enzymatic cleavage of the substrate separates the donor/acceptor pair and a change in fluorescence is observed due to a decrease in quenching.
• Cleavable donor/acceptor substrates have been developed for chymotrypsin (Li et al. Bioconj. Chem., 10: 241-245 (1999)), aminopeptidase P (Hawthorne et al, Anal.
• oligonucleotides having a covalently attached minor groove binder (MGB) are more sequence specific for their complementary targets than unmodified oligonucleotides.
• MGB-ODNs show substantial increase in hybrid stability with complementary DNA target strands when compared to unmodified oligonucleotides, allowing hybridization with shorter oligonucleotides.
• Reagents for fluorescent labeling of oligonucleotides are critical for efficient application of the FRET assays described above. Other applications such as DNA micro arrays also use fluorescently labeled DNA probes or primers, and there is a need for improved reagents which facilitate synthesis of fluorescent DNA.
• phosphoramidite reagents and solid supports are widely used on ODN synthesis.
• Linker groups to attach different ligand groups to ODNs play an important role in the synthesis of oligonucleotide conjugates.
• a method for the synthesis of 3'-aminohexyl-tailed oligonucleotides (Petrie et al, Bioconj. Chem., 3: 85-87 (1992)), the use of a trifunctional trans-4-hydroxy-L-prolinol group (Reed et al, Bioconjug.
• reporter fluorophores are available to be used in reporter/quencher pairs, most suffer from having some undesirable characteristic, mixtures difficult to separate, positively charged, difficult to synthesize, unstable during oligonucleotide synthesis or having overlapping emission wavelengths with other desirable reporters.
• the present invention provides reagents for oligonucleotide probes that address these unfavorable characteristics and overcome some or all of the difficulties.
• the present invention provides quencher molecules based on the 4- [4- nitrophenyl)diazinyl]phenylamine and/or the 4-[4-nitrophenyl)diazinyl]- naphthylamine structure.
• the quencher chromophores of the present invention are non- fluorescent, easily incorporated into DNA synthesis reagents, stable during automated DNA synthesis and during storage and compatible with no adverse effects on hybridization properties.
• the present invention offers considerable advantages over the use of dabcyl (Nazerenko et al, Nucl. Acids Res., 25: 2516-21 (1997)) as a quenching dye, as used in the prior art.
• the quenchers based on the 4-[4-nitrophenyl)diazinyl]phenylamine (and/or the 4-[4- nitrophenyl)diazinyl]naphthylamine structure) are modified with linker structures that allow their easy incorporation into fluorogenic DNA probes during automated DNA synthesis.
• the invention includes synthesis of phosphoramidites derived from the novel quencher molecules for incorporation of the quencher moieties into oligonucleotides during automated synthesis, and also synthesis of reagents derived from the novel quencher molecules for post solid-phase support attachment to amino-tailed oligonucleotides.
• the novel quencher molecules are introduced into oligonucleotides using pyrazolo-[5,4-d]pyrimidines and pyrimidines phosphoramidites containing the quenchers attached at the 3'- and 5 '-positions, respectively.
• three different fluorescent reagent types that are compatible with DNA synthesis are synthesized or selected and converted into phosphoramidite reagents suitable for incorporation onto ODNs.
• red fluorescent dyes based on 7-hydroxyphenoxazin-3-one (resorufin) and blue fluorescent dyes based on the structure of coumarin are incorporated into phosphoramidite reagents.
• fluorescent dyes have excellent properties for multicolor fluorescent analysis in combination with other dyes (eg. fluorescein). These reagents are valuable for a variety of analytical methods that use either direct detection of fluorescence or FRET detection formats.
• the PPT-, coumarin- and resorufin-based fluorophores are converted into novel reagents suitable for "post-oligonucleotide-synthesis" covalent attachment at the 5 '-end of ODNs.
• the new fluorescent dyes are incorporated into oligonucleotides using pyrazolo-[5,4- djpyrimidines and pyrimi dines phosphoramidites which contain the fluorophores attached at the 3- and 5-positions, respectively.
• ODNs covalently linked with the novel quencher structures of the invention, paired with a covalently attached fluorescent moieties, are prepared.
• the resulting FL-ODN-Q conjugate may also include a minor groove binder (MGB) that improves the binding and discrimination characteristics of the resulting FL- ODN-Q-MGB conjugate in diagnostic assays, particularly in the TaqMan PCR assay of single nucleotide polymorphism (and the like) where allele- specific discrimination not only requires probes with different fluorescent reporter molecules but efficient quenchers.
• MGB minor groove binder
• the quenchers used in accordance with the invention in the FL-ODN-Q-MGB conjugates provide broad quenching wavelength range, and certain novel reporter labeling reagents in accordance with the invention have distinctive emission wavelengths for improved multicolor analysis.
• fluorogenic probes are prepared using a universal "3'-hexanol" solid support (available in accordance with Gamper et al. Nucleic Acids Res., 21: 145-150 (1993) expressly incorporated herein by reference), where a quencher phosphoramidite of the invention is added at the first coupling step (3 '-end) of the ODN sequence and a fluorophore (FL) was attached at the final coupling step, yielding 5'-FL-ODN-Q-hexanol conjugate probes.
• a quencher phosphoramidite of the invention is added at the first coupling step (3 '-end) of the ODN sequence and a fluorophore (FL) was attached at the final coupling step, yielding 5'-FL-ODN-Q-hexanol conjugate probes.
• oligonucleotide probe is prepared from a MGB modified solid support substantially in accordance with the procedure of Lukhtanov et al.
• oligonucleotides such as the array-based analysis of gene expression (Eisen, Methods of Enzym., 303: 179-205 (1999)).
• array-based analysis of gene expression Eisen, Methods of Enzym., 303: 179-205 (1999)
• an ordered array of oligonucleotides or DNAs that correspond to all, or a large fraction of the genes in many organism is used as a platform for hybridization.
• Microarray-based methods are used in assays to measure the relative representation of expressed RNA species. The quantitation of differences in abundance of each RNA species is achieved by directly comparing two samples by labeling them with spectrally distinct fluorescent dyes and mixing the two probes for simultaneous hybridization to one array.
• compositions and methods of present invention relate to the detection of nucleic acids, it includes but is not limited to methods where FRET is involved, such as 5'-nuclease, universal energy transfer primers or beacon assays. These methods are usually directed to, but are not limited to the detection of PCR-generated nucleic acid sequences. Some of these methods involve simultaneous detection of more than one nucleic acid sequence in the same assay. Similarly, the invention relates to methods where FRET is involved in the detection of protein concentration or enzyme activity.
• Still other applications of the invention relate to the labeling with luminescent PPT- , coumarin- and resorufin-based dyes of nucleic acids, proteins and other materials including, drugs, toxins, cells, microbial materials, particles, glass or polymeric surfaces and the like, at a reactive group such as an amino, hydroxyl or sulfhydryl group.
• the present invention may be used in single- and two-step labeling processes.
• a primary component such as an oligonucleotide is labeled with the reagent capable of introducing the novel fluorophore PPT- , coumarin- and resorufin-based dyes, by reaction with a reactive group of the ODN (such as an amine, hydroxyl, carboxyl, aldehyde or sulfhydryl group) and the label is used to probe for a secondary component, such as an oligonucleotide target.
• a reactive group of the ODN such as an amine, hydroxyl, carboxyl, aldehyde or sulfhydryl group
• Figure 1 is a schematic representation of real-time 5'-nuclease PCR assay.
• Figure 2 is a graph showing the UV spectra of Dabcyl- and Redl3 dye-modified DNA probes.
• Figure 3 is a graph showing the performance of fluorogenic MGB probes in a "real-time" PCR assay.
• Figure 4 is a graph showing the fluorescent spectra of violet, F AM and resorufin dye containing DNA probes .
• QUENCHER REAGENTS FOR OLIGONUCLEOTIDE SYNTHESIS Two types of reagents for introducing the substituted 4- (phenyldiazenyl)phenylamine quencher moieties into oligonucleotides using an automated DNA synthesizer are exemplified in the disclosure below.
• MGB, FL, Q, CPG and ODN stand for "minor groove binder”, “fluorescent or fluorophor”, “quencher” "controlled pore glass” and “oligonucleotide” moieties or molecules, respectively, and in a manner which is apparent from context.
• the first type of reagents disclosed herein are phosphoramidites that bear the quencher molecule (Q) as well as a dimethoxytrityl (DMTr) (methoxyfrityl, trityl or the like acid labile blocking group) protected primary alcohol that provides an attachment point for the growing oligodeoxynucleotide (ODN) chain during subsequent oligonucleotide synthesis.
• Q quencher molecule
• DMTr dimethoxytrityl
• ODN oligodeoxynucleotide
• Examples of these reagents are depicted in Formulas 1, 2, and 3, and in Reaction Schemes 1 and 2.
• the starting compound is a substituted 4- (phenyldiazenyl)phenylamine 1 that has a primary hydroxyl group.
• Such starting material is commercially available or can be synthesized in accordance with methods known in the art, applying routine skill available to the practicing organic chemist.
• 4-nitrobenzendiazonium salt is reacted with 2-(2-chloroanilino)ethanol to yield 2-[2-chloro-4-(4- nitrophenylazo)anilino]ethanol in accordance with the teachings of United States Patent No. 2,264,303.
• 2-[2-chloro-4-(4-nitrophenylazo)anilino]ethanol is within the scope of compound 1 as depicted in Reaction Scheme 1.
• the pyrrolidinediol is a trifunctional reagent that has an amino, a primary and a secondary hydroxyl group.
• the diol 3 is reacted first with dimethoxy trityl chloride (DMTrCl) to block the primary hydroxyl group of the trifunctional reagent and yield intermediate 4.
• DMTrCl dimethoxy trityl chloride
• the intermediate 4 still having a free secondary hydroxyl group in the trifunctional reagent, is then reacted with 2-cyanoethyl diisopropylchlorophosphoramidite to give the dimethoxy trityl protected phosphoramidite reagent 5.
• the symbols are defined as follows.
• the dimethoxytrityl protected phosphoramidite reagent 5 is suitable for attachment to oligonucleotides in steps otherwise known in routine ODN synthesis.
• Formula Formula 2 Reaction Scheme 2 discloses the synthesis of another exemplary phosphoramidite reagent 10 bearing the substituted 4-(phenyldiazenyl)- phenylamine quencher moiety and including the trifunctional pyrrolidinediol moiety.
• the starting material is a substituted 4- (phenyldiazenyl)phenylamine compound 6 that has a free carboxyl group.
• a second class of compounds or reagents suitable for introducing the quencher molecules into ODNs constitute a composition that has a solid support of the type used for ODN synthesis (for example controlled pore glass (CPG)), and linker attaching the quencher to the solid support.
• the linker has a hydroxyl function that is protected, usually by a dimethoxytrityl group which is removed during the synthesis when the first nucleotide is attached to the linker.
• CPG controlled pore glass
• the secondary hydroxyl group of the intermediate 4 (shown in Scheme 1) is reacted with succinic anhydride, and thereafter pentafluorophenyl trifluoroacetate to provide the active ester 11.
• the active ester 11 is then reacted with the free amino group attached to the solid support (CPG bead) to provide the modified solid support 12.
• modified solid support 12 includes the "trifunctional linker" derived from pyrrolidine diol
• analogous modified solid supports including other linkers and related structures such as the linkers shown in Formulas 1, 2 and 3 can also be made substantially in accordance with Reaction Scheme 3, resulting in modified solid support compositions including the quencher moiety, such the ones shown in Formula 4 and Formula 5.
• the modified solid support compositions including the quencher moiety of structure 12 and of Formula 4 and 5 are used for preparing 3'- quencher conjugates, allowing the introduction of a fluorophore at the 5 '-end with the appropriate phosphoramidite, or post- synthetically with a fluorophore containing a reactive group.
• a minor groove binder is attached to controlled pore glass (CPG) through a cleavable linker.
• a quencher moiety based on the 4-(phenyldiazenyl)phenylamine structure, is attached through a linker molecule to the MGB.
• the linker molecule also contains a hydroxyl group blocked with DMTr (or like) blocking group. After removal of the DMTr group, an oligonucleotide is synthesized on an automated oligonucleotide synthesizer by step-wise attachment of nucleotide units to the hydroxyl group.
• a fluorophore is introduced at the 5 '-end with the appropriate phosphoramidite, or post-synthetically with a fluorophore containing a reactive group, to yield an ODN having an attached fluorescent moiety (FL), quencher (Q) and MGB (FL-ODN-Q-MGB).
• FL fluorescent moiety
• Q quencher
• MGB MGB
• FL-ODN-Q-MGB MGB
• the MGB is 3- ⁇ [3-(pyrrolo[4,5-e]indolin-7- ylcarbonyl)pyrrolo[4,5 -e] indolin-7-yl] carbonyl ⁇ pyrrolo [3 ,2-e] indoline-7- carboxylic acid (DPI 3 ).
• DPI 3 pyrrolo [3 ,2-e] indoline-7- carboxylic acid
• the first phase is the synthesis of an intermediate, 2-(4- nitrophenyl)ethyl 3-(pyrrolo[4,5-e]indoline-7-carbonyl)pyrrolo[4,5- e]indoline-7-carboxylate (DPI 2 -NPC) 17.
• the second phase is the synthesis of Q-DMTr-DPI-C0 2 PFP 24 where a quencher is coupled through a linker to a pyrrolo[3,2-e]indoline-7-carboxylic acid unit (DPI ).
• DPI pyrrolo[3,2-e]indoline-7-carboxylic acid unit
• PFP stands for the pentafluorophenyl or pentafluorophenyloxy group, as the context requires.
• DMTr-Q-DPI 3 -PFP 25a is synthesized from 17 and 24.
• the fourth phase 25a is coupled to CPG to yield a DMTr-Q- DPI 3 -CPG 29, and in the fifth phase 29 is used on an automated oligonucleotide synthesizer to stepwise attach nucleotide units and to provide, after removal from the CPG, the product FL-5'-ODN-3'-Q-DPI 3 30.
• the fourth and fifth phases of these synthetic process are shown in Reaction Scheme 7. Experimental conditions for this sequence (phases 1 through 5) are described below.
• Q-DPI 3 moiety 25 (phase 3) is synthesized by the reaction of two intermediates, 17 and 24 as shown in Reaction Scheme 6.
• the first intermediate DPI 2 -NPE 17 is made as shown in Scheme 4.
• DPI-tBoc 13 was reacted with p- nitrophenylethanol in the presence of diethylazodicarboxylate (DEAD) and triphenylphosphine to yield the di-ester 14.
• a substituted nitroaniline 18 (available commercially or in accordance with the chemical literature) is diazotized in the presence of nitrous acid and is coupled to a substituted aniline 19 (available commercially or in accordance with the chemical literature) to form the azo intermediate quencher molecule 20.
• Alkaline hydrolysis of the ethyl ester 20 followed by the treatment with DMTrCl gives the DMTr-Q 21, that is subsequently activated with pentafluorophenyl trifluoroacetate to yield 22.
• Reaction of 22 with DPI-methyl ester gives the Q-DMTr-DPI methylester 23.
• DMTr-Q-DPI 3 -PFP 25a (third phase) is synthesized first by reacting the activated quencher 24 (DMTr-Q-DPI PFP) with DPI 2 -NPC 17 to yield the p-nitrophenylethyl ester 25, which is converted to the active ester 25a, first by treatment with base such as 1 ,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to remove the p-nitrophenylethyl moiety and then treatment with 2,3,4,5,6-pentafluorophenyl trifluoroacetate (PFP-TFA).
• base such as 1 ,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
• Reaction of 27 with long chain aminoalkyl CPG in the presence of activating agents yields the MMT- diglycolate-CPG 28, that is converted after detritylation and reaction with 25a to DMTrO-Q-DPI 3 -CPG 29.
• activating agents HOBT and HBTU
• MMT- diglycolate-CPG 28 that is converted after detritylation and reaction with 25a to DMTrO-Q-DPI 3 -CPG 29.
• oligonucleotide synthesis is performed with the aid of an automated DNA synthesizer, and a fluorophore is attached at the 5 '-end of the ODN, using either a fluorphore- phosphoramidite or a fluorophore containing a reactive group, to yield the FL- ODN-Q-DPI 3 30 conjugate.
• the FL-ODN-Q-DPI 3 30 conjugate can also be synthesized by an alternative synthetic route which is not specifically illustrated in the reaction schemes.
• DPI 3 -methyl ester obtained in accordance with Boger et al, J. Org. Chem., 52: 1521- (1987) incorporated herein by reference
• Q- DPI 3 -methyl ester and Q-DPI 3 -COOH respectively.
• the latter compound is then activated with pentafluorophenyl trifluoroacetate, to yield 25a, which is then used in the reactions shown in Scheme 7, to yield 30.
• FL-ODN-0 and FL-ODN-O-MGB Probes A general structure of a preferred embodiment of Fl-ODN-Q-DPI 3 conjugates is shown in Formula 6 where: FL is a fluorophore with emission wavelengths in the range of about 300 to about 800 nm and more preferably 400 to 700 nm; K is a linker containing between 1 and 30 atoms, which include any of C, O, N, S, P and H; [A-B] n symbolizes a DNA, RNA or PNA or any combination thereof, where A is the sugar phosphate backbone (including modified sugars and modified phosphates), B is the heterocyclic base, and n is the number of nucleotide units.
• B can independently be any of the purine- and pyrimidine-; pyrazolo[3,4-d]pyrimidine-, 7-substituted pyrazolo[3,4-d]pyrimidine- , 7- deazapurines, 7-substiuted 7-deazapurines, modified purine- and pyrimidine- bases, and the oligonucleotide or nucleic acid can include any combinations of these bases.
• Conjugate probes of the present invention containing a fluorescent reporter-quencher pair are, generally speaking, used in conjunction with the amplification of target polynucleotides, frequently in methods utilizing PCR, as described for example by Holland et al. Proc. Acad. Sci., 88: 7276- 7280(1991) and Wittwer et al, Biotechniques, 22: 176-181 (1997) which are incorporated herein by reference.
• the binding site of the conjugate probe is located between the PCR primers used to amplify the target polynucleotide.
• the conjugate oligonucleotide probes according to the present invention for detection of target oligonucleotide sequences provides several advantages over prior-art reporter quencher groups and combinations.
• the quenchers including the 4-[4-nitrophenyl)diazinyl]phenylamine structure in accordance with the present invention gave larger signal to noise ratios (S/N) in probes with either FAM or TAMRA serving as reporters than dabcyl as a quencher.
• the quenchers in accordance with the invention show a broader absorbance range than dabcyl, allowing efficient quenching of a broad range of fluorophores.
• an improved quencher showed about 30-fold increase in S/N ratio with TAMRA compared to a standard probe (no DPI 3 ) with dabcyl.
• Reagents of the present invention allow the introduction of the quencher during automated oligonucleotide synthesis.
• Dabcyl phosphoramidite is commercially available; (Glen Research, Sterling, VA)
• an oligonucleotide comprises a plurality of nucleotide units, a 3' end and a 5 'end.
• oligonucleotide may contain one or more modified bases other than the normal purine and pyrimidine bases, as well as modified internucleotide linkages capable of specifically binding target polynucleotide through Watson-Crick base pairing, or the like.
• oligonucleotides may include peptide oligonucleotides (PNAs) or PNA DNA chimeras, the synthesis of which is known and can be performed for example in accordance with the publications Uhlmann et al, Angew. Chem. Inter. Ed., 37:2796- 2823 (1998) and Mayfield et al, Anal. Biochem., 401-404 (1998); all of which are expressly incorporated herein by reference.
• PNAs peptide oligonucleotides
• the oligonucleotide probes of the invention will have a sufficient number of phophodiester linkages adjacent to the 5' end to allow 5 '-3' exonuclease activity to allow efficient cleavage between the quencher and fluorophore molecules in the probe.
• An adequate number in this regards is approximately between 1 and 100.
• the reagent 34 includes a covalently linked coumarin chromophore which emits light at 458 nm. DNA probes containing this coumarin chromophore were prepared and gave the desired fluorescent emission properties.
• Reaction of 32 first with DMTrCl and then with trimethylacetic anhydride followed by the removal of the DMTr blocking group gives a pivaloate derivative 33, in the specific example 33a where Rg is -OC( 0)CH(CH 3 ) 2 and R 9 is -H.
• the reagent 34 is used for incorporating the coumarin fluorophore into the 5 '-terminus of DNA probes.
• Resorufin Phosphoramidite Another new class of DNA synthesis reagents are based on the 7- hydroxy-3H-phenoxazin-3-one chromophore present in the parent compound (resorufin) and have emission wavelength (595 nm) that is easily distinguished from FAM emission.
• the chromophore is synthesized in such a way as to incorporate a linker structure for further functionalization to the desired phosphoramidite reagents.
• R 14 in the scheme is H or DMTr.
• resorufin derivative 35 (specifically 35a where R 10 is OH, and R ⁇ is H) contaminated with 2,3,4-trihydro-2H- pyrano[3,2-b]phenoxazin-9-one as major impurity.
• the latter mixture was treated with DMTrCl and pyridine, and then with trimethylacetic acid anhydride.
• PPT Phosphoramidite The synthesis of a phosphoramidite reagent incorporating a purple fluorescent dye PPT 44 having excitation and emmision wavelengths of 384 and 400 nm, respectively is shown in Reaction Scheme 10 and in Example X.
• 6-chloro-3-n-butyluracil 38 and 2-(4- aminophenyl)ethanol 39 are reacted to yield the phenyl substituted uracil derivative 40.
• the compounds 38 and 39 can be obtained in accordance with the state-of-the-art and the chemical literature. Reaction of 40 with 5- formyl-4,6-dichloro pyrimidine in DMF at room temperature affords the tricyclic heterocycle 41.
• the fluorophores coumarin, resorufin and PPT substituted with an alkylcarboxyl group are either commercially available or can be synthesized in accordance with the state-of-the-art. These compounds are activated on the alkylcarboxyl group as the pentafluorophenyl esters. The activated esters are used to attach these dyes to amine modified oligonucleotides. Similarly, in still other embodiments, dUTP-labeled quenchers or fluorophores are obtained for example in accordance with the teachings of United States Patent No. 5,328,824).
• phosphoramidite of 7- labeled pyrazolo[3,4-d]pyrimide-labeled quenchers or fluorophores are synthesized according to the teaching of 5,824,796 (incorporated herein by reference) and can be used for labeling of oligonucleotides.
• the red dye 13 quencher is attached to the 3- position of pyrazolo[5,4-d]pyrimidines (PP) or the 5-position of a pyrimidine.
• PP pyrazolo[5,4-d]pyrimidines
• the starting material is 5-(4-amino-3-iodopyrazolo[5,4-d]pyrimidinyl)-2-(hydroxymethyl)oxolan-3-ol 45 which is available in accordance with the publication Seela et al. J. Chem. Soc, Perkin. Trans., 1 (1999, 479-488) incorporated herein by reference.
• pyrazolopyrimidine-Red-13- or uridine-Red 13-based phosphoramidites within the scope of this invention may contain various linkers between the pyrazolopyrimidine and uracil bases and the Red 13 quenchers, to the full extent such linkers are available in accordance with the state of the art and this disclosure.
• the methods and compositions of the present invention can be used with a variety of techniques, both currently in use and to be developed, in which hybridization of an oligonucleotide to another nucleic acid is involved. These include, but are not limited to, techniques in which hybridization of an oligonucleotide to a target nucleic acid is the endpoint; techniques in which hybridization of one or more oligonucleotides to a target nucleic acid precedes one or more polymerase-mediated elongation steps which use the oligonucleotide as a primer and the target nucleic acid as a template; techniques in which hybridization of an oligonucleotide to a target nucleic acid is used to block extension of another primer; techniques in which hybridization of an oligonucleotide to a target nucleic acid is followed by hydrolysis of the oligonucleotide to release an attached label; and techniques in which two or more oligonucleotides are hybridized to a target nu
• one or more FL- oligonucleotide conjugates are used as probe(s) to identify a target nucleic acid by assaying hybridization between the probe(s) and the target nucleic acid.
• a probe may be labeled with any detectable label of the present invention, or it may have the capacity to become labeled either before or after hybridization, such as by containing a reactive group capable of association with a label or by being capable of hybridizing to a secondary labeled probe, either before or after hybridization to the target.
• conditions for hybridization of nucleic acid probes are well- known to those of skill in the art. See, for example, Sambrook et al.
• Hybridization can be assayed (i.e., hybridized nucleic acids can be identified) by distinguishing hybridized probe from free probe by one of several methods that are well-known to those of skill in the art. These include, but are not limited to, attachment of target nucleic acid to a solid support, either directly or indirectly (by hybridization to a second, support- bound probe or interaction between surface-bound and probe-conjugated ligands) followed by direct or indirect hybridization with probe, and washing to remove unhybridized probe; determination of nuclease resistance; buoyant density determination; affinity methods specific for nucleic acid duplexes (e.g., hydroxyapatite chromatography); interactions between multiple probes hybridized to the same target nucleic acid; and other known techniques.
• attachment of target nucleic acid to a solid support either directly or indirectly (by hybridization to a second, support- bound probe or interaction between surface-bound and probe-conjugated ligands) followed by direct or indirect hybridization with probe,
• oligonucleotide conjugates containing a fluorophore and quencher are found in assays in which a labeled probe is hybridized to a target and/or an extension product of a target, and a change in the physical state of the label is effected as a consequence of hybridization.
• a probe is a nucleic acid molecule that is capable of hybridizing to a target sequence in a second nucleic acid molecule.
• the hydrolyzable probe assay takes advantage of the fact that many polymerizing enzymes, such as DNA polymerases, possess intrinsic 5 '-3' exonucleolytic activities.
• a polymerizing enzyme that has initiated polymerization at an upstream amplification primer is capable of exonucleo lyrically digesting the probe.
• Any label attached to such a probe will be released, if the probe is hybridized to its target and if amplification is occurring across the region to which the probe is hybridized. Released label is separated from labeled probe and detected by methods well-known to those of skill in the art, depending on the nature of the label.
• radioactively labeled fragments can be separated by thin-layer chromatography and detected by autoradiography; while fluorescently-labeled fragments can be detected by irradiation at the appropriate excitation wavelengths with observation at the appropriate emission wavelengths.
• This basic technique is described for example in United States Patent No. 5,210,015 incorporated herein by reference.
• a probe contains both a fluorescent label and a quenching agent, which quenches the fluorescence emission of the fluorescent label.
• the fluorescent label is not detectable until its spatial relationship to the quenching agent has been altered, for example by exonucleolytic release of the fluorescent label from the probe.
• the dual fluorophore/quencher labeled probe does not emit fluorescence.
• the fluorophore/quencher-labeled probe becomes a substrate for the exonucleolytic activity of a polymerizing enzyme which has initiated polymerization at an upstream primer.
• Exonucleolytic degradation of the probe releases the fluorescent label from the probe, and hence from the vicinity of the quenching agent, allowing detection of a fluorescent signal upon irradiation at the appropriate excitation wavelengths.
• This method has the advantage that released label does not have to be separated from intact probe.
• Multiplex approaches utilize multiple probes, each of which is complementary to a different target sequence and carries a distinguishable label, allowing the assay of several target sequences simultaneously.
• the use of FL-ODN-Q-DPI 3 conjugates in this and related methods allows greater speed, sensitivity and discriminatory power to be applied to these assays.
• the enhanced ability of MGB-oligonucleotide conjugates to allow discrimination between a perfect hybrid and a hybrid containing a single-base mismatch facilitates the use of hydrolyzable probe assays in the identification of single-nucleotide polymorphisms and the like, as described in the publication WO 995162 A2, incorporated herein by reference.
• Examples 13 and 14 illustrate the utility of FL-ODN-Q- DPI 3 conjugates in this type of assay. It will be clear to those of skill in the art that compositions and methods, such as those of the invention, that are capable of discriminating single-nucleotide mismatches will also be capable of discriminating between sequences that have multiple mismatches with respect to one another.
• Another application embodiment uses a self-probing primer with an integral tail, where the quencher/fluorophore is present in the hairpin, that can probe the extension product of the primer and after amplification hybridizes to the amplicon in a form that fluoresces. The probing of a target sequence can thereby be converted into a unimolecular event (Whitcombe, D. et al., Nat. Biotech., 17: 804-807 (1999)).
• oligonucleotide conjugates containing a fluorophore/quencher pair are used in various techniques which involve multiple fluorescent-labeled probes.
• changes in properties of a fluorescent label are used to monitor hybridization.
• fluorescence resonance energy transfer FRET
• FRET fluorescence resonance energy transfer
• two probes are used, each containing a fluorescent label and a quencher molecule respectively.
• the fluorescent label is a donor
• the quencher is an acceptor, wherein the emission wavelengths of the donor overlap the absorption wavelengths of the acceptor.
• the sequences of the probes are selected so that they hybridize to adjacent regions of a target nucleic acid, thereby bringing the fluorescence donor and the acceptor into close proximity, if target is present. In the presence of target nucleic acid, irradiation at wavelengths co ⁇ esponding to the absorption wavelengths of the fluorescence donor will result in emission from the fluorescence acceptor.
• These types of assays have the advantage that they are homogeneous assays, providing a positive signal without the necessity of removing unreacted probe.
• European Patent Publication 070685 See, for example, European Patent Publication 070685; and the publication C ⁇ rdullo et ⁇ l. (1988) Proc.
• the minor groove binder, DPI 3 is coupled to a quencher in a FL-ODN-Q- CDPI 3 conjugate to improve signal to noise ratios (See Table 2).
• Preferred quenchers are the quenchers of Formula 6 and more preferred the quenchers are 8-11, 12-16 and 30.
• Additional quenchers suitable for use in combination with the novel fluorophores (34, 37 and 44) of the invention include dabcylnitrothiazole, TAMRA, 6-(N-[7-nitrobenz-2-oxa-l,3-diazol-4-yl]amino) hexanoic acid , 6- carboxy-X-rhodamine (Rox) and QSY-7 .
• Another application of the novel fluorophore/quencher pairs of the invention is to incorporate the pair into enzyme substrates, where fluorescence is quenched because of the proximity of the fluorophore and quencher.
• Oligonucleotide A ⁇ ays are utilized in procedures employing arrays of oligonucleotides.
• Examples for this technique that is per se known in the art include sequencing by hybridization and array-based analysis of gene expression.
• an ordered array of oligonucleotides of different known sequences is used as a platform for hybridization to one or more test polynucleotides, nucleic acids or nucleic acid populations. Determination of the oligonucleotides which are hybridized and alignment of their known sequences allows reconstruction of the sequence of the test polynucleotide.
• United States Patent Nos see for example, United States Patent Nos.
• oligonucleotide, polynucleotide and nucleic acid are used interchangeably to refer to single- or double-stranded polymers of DNA or RNA (or both) including polymers containing modified or non-naturally- occurring nucleotides, or to any other type of polymer capable of stable base- pairing to DNA or RNA including, but not limited to, peptide nucleic acids which are disclosed by Nielsen et al. (1991) Science 254:1497-1500; bicyclo DNA oligomers (Bolli et al. (1996) Nucleic Acids Res. 24:4660-4667) and related structures.
• MGB moieties and/or one or more fluorescent labels, and quenching agents can be attached at the 5' end, the 3' end or in an internal portion of the oligomer.
• a preferred MGB in accordance with the invention is DPI 3 and the prefe ⁇ ed quencher is red 13 amide.
• Preferred in the present invention are DNA oligonucleotides that are single-stranded and have a length of 100 nucleotides or less, more preferably 50 nucleotides or less, still more preferably 30 nucleotides or less and most preferably 20 nucleotides or less with a lower limit being approximately 5 nucleotides.
• Oligonucleotide conjugates containing a fluorophore/quencher pair with or without an MGB may also comprise one or more modified bases, in addition to the naturally-occurring bases adenine, cytosine, guanine, thymine and uracil.
• Modified bases are considered to be those that differ from the naturally-occurring bases by addition or deletion of one or more functional groups, differences in the heterocyclic ring structure (i.e., substitution of carbon for a heteroatom, or vice versa), and/or attachment of one or more linker arm structures to the base.
• the modified nucleotides which may be included in the ODN conjugates of the invention include 7-deazapurines and their derivatives and pyrazolopyrimidines (described in PCT WO 90/14353 incorporated herein by reference); and in co-owned and co-pending application serial number 09/054,630.
• Preferred base analogues of this type include the guanine analogue 6-amino-lH-pyrazolo[3,4-d]pyrimidin-4(5H)-one (ppG or PPG) and the adenine analogue 4-amino-lH-pyrazolo[3,4-d]pyrimidine (ppA or PPA).
• ppX xanthine analogue lH-pyrazolo[5,4-d]pyrimidin-4(5H)- 6(7H)-dione
• Sugar modifications include, but are not limited to, attachment of substituents to the 2', 3' and/or 4' carbon atom of the sugar, different epimeric forms of the sugar, differences in the ⁇ - or ⁇ - configuration of the glycosidic bond, and other anomeric changes.
• Sugar moieties include, but are not limited to, pentose, deoxypentose, hexose, deoxyhexose, ribose, deoxyribose, glucose, arabinose, pentofuranose, xylose, lyxose, and cyclopentyl. Modified internucleotide linkages can also be present in oligonucleotide conjugates of the invention.
• Such modified linkages include, but are not limited to, peptide, phosphate, phosphodiester, phosphotriester, alkylphosphate, alkanephosphonate, thiophosphate, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, substituted phosphoramidate and the like.
• bases, sugars and/or internucleotide linkages, that are compatible with their use in oligonucleotides serving as probes and/or primers will be apparent to those of skill in the art.
• Certain prefe ⁇ ed embodiments of the invention involve the synthesis of numerous phophoramidites with various quencher chromophores and linkers and their incorporation at the 3 '-end of fluorogenic MGB ODNs as shown in Reaction Scheme 3.
• Different fluorescent reporter groups shown in Reaction Scheme 7
• the fluorogenic properties of these ODN conjugates are described in Table 2.
• MGB molecules due to their desirable improved hybridization properties, were incorporated into oligonucleotides containing both a fluorophore and a quencher, without loss in hybridization specificity, fluorescent quenching and fluoresent signal.
• the quenchers incorporated in the compounds represented by Formulas 7-16 are the commercially available 2-[4-(4-nitrophenylazo)-N- ethylphenylaminojethanol (Disperse Red 1), 2-[4-(2-chloro-4- nitrophenylazo)-N-ethylphenylamino]ethanol (Disperse Red 13) and 2-[4- (dimethylamino)phenylazo]benzoic acid, identified in this invention as Redl, Red 13 and dabcyl respectively.
• Figure 2 shows the absorbance properties of the red 13 chromophore (Formula 8, without DPI 3 ) in comparison to dabcyl (Formula 7, without DPI 3 when incorporated at the 3 '-end of an otherwise unmodified DNA probe.
• the broader absorbance (especially at long wavelengths) of the redl 3 chromophore is a clear advantage.
• the 8 max for redl 3 is at 522 nm whereas the 8 max for dabcyl is 479 nm.
• S/N Signal to noise
• the ODN sequence was 5 ' -gagggatgtaaaat (SEQUENCE Id. No. 1).
• the fluorophores (R ⁇ studied here is either 6- carboxyfluorescein (6-FAM) or 6-carboxytetramethylrhodamine (TAMRA).
• red 13 chromophore and the closely related redl chromophore are better quenchers for both FAM and TAMRA with a variety of linkers than dabcyl.
• the linker can affect quenching by the redl 3 chromophore.
• Formula 14 and Formula 15 worked well with FAM, but had poor quenching efficiency for TAMRA. It is somewhat su ⁇ rising that dabcyl worked so well, especially for the TAMRA probes.
• effective FRET quenching by dabcyl is a specific case for MGB probes.
• DPI 3 redl 3 amide 48 97 x Signal to noise (S/N) was determined using the phosphodiesterase assay described above.
• the ODN sequence was 5 '-gagggatgtaaaaat (SEQUENCE Id. No. 1).
• the linker structure of the dabcyl or redl 3 quenchers (Q) is shown in Formulas 7 and 8 respectively.
• redl 3 This improved quenching by redl 3 is consistent with the increased spectral overlap presented in Figure 2 and a standard FRET mechanism.
• the increased S/N of both 8 and 30 when inco ⁇ orated into the DPI 3 probes is dramatic and su ⁇ rising.
• the combination of the redl 3 quencher and the DPI 3 resulted in a 10-fold increase in S/N for FAM quenching and a 28-fold increase in S/N for TAMRA quenching. It is su ⁇ rising and that the DPI 3 residue helps improve fluorescent quenching by the dabcyl and redl 3 chromophores.
• DPI 3 probes prepared with 5 '-fluorescein and the redl 3 amide linker were tested in the 5'-nuclease assay to see if the hybridization properties were compatible with the linker system.
• both dabcyl and Redl 3 worked as quenchers for fluorescein in the 5'-nuclease assay when used in MGB probes.
• Red 13 performed better than dabcyl as evidenced by the lower initial fluorescence (background) and the higher plateau after PCR.
• the 5'-fluorophore-ODN-Q- MGB conjugates of the instant invention have improved performance in assays designed to detect DNA targets by direct hybridization. A basic description of this method is found in United States Patent No. 5,876,930, inco ⁇ orated herein by reference. In this assay format, the non-hybridized probes (quenched by FRET) become fluorescent upon forming a rigid duplex structure, thereby separating the quencher and fluorophore.
• Red 13 Chromophore Quenches a Broad Range of Fluorescent Reporter Groups A series of DPI 3 probes with the redl 3 amide were prepared with several different fluorescent reporter groups to examine the effective range of quenching. Probes were digested with PDE as usual and showed good S/N for dyes which emit from 458 -665 nm. Table 3. Performance of Fluorogenic DPI 3 Probes with Various Fluorophores.
• the structure of the fluorogenic probes was FL-ODN-Q-CDPI 3 where Q is the redl 3 amide and the ODN sequence was 5 '- GTC CTG A TT TTA C (SEQUENCE Id. No. 2).
• the fluorophores FAM, TAMRA, cy3 and cy5 were incorporated using commercially available phosphoramidite reagents.
• the coumarin and resorufin fluororophores were incorporated using phosphoramidites 34 and 37 which were prepared as described below.
• the fluorescent emission is well separated from FAM, as shown in the overlayed spectra in Figure 4. As shown in Table 3, the resorufin fluorescence is also quenched by the redl 3 chromophore.
• the resorufin phosphoramidite has excellent properties for use in FRET probes and in combination with FAM for multicolor analysis. As shown in Table 3, the coumarin fluorescence is also quenched by the redl 3 chromophore. Thus, the coumarin phosphoramidite reagent can be inco ⁇ orated in FRET probes and particularly in combination with FAM for multicolor analysis.
• the improved quencher molecules can be used in other FRET based assay systems.
• a quencher molecule and fluorophore are attached to an enzyme substrate, which through its catalytic action on this Q-substrate-fluorophore conjugates cleaves and separates the Q and fluorophore molecules.
• the pentafluorophenyl activated ester 11 shown in Reaction Scheme 3 can be used for labeling lysine residues of peptides for studying proteolytic enzymes.
• the pentafluorophenyl ester (11) is synthesized by the same method used for the synthesis of Compound 22 as described in Example 4 and Reaction Scheme 5.
• RED 13-pyrrolidine-DMTr-CPG 10 g of LC AA CPG was combined with 5 ml of a 0.3 M solution of 11 in DMF and agitated gently overnight, when it was filtered and washed with 2x 100 mL of DMF, 2x 100 mL of acetonitrile, and 2x 100 mL of ether. Traces of ether were removed in vacuo (oil pump). Unreacted amino groups were acetylated by treating the CPG with 40 mL of dry pyridine and 5 mL of acetic anhydride. After swirling for 1.5 h, the CPG was filtered and washed 1 with 2x 100 mL of DMF, 2x 100 mL of acetonitrile, and 2x 100 mL of ether.
• reaction is allowed to proceed for an hour, at 5 which time, a TLC analysis is done (2: 1 hexanes/ethyl acetate) examined by 6 UV (254 nm) to determine whether the reaction is complete. If it is complete, 7 then the baseline spot (bluish) will disappear and the product, with an R f of 8 0.55, will appear as a dark spot. Often, especially if the reactants are not 9 entirely dry, another portion of triphenylphosphine and DEAD are required. 0 If so, a tenth of the original amounts is usually sufficient, i.e., 1.73 g of triphenylphosphine and 1.04 mL of DEAD. These can be added neat to the stirred solution.
• Ethyl 4-[(3-hydroxypropyl)phenylamino]butanoate (19) A mixture of 3-(phenylamino)propan-l-ol (Huang, Yande; Arif, Atta M.; Bentrude, Weseley G.; J.Org.Chem.; 58(23) 1993; 6235-6246) (65.6 g, 0.43 mol), ethyl 4-bromobutyrate (104.5 g, 0.54 mol) and 100 mL of ethyldiisopropylamine is stirred at 100°C for 1 h. The reaction is cooled to room temperature and partitioned between water 400 mL and ethyl acetate (500 mL).
• the flask is fitted with a dropping funnel, and a solution of 1.51 g (21.9 mmol) sodium nitrite in 3-4 mL of water is added to the dropping funnel and slowly added to the solution in the flask with stirring, over about 20 minutes.
• 0.6 g (-21 mmol) of urea is added followed by 2.73 g of ethyl 4-[(3- hydroxypropyl)phenylamino]butanoate as a solution in 8.2 mL acetic acid.
• 20 g of sodium acetate in ⁇ 50 mL of water is added. The mixture is allowed to stir for an hour at room temperature. Most of the product is separated as an emulsion.
• the mixture is partitioned between ethyl acetate and water.
• the organic layer is washed with NaHC0 3 (3x 50 ml), brine and dried over anhydrous sodium sulfate. Then the organic solvents are evaporated to a syrup.
• the crude product is chromatographed on silica gel (1.5 x 20 inches) eluting with 50% ethyl acetate/hexane. The appropriate fractions are collected, combined, evaporated (30-40 degrees), and dried in a vacuum. The product is a dark oil.
• pyridine is evaporated and the resulting syrup is dissolved in a few milliliters of 18: 1 : 1 methylene chloride/methanol/ triethylamine.
• a silica gel column ( ⁇ 1.5" x 20") is prepared with an eluent of 18:1 : 1 methylene chloride/methanol/ triethylamine and the product is run through the column, collecting and combining the appropriate fractions. After the solvents are removed by evaporation the resulting amo ⁇ hous solid contains some triethylammonium salts in addition to the desired product. The impurity does not interfere with the next step and the product is used without additional purification.
• PFP ester preparation The product obtained in the previous step is dissolved in 100 mL anhydrous DMF. Triethylamine (2 mL) is added followed by PFP-TFA (2 mL, 4.4 mmol). The reaction is stirred for 30 min and analyzed by HPLC. No starting material, free acid should be observed. DMF is evaporated and the residue, deep pu ⁇ le syrup is suspended in 100 mL MeOH. After stirring for 30 min, a dark precipitate is formed which is collected by filtration on a sintered glass funnel, washed with methanol (3x20 mL) and dried under vacuum (15 - 30 h). This procedure yields 2.7 g (94%) of the desired product as a pu ⁇ le solid.
• the product is dried in vacuo for an hour or two before it is used in the next step.
• the material is dissolved in 20 mL of DMF in a 100 mL flask and stirred to dissolve. Then 0.6 mL (4.3 mmol) of triethylamine is added, followed by 0.6 mL of PFP-TFA.
• the reaction mixture is stirred under argon overnight, and then evaporated to a gum and a ⁇ 10 mL of DMF is added, followed by ⁇ 80 mL of methanol. This mixture is swirled and sonicated, and then the product, which precipitates out, is filtered off and dried in vacuo. Yield is 85-90%. !
• Example 6 DMTrO-Red 13-amide-CDPI 3 -CPG (29) (Reaction Scheme 7) 3-[(4-Methoxyphenyl)diphenylamino]propan-l-ol (26). 4 g (53 mmol) of 3 -aminopropanol was dissolved by stirring in 50 mL of methylene chloride in an oven dried 250 mL round bottom flask. This solution was stoppered and set aside. 7.7 g (24.9 mmol) of monomethoxytrityl chloride (MMT-Cl, Aldrich reagent grade) was dissolved in another 50 ml of methylene chloride.
• MMT-Cl monomethoxytrityl chloride
• MMT loading (:mol / g ) A 72 x volume (in mL) x 14.3 ⁇ wt of CPG (mg) Synthesis of CPG 29.
• the CPG was filtered and washed with 2x 50 mL of DMF, 2x 50 mL of acetonitrile, and 2x 50 mL of ether. Traces of ether were removed in vacuo (oil pump).
• oligonucleotides were synthesized on the CPG 29 using standard phosphoramidite coupling chemistry except that the standard 0.1 I 2 oxidizing solution was diluted to 0.01-0.015 to avoid iodination of the MGB moiety.
• FAM and TET were inco ⁇ orated at the 5 'end using the corresponding phosphoramidites available from Glen Research.
• Example 8 4- ⁇ [N-(6- ⁇ [Bis(methylethyl)amino](2- cyanoethoxy)phosphinooxy ⁇ hexyl)carbamoyl]methyl ⁇ -2-oxo-2H- chromen-7-yl 2,2-dimethylpropanoate (34a). N-(6-Hydroxyhexyl)-2-(7-hydroxy-2-oxo(2H-chromen-4-yl))acetamide (32a). (7-Hydroxy-2-oxo-2H-chromen-4-yl)-acetic acid methyl ester (1) was synthesized according to Baker et al. (J. Chem. Soc; 1950; 170, 173.).
• the DMT derivative was dissolved in 100 mL of 10% MeOH in CH 2 C1 2 and treated with 0.5 mL of trifluoroacetic acid. After being stirred for 1 h, the reaction mixture was neutralized with triethylamine (0.7 mL) and concentrated. The resultant viscous oil was partitioned between ethyl acetate and water. The organic layer was dried over Na 2 S0 4 and concentrated. The solid obtained was suspended in ether (50 mL) and stirred for 30 min. The desired product was the insoluble material, and was collected by filtration, washed with ether and dried. The yield of the title product 33 was 1.6 g (64%).
• Example 9 8-(3- ⁇ [bis(methylethyl)amino](2-cyanoethoxy)phosphinooxy ⁇ propyl)-7- oxophenoxazin-3-yl 2,2-dimethylpropanoate (37a).
• (Reaction Scheme 9) 7-Hydroxy-2-(3-hydroxypropyl)phenoxazin-3-one (35a).
• a suspension of 4-nitrosorecorcinol 4.5 g, 32.4 mmol
• 4-(3- hydroxypropyl)benzene- 1,3 -diol Formulachiassin, M.; Russo, C, J. Heterocyc. Chem.
• Zinc dust (2.0 g) was added and the suspension was stirred for 20 min.
• the resultant pu ⁇ le mixture was filtered, the filtrate was vigorously stirred on air to oxidize the leuco resorufin, the product of partial over reduction.
• the reaction was acidified with acetic acid, the brown solid formed was collected by filtration washed with water and dried. The yield was 2.1 g.
• the material contained -50% of 2,3,4-trihydro- 2H-pyrano[3,2-b]phenoxazin-9-one, product of intramolecular cyclization which had been carried over from the first step. The rest of the material was the desired title compound 35.
• Residual pyridine was removed by co-evaporation with triethylamine and xylene.
• the resultant crude product 36a was chromatographed on silica eluting with 50% ethyl acetate/hexane.
• the DMT derivative was dissolved in 100 mL of 10% MeOH/CH 2 Cl 2 and treated with 0.5 mL of trifluoroacetic acid. After 1 h, triethylamine (2 mL) was added and the solution was concentrated. Chromatography on silica (ethyl acetate) and drying afforded 0.38 g of the desired product as an orange solid.
• 3-n-Butyl-6-[4-(2-hydroxyethyl)aminophenyl]uracil 40 A mixture of 6-chloro-3-n-butyluracil (10.4 g, 51.3 mmol), 2-(4- aminophenyl)ethanol (10.0 g, 72.9 mmol) and ethyldiisopropylamine (18 ml, 0.1 mol) was heated with stirring under argon on a 150° oil bath for 1 hr 20 min. The mixture was cooled to room temperature, diluted with 50 ml of water, treated with 10 ml of acetic acid and stirred for crystallization overnight.
• a precipitated solid was filtered, washed with 2% acetic acid, dried on filter and dissolved in 100 ml of hot 96% ethanol. To the solution 100 ml of hot water was added followed by 1.0 g of charcoal. The mixture was filtered hot and crystallized on ice. Yellow solid was collected by filtration and dried in vacuum to yield 10.7 g of 40, mp 207-208 °C.
• Solid 42 (1.2 g, 2.82 mmol) was evaporated with pyridine (10 ml), suspended in pyridine (13 ml), treated with Me 3 SiCl (2.2 ml, 17.3 mmol) and stirred under argon at ambient temperature for 30 min.
• the reaction mixture was cooled with ice and treated slowly with toluoyl chloride (5 ml, 28.8 mmol). Stirring was continued at room temperature for 2 hr, and the solvent evaporated.
• the residue was treated with acetic acid (10 ml) followed by addition of water (10 ml). Precipitated oil was extracted with hexanes (3x50 ml), and the residue that was insoluble in hexanes was evaporated with water.
• the reaction mixture was stirred under argon for 1 hr, treated with methanol (1 ml), taken into EtOAc (100 ml), washed with sat. NaCl solution and dried over Na 2 S0 4 .
• the solution was evaporated, purified by HPLC on silica gel column using a gradient system 0-50% B; CH 2 Cl 2 -hexanes-NEt 3 (15:30: 1) (A); EtOAc (B); detected at 320 nm.
• the main fraction was evaporated giving a colorless foam, 0.79 g (33%) of AG1 phosphoramidite 44.
• Dimethoxytrityl chloride (0.42 g) was added to a solution of 49 (0.75 g, 1.03 mmol) in 10 ml of dry pyridine. The solution was stirred for 4.0 hr under argon and then poured into 200 ml of 5% sodium bicarbonate solution. The product was extracted with 300 ml of ethyl acetate. The extract was dried over sodium sulfate and evaporated. The residue was purified by silica gel chromatography eluting with 10% methanol in ethyl acetate (1% triethylamine). The product fractions were evaporated affording a foam: 556 mg (57%>) yield.
• Example 12 Synthesis of fluorogenic oligodeoxynucleotide probes.
• the 3'- DPI 3 probes were prepared by automated DNA synthesis from a DPI 3 -modified glass support using methods described earlier (Lukhtanov et al. Biorg. Chem., 7: 564-567 (1996)). Oligonucleotide synthesis was performed on an ABI 394 synthesizer according to the protocol supplied by the manufacturer except that 0.015 M (instead of the standard 0.1 M) iodine solution was utilized in the oxidation step to avoid iodination of the CDPI 3 moiety.
• probes without 3'- CDPI 3 were prepared with the 3'-hydroxyhexyl phosphate as previously described (Gamper et al. Biochem. 36: 14816-14826 (1997)).
• the quencher phosphoramidites were added to the CPG and standard ⁇ - cyanoethylphosphoramidites and reagents (Glen Research, Sterling, VA) were used in oligonucleotide synthesis.
• 6-Carboxyfluorescein (6-FAM) phosphoramidite was used to introduce the 5 '-reporter dyes.
• TAMRA-dU phosphoramidite (Glen Research), cy3 or cy5 phosphoramidite (Glen Research), resorufin phosphoramidite, coumarin phosphoramidite, or AG phosphoramidite was used to introduce the indicated 5 '-fluorophore.
• 5'-Hexylamine phosphoramidite (Glen Research) was inco ⁇ orated into certain ODNs for post-synthetic conjugation of the 3'- quencher dye tetramethylrhodamine (TAMRA).
• TAMRA NHS ester (Glen Research) was used to acylate the hexylamine linkers in certain ODNs according to the protocol supplied by the manufacturer.
• the resulting CDPI 3 -probes with two conjugated dyes were purified by denaturing gel electrophoresis using 8% polyacrylamide. The desired bands were excised and the gel slices were incubated overnight at 37°C in 10 mL of 100 mM Tris-HCl, lOmM triethylammonium chloride, 1 mM EDTA (pH 7.8).
• the products were isolated from the extract by reverse phase HPLC, butanol concentration and sodium perchlorate precipitation.
• Example 14 Digestion of oligonucleotides by Snake Venom Phosphodiestaerase. Oligonucleotides were digested with snake venom phosphodiesterase (PDE) to study the fluorescence quenching potential of various quenchers. 200 nM of oligonucleotide was taken in a buffer containing 40 mM of NaCl, 20 mM of Tris (pH 8.9) , 5mM of MgCl 2 and 0.025%) of BSA.
• PDE snake venom phosphodiesterase
• Example 15 5' Nuclease PCR Assay.
• CDPI 3 -conjugated oligonucleotides were conjugated with a fluorophore, FAM at the 5' end and various quenchers were conjugated through a linker at the 3' end by the methods discussed above.
• 5' nuclease assays were performed with the above oligonucleotides to determine the quenching ability of the various quenchers under investigation. Fluorescent monitoring was performed in an Idaho Technologies LC-24 LightCycler.
• Each reaction contained PCR buffer (40 mM NaCl, 20 mM Tris HCl, pH 8.9, 5 mM MgS0 4 , 0.05% bovine serum albumin), 125 mM each dNTP, 0.5 mM each primer, 0.1 mM fluorescent CDPI 3 probe, 0.5 U/10 mL Taq polymerase and 0.1 ng/10 mL of synthetic DNA as template.
• the cycling program was 50 cycles (or as indicated) of 2 sec at 95EC, then 30 sec at the extension temperature (55-70E).

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