Non-fluorescent quencher compounds and biomolecular assays

Abstract

Bis-diazo,triaryl and aryldiazo-N-arylphenazonium quencher moieties, substituted with electron-withdrawing and electron-donating substituents which induce polarity in the delocalized aryl/diazo ring systems, are useful as labels when attached to biomolecules such as polynucleotides, nucleosides, nucleotides, and polypeptides. The quencher moieties are non-fluorescent and accept energy from fluorescent reporter labels by any energy-transfer mechanism, such as FRET. Fluorescence quencher compositions are useful in preparing quencher labelled biomolecules for various molecular biology assays based on fluorescence detection.

We claim:  
     
         1 . A fluorescence quencher composition having the structure:  
       
         
           
           
               
               
           
         
         wherein Y is selected from N and CR, where R is H, C 1 -C 6  alkyl or C 5 -C 14  aryl;  
         L 1 , L 2 , and L 3  are independently selected from a bond, C 1 -C 12  alkyldiyl, C 1 -C 12  alkoxyldiyl, C 1 -C 12  alkylaminodiyl, C 1 -C 12  alkylamidediyl, C 5 -C 14  aryldiyl, and 1-20 ethyleneoxy units;  
         X is an amino acid, a polypeptide, a nucleoside, a nucleotide, a polynucleotide, or a protected form thereof; or X is an acid-labile protecting group;  
         Z is selected from H, CO 2 H, OH, NH 2 , NHR, NR 2 , SH, an ester, a cleavable linker, a solid support, a reactive linking group, and a label selected from a fluorescent dye, a hybridization-stabilizing moiety, a chemiluminescent dye, and an affinity ligand; and  
         Q is selected from the diazo structures:  
         
           
             
             
                 
                 
             
           
         
         wherein Ar is C 5 -C 14  aryl; one of the aryl carbons of the diazo structures is the site of attachment to L 1 ; at least one aryl carbon of each diazo structure is substituted with an electron-withdrawing group and at least one aryl carbon of each diazo structure is substituted with an electron-donating group.  
       
     
     
         2 . The fluorescence quencher composition of  claim 1  wherein the electron-withdrawing groups are selected from NO 2 , CN, CF 3 , CO 2 H, CO 2 R, C(O)NH 2 , C(O)NHR, C(O)NR 2 , CHO, C(O)R, SO 2 R, SO 2 CF 3 , SO 2 OR, SO 3 H, NO, and C 5 -C 14  aryl, where R is H, C 1 -C 12  alkyl or C 5 -C 14  aryl.  
     
     
         3 . The fluorescence quencher composition of  claim 2  wherein a NO 2  is para to a diazo group.  
     
     
         4 . The fluorescence quencher composition of  claim 1  wherein the electron-donating groups are selected from O − , S − , NR 2 , NHR, NH 2 , NHC(O)R, OR, OH, OC(O)R, SR, SH, Br, I, Cl, F, R, and C 5 -C 14  aryl, where R is H, C 1 -C 12  alkyl or C 5 -C 14  aryl.  
     
     
         5 . The fluorescence quencher composition of  claim 4  wherein a OCH 3  is ortho or meta to a diazo group.  
     
     
         6 . The fluorescence quencher composition of  claim 1  where Z is OH.  
     
     
         7 . The fluorescence quencher composition of  claim 1  where Z is an ester selected from the structures:  
       
         
           
           
               
               
           
         
       
     
     
         8 . The fluorescence quencher composition of  claim 1  selected from the structures:  
       
         
           
           
               
               
           
         
       
       where n is 1 to 12.  
     
     
         9 . The fluorescence quencher composition of  claim 1  wherein X is selected from DMT, MMT, trityl, substituted trityl, pixyl, and trialkylsilyl.  
     
     
         10 . The fluorescence quencher composition of  claim 1  having the structure:  
       
         
           
           
               
               
           
         
         wherein A is a cleavable linker selected from the structures:  
         
           
             
             
                 
                 
             
           
         
         where R′ is H, C 1 -C 12  alkyl or C 1 -C 12  alkoxy;  
         L 4  is selected from a bond, C 1 -C 12  alkyldiyl, C 1 -C 12  alkoxyldiyl, C 1 -C 12  alkylaminodiyl, C 1 -C 12  alkylamidediyl, C 5 -C 14  aryldiyl, and 1-20 ethyleneoxy units; and  
         
           
             
             
                 
                 
             
           
         
         is a solid support.  
       
     
     
         11 . The fluorescence quencher composition of  claim 10  wherein X is a nucleotide.  
     
     
         12 . The fluorescence quencher composition of  claim 10  wherein the solid support is selected from polystyrene, controlled-pore-glass, silica gel, silica, polyacrylamide, polyacrylate, hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy, and copolymers and grafts of such.  
     
     
         13 . The fluorescence quencher composition of  claim 10  wherein the form of the solid support is selected from a particle, a bead, a membrane, a frit, a fiber, a tube, a capillary, a slide, a plate, a micromachined chip, an alkanethiol-gold layer, a magnetic bead, a non-porous surface, an addressable array, and polynucleotide-immobilizing medium.  
     
     
         14 . The fluorescence quencher composition of  claim 1  having the structure:  
       
         
           
           
               
               
           
         
       
       wherein A is a cleavable linker selected from the structures:  
       
         
           
           
               
               
           
         
         where R is H, C 1 -C 12  alkyl or C 1 -C 12  alkoxy;  
         L 4  and L 5  are independently selected from a bond, C 1 -C 12  alkyldiyl, C 1 -C 12  alkoxyldiyl, C 1 -C 12  alkylaminodiyl, C 1 -C 12  alkylamidediyl, C 5 -C 14  aryldiyl, and 1-20 ethyleneoxy units;  
         G is a hybridization-stabilizing moiety; and  
         
           
             
             
                 
                 
             
           
         
         is a solid support.  
       
     
     
         15 . The fluorescence quencher composition of  claim 14  in which G comprises:  
       
         
           
           
               
               
           
         
         where L are the sites of attachment to L 3  and L 5 .  
       
     
     
         16 . The fluorescence quencher composition of  claim 14  wherein the solid support is selected from polystyrene, controlled-pore-glass, silica gel, silica, polyacrylamide, magnetic beads, polyacrylate, hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy, and copolymers and grafts of such.  
     
     
         17 . The fluorescence quencher composition of  claim 14  wherein the form of the solid support is selected from a particle, a bead, a membrane, a frit, a fiber, a tube, a capillary, a slide, a plate, a micromachined chip, an alkanethiol-gold layer, a magnetic bead, a non-porous surface, an addressable array, and polynucleotide-immobilizing medium.  
     
     
         18 . The fluorescence quencher composition of  claim 1  having the structure:  
       
         
           
           
               
               
           
         
         wherein X is an acid-labile protecting group; R 1  and R 2  are individually selected from isopropyl, morpholino, methyl, ethyl and C 5 -C 14  aryl; R 1  and R 2  taken together are C 4 -C 11  cycloalkyl or morpholino; and R 3  is C 1 -C 6  alkyl or C 5 -C 14  aryl.  
       
     
     
         19 . The fluorescence quencher composition of  claim 18  wherein R 1  and R 2  are each isopropyl and R 3  is cyanoethyl.  
     
     
         20 . The fluorescence quencher composition of  claim 18  wherein X is selected from DMT, MMT, trityl, substituted trityl, pixyl, and trialkylsilyl.  
     
     
         21 . The fluorescence quencher composition of  claim 11  having the structure:  
       
         
           
           
               
               
           
         
         wherein X is an acid-labile protecting group; B is a nucleobase; and R 3  is selected from H, C 1 -C 6  alkyl, and C 5 -C 14  aryl.  
       
     
     
         22 . The fluorescence quencher composition of  claim 11  having the structure:  
       
         
           
           
               
               
           
         
         wherein X is an acid-labile protecting group; B is a nucleobase; and R 3  is selected from H, C 1 -C 6  alkyl, and C 5 -C 14  aryl.  
       
     
     
         23 . The fluorescence quencher composition of  claim 1  where X is a polynucleotide.  
     
     
         24 . The fluorescence quencher composition of  claim 23  wherein the polynucleotide comprises one or more N-[2-(aminoethyl)]glycine units having a nucleobase attached to nitrogen through a methylene carbonyl linkage.  
     
     
         25 . The fluorescence quencher composition of  claim 23  wherein the polynucleotide comprises one or more 2′-4′ or 3′-4′ bicyclic sugar modifications.  
     
     
         26 . A labelled nucleoside or nucleotide having the formula:  
       
         
           
           
               
               
           
         
         wherein Q is a quencher moiety selected from the diazo structures:  
         
           
             
             
                 
                 
             
           
         
         wherein Ar is C 5 -C 14  aryl; one of the aryl carbons of the diazo structures is the site of attachment to L; at least one aryl carbon of each diazo structure is substituted with an electron-withdrawing group and at least one aryl carbon of each diazo structure is substituted with an electron-donating group;  
         B is a nucleobase;  
         R 19  is H, monophosphate, diphosphate, triphosphate, thiophosphate, phosphate analog, or acid-labile protecting group;  
         R 20  and R 21 , when taken alone, are each independently H, HO, F, or a moiety which terminates polymerase-mediated target-directed polymerization; or when taken together form 2′-3′-didehydroribose; and  
         L is a linker comprising an alkynyl, propargyl, propargylethoxyamido, vinyl, or allyl group.  
       
     
     
         27 . The labelled nucleoside or nucleotide of  claim 26  in which L comprises:  
       
         
           
           
               
               
           
         
         wherein n is 0, 1, or 2.  
       
     
     
         28 . The labelled nucleoside or nucleotide of  claim 26  which is enzymatically incorporatable.  
     
     
         29 . The labelled nucleoside or nucleotide of  claim 26  which is enzymatically extendable.  
     
     
         30 . The labelled nucleoside or nucleotide of  claim 26  which is a terminator.  
     
     
         31 . The labelled nucleoside or nucleotide of  claim 26  wherein Q further comprises a fluorescent dye, wherein the fluorescent dye and quencher moiety are covalently attached by a linker; and the fluorescent dye is selected from a fluorescein dye, a rhodamine dye, a benzophenoxazine, and a cyanine dye.  
     
     
         32 . A nucleobase-labelled polynucleotide having the formula:  
       
         
           
           
               
               
           
         
       
       comprising 2 to 100 nucleotides, wherein 
 Q is a quencher moiety selected from the diazo structures:  
                     
 wherein Ar is C 5 -C 14  aryl; one of the aryl carbons of the diazo structures is the site of attachment to L; at least one aryl carbon of each diazo structure is substituted with an electron-withdrawing group and at least one aryl carbon of each diazo structure is substituted with an electron-donating group;  
 B is a nucleobase;  
 R 21  is H, OH, halide, azide, amine, C 1 -C 6  aminoalkyl, C 1 -C 6  alkyl, allyl, C 1 -C 6  alkoxy, —OCH 3 , or —OCH 2 CH═CH 2 ;  
 R 22  is H. phosphate, internucleotide phosphodiester, or internucleotide analog;  
 R 23  is H, phosphate, internucleotide phosphodiester, or internucleotide analog; and  
 L is a linker comprising an alkynyl, propargyl, propargylethoxyamido, vinyl, allyl, or C 1 -C 12  alkyldiyl group.  
 
     
     
         33 . The nucleobase-labelled polynucleotide of  claim 32  in which L comprises:  
       
         
           
           
               
               
           
         
         wherein n is 0, 1, or 2.  
       
     
     
         34 . The nucleobase-labelled polynucleotide of  claim 32  which further comprises one or more N-[2-(aminoethyl)]glycine units having a nucleobase attached to nitrogen through a methylene carbonyl linkage.  
     
     
         35 . The nucleobase-labelled polynucleotide of  claim 32  which further comprises one or more 2′-4′ or 3′-4′ bicyclic sugar modifications.  
     
     
         36 . The nucleobase-labelled polynucleotide of  claim 32  wherein Q further comprises a fluorescent dye, wherein the fluorescent dye and quencher moiety are covalently attached by a linker; and the fluorescent dye is selected from a fluorescein dye, a rhodamine dye, a benzophenoxazine, and a cyanine dye.  
     
     
         37 . A method of labelling a polypeptide comprising the step of reacting a linking moiety of a fluorescence quencher with a polypeptide to form a labelled quencher-polypeptide conjugate, 
 wherein the linking moiety is selected from the group consisting of an azido, a monosubstituted primary amine, a disubstituted secondary amine, a thiol, an hydroxyl, a halide, an epoxide, an N-hydroxysuccinimidyl ester, a carboxyl, and an activated ester;    whereby the quencher moiety is attached to a location of the polypeptide selected from the amino terminus, the carboxyl terminus, and an amino acid side-chain.    
     
     
         38 . A method of polynucleotide labelling comprising: 
 a) providing the fluorescence quencher composition of  claim 1  wherein Z is a solid support, L 2  is C 1 -C 12  alkoxydiyl, and X is an acid-labile protecting group;    b) reacting the labelled solid-support with acid to remove X;    c) adding a 3′-phosphoramidite, 5′ protected nucleoside and an activator, thereby forming a bond between L 2  and the 3′ terminus of the nucleoside;    d) adding an oxidizing reagent; and    e) repeating steps b) to d) until a labelled polynucleotide is synthesized.    
     
     
         39 . The method of polynucleotide labelling of  claim 38  further comprising capping any unreacted sites on the solid-support after step c).  
     
     
         40 . The method of polynucleotide labelling of  claim 38  wherein the 5′ terminus is attached to a fluorescent dye by a linkage, wherein the fluorescent is selected from a fluorescein, a rhodamine, a benzophenoxazine, and a cyanine.  
     
     
         41 . The method of polynucleotide labelling of  claim 38  wherein a nucleobase of the polynucleotide is labelled with a fluorescent dye, selected from a fluorescein, a rhodamine, a benzophenoxazine, and a cyanine, by a linkage at a position on the polynucleotide selected from the 8-position of a purine nucleobase, the 7- or 8-position of a 7-deazapurine nucleobase, and the 5-position of a pyrimidine nucleobase.  
     
     
         42 . The method of polynucleotide labelling of  claim 38  further comprising deprotecting the labelled polynucleotide.  
     
     
         43 . The method of polynucleotide labelling of  claim 38  wherein the solid-support is selected from polystyrene, controlled-pore-glass, silica gel, silica, polyacrylamide, magnetic beads, polyacrylate, hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy, and copolymers and grafts of such.  
     
     
         44 . The method of polynucleotide labelling of  claim 38  wherein the form of the solid support is selected from a particle, a bead, a membrane, a frit, a fiber, a tube, a capillary, a slide, a plate, a micromachined chip, an alkanethiol-gold layer, a magnetic bead, a non-porous surface, an addressable array, and polynucleotide-immobilizing medium.  
     
     
         45 . The method of polynucleotide labelling of  claim 38  wherein a plurality of polynucleotides covalently attached to a solid support in an addressable array are synthesized.  
     
     
         46 . A method of polynucleotide labelling comprising coupling a polynucleotide with the fluorescence quencher composition of  claim 18 , whereby a 5′ quencher labelled polynucleotide is formed.  
     
     
         47 . The method of polynucleotide labelling of  claim 46  wherein the 3′ terminus of the polynucleotide is covalently attached to a solid support.  
     
     
         48 . A 5′ quencher labelled polynucleotide having the formula:  
       
         
           
           
               
               
           
         
       
       comprising 2 to 100 nucleotides, wherein Q is a quencher moiety selected from the diazo structures:  
       
         
           
           
               
               
           
         
         wherein Ar is C 5 -C 14  aryl; one of the aryl carbons of the diazo structures is the site of attachment to L; at least one aryl carbon of each diazo structure is substituted with an electron-withdrawing group and at least one aryl carbon of each diazo structure is substituted with an electron-donating group;  
         B is a nucleobase;  
         X is O, NH, or S;  
         R 21  is H, OH, halide, azide, amine, C 1 -C 6  aminoalkyl, C 1 -C 6  alkyl, allyl, C 1 -C 6  alkoxy, —OCH 3 , or —OCH 2 CH═CH 2 ;  
         R 22  is internucleotide phosphodiester or internucleotide analog; and  
         L is C 1 -C 12  alkyldiyl, aryldiyl, or polyethyleneoxy.  
       
     
     
         49 . A 3′ quencher labelled polynucleotide having the formula:  
       
         
           
           
               
               
           
         
       
       comprising 2 to 100 nucleotides, wherein Q is a quencher moiety selected from the diazo structures:  
       
         
           
           
               
               
           
         
         wherein Ar is C 5 -C 14  aryl; one of the aryl carbons of the diazo structures is the site of attachment to L; at least one aryl carbon of each diazo structure is substituted with an electron-withdrawing group and at least one aryl carbon of each diazo structure is substituted with an electron-donating group;  
         B is a nucleobase;  
         X is O, NH, or S;  
         R 21  is H, OH, halide, azide, amine, C 1 -C 6  aminoalkyl, C 1 -C 6  alkyl, allyl, C 1 -C 6  alkoxy, —OCH 3 , or —OCH 2 CH═CH 2 ;  
         R 23  is internucleotide phosphodiester or internucleotide analog; and  
         L is C 1 -C 12  alkyldiyl, aryldiyl, or polyethyleneoxy.  
       
     
     
         50 . A method of primer extension comprising: 
 annealing a polynucleotide primer to a target polynucleotide; and    extending the primer by polymerase-mediated incorporation of a 2′-deoxynucleotide 5′-triphosphate;    wherein the primer or the nucleotide 5′-triphosphate is covalently attached by a linkage to an aryl carbon of a quencher moiety selected from the diazo structures:                          wherein Ar is C 5 -C 14  aryl; at least one aryl carbon of each diazo structure is substituted with an electron-withdrawing group and at least one aryl carbon of each diazo structure is substituted with an electron-donating group;    whereby a labeled polynucleotide is formed.    
     
     
         51 . The method of  claim 50  further comprising amplifying the target polynucleotide with nucleotide 5′-triphosphates, a polymerase, and two or more primers; wherein the primers are complementary to the target polynucleotide sequence and at least one primer is covalently attached by a linkage to an aryl carbon of a quencher moiety.  
     
     
         52 . The method of  claim 50  further comprising amplifying the target polynucleotide with nucleotide 5′-triphosphates, a polymerase, and two or more primers; wherein the primers are complementary to the target polynucleotide sequence and at least one nucleotide 5′-triphosphate is covalently attached by a linkage to an aryl carbon of a quencher moiety.  
     
     
         53 . The method of  claim 50  further comprising amplifying the target polynucleotide with nucleotide 5′-triphosphates, a polymerase, two or more primers; wherein the primers are complementary to the target polynucleotide sequence, and a detectable probe; wherein the detectable probe is complementary to the target polynucleotide and is covalently attached to a fluorescent dye and a quencher moiety.  
     
     
         54 . The method of  claim 53  further comprising detecting a signal from the fluorescent dye of said detectable probe.  
     
     
         55 . The method of  claim 54  wherein the signal is detected at each thermal cycle during amplification.  
     
     
         56 . The method of  claim 53  wherein said polymerase cleaves the detectable probe during amplification; whereby the fluorescent dye and the quencher moiety are separated.  
     
     
         57 . The method of  claim 56  further comprising detecting a signal from the fluorescent dye of said cleaved, detectable probe.  
     
     
         58 . The method of  claim 57  wherein the signal is detected at each thermal cycle during amplification.  
     
     
         59 . The method of  claim 53  wherein said fluorescent dye is attached to the 5′ terminus or 3′ terminus of the detectable probe.  
     
     
         60 . The method of  claim 53  wherein said quencher moiety is attached to the 5′ terminus or 3′ terminus of the detectable probe.  
     
     
         61 . The method of  claim 53  wherein the detectable probe is further labelled with a hybridization-stabilizing moiety.  
     
     
         62 . The method of  claim 61  wherein the hybridization-stabilizing moiety comprises the structure:  
       
         
           
           
               
               
           
         
         where L is an attachment site to the detectable probe.  
       
     
     
         63 . The method of  claim 50  further comprising: 
 forming one or more labeled polynucleotide fragments by polymerase-directed primer extension of a primer;  
 resolving the one or more labeled polynucleotide fragments; and  
 detecting the resolved labeled polynucleotide fragments.  
 
     
     
         64 . The method of  claim 63  wherein the resolving step is an electrophoretic size-dependent separation process and the one or more labeled polynucleotide fragments are detected by fluorescence.  
     
     
         65 . The method of  claim 64  wherein the primer is covalently attached by a linkage to an aryl carbon of a quencher moiety.  
     
     
         66 . The method of  claim 64  wherein a nucleotide 5′-triphosphate is covalently attached by a linkage to an aryl carbon of a quencher moiety.  
     
     
         67 . A method of oligonucleotide ligation comprising annealing two probes to a target sequence and forming a phosphodiester bond with a ligase enzyme between the 5′ terminus of one probe and the 3′ terminus of the other probe; wherein one probe is covalently attached to a fluorescent dye and the other probe is covalently attached by a linkage to an aryl carbon of a quencher moiety selected from the diazo structures:  
       
         
           
           
               
               
           
         
         wherein Ar is C 5 -C 14  aryl; at least one aryl carbon of each diazo structure is substituted with an electron-withdrawing group and at least one aryl carbon of each diazo structure is substituted with an electron-donating group;  
         whereby an oligonucleotide ligation product is formed.  
       
     
     
         68 . A method of hybridization detection comprising annealing a probe to a target polynucleotide sequence, wherein the probe is covalently attached to a fluorescent dye and a quencher moiety selected from the diazo structures:  
       
         
           
           
               
               
           
         
         wherein Ar is C 5 -C 14  aryl; at least one aryl carbon of each diazo structure is substituted with an electron-withdrawing group and at least one aryl carbon of each diazo structure is substituted with an electron-donating group; and  
         detecting a signal from the fluorescent dye.  
       
     
     
         69 . The method of hybridization detection of  claim 68  wherein the probe comprises one or more N-[2-(aminoethyl)]glycine units having a nucleobase attached to nitrogen through a methylene carbonyl linkage.  
     
     
         70 . The method of hybridization detection of  claim 68  wherein the probe comprises one or more 2′-4′ or 3′-4′ bicyclic sugar modifications.  
     
     
         71 . The method of hybridization detection of  claim 68  wherein the probe sequence comprises a self-complementary hairpin sequence, whereby an increase in fluorescence signal is detectable when the probe is annealed to the target sequence.  
     
     
         72 . A kit for primer extension comprising one or more nucleotide 5′-triphosphates and one or more primers wherein at least one primer is covalently attached by a linkage to an aryl carbon of a quencher moiety selected from the diazo structures:  
       
         
           
           
               
               
           
         
         wherein Ar is C 5 -C 14  aryl; at least one aryl carbon of each diazo structure is substituted with an electron-withdrawing group and at least one aryl carbon of each diazo structure is substituted with an electron-donating group.  
       
     
     
         73 . The kit of  claim 72  further comprising a polymerase.  
     
     
         74 . The kit of  claim 72  further comprising a chain-terminating nucleotide analog.  
     
     
         75 . A kit for nucleic acid amplification comprising two or more primers, and a detectable probe covalently attached to a fluorescent dye and a quencher moiety; wherein the detectable probe is covalently attached by a linkage to an aryl carbon of a quencher moiety selected from the diazo structures:  
       
         
           
           
               
               
           
         
         wherein Ar is C 5 -C 14  aryl; at least one aryl carbon of each diazo structure is substituted with an electron-withdrawing group and at least one aryl carbon of each diazo structure is substituted with an electron-donating group.