Quenching oligonucleotides
First Claim
Patent Images
1. A method for decreasing detectable luminescence of nucleic acid stain molecules that are noncovalently associated with an oligonucleotide, said method comprising:
- a) preparing the oligonucleotide by covalently attaching a quenching moiety to the oligonucleotide, wherein said quenching moiety is essentially nonfluorescent;
b) combining said oligonucleotide with a plurality of nucleic acid stain molecules such that the nucleic acid stain molecules associate noncovalently with said oligonucleotide;
wherein said nucleic acid stain molecules normally exhibit a luminescence when associated noncovalently with nucleic acids, and said nucleic acid stain molecules undergo energy transfer to said quenching moiety, such that said luminescence is detectably decreased.
3 Assignments
0 Petitions
Accused Products
Abstract
The invention relates to oligonucleotides labeled with an energy transfer acceptor useful in conjunction with fluorescent nucleic acid stains. The resulting oligonucleotides are useful for decreasing background fluorescence during amplification assays and in ligation assays, and for detecting hybridization.
-
Citations
64 Claims
-
1. A method for decreasing detectable luminescence of nucleic acid stain molecules that are noncovalently associated with an oligonucleotide, said method comprising:
-
a) preparing the oligonucleotide by covalently attaching a quenching moiety to the oligonucleotide, wherein said quenching moiety is essentially nonfluorescent;
b) combining said oligonucleotide with a plurality of nucleic acid stain molecules such that the nucleic acid stain molecules associate noncovalently with said oligonucleotide;
wherein said nucleic acid stain molecules normally exhibit a luminescence when associated noncovalently with nucleic acids, and said nucleic acid stain molecules undergo energy transfer to said quenching moiety, such that said luminescence is detectably decreased. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 58, 59, 60)
-
-
11. A method of amplifying a target nucleic acid sequence, comprising:
-
a) preparing a reaction mixture comprising one or more polymerase enzymes, the target nucleic acid sequence, and a quenching primer;
wherein said quenching primer is an oligonucleotide having 6-60 nucleotides that is covalently bound to a quenching moiety;
b) incubating said reaction mixture under conditions such that the target nucleic acid sequence is amplified to produce an amplification product mixture;
c) adding a nucleic acid stain before, during, or after said incubating step, such that the nucleic acid stain associates noncovalently with the nucleic acids present in said amplification product mixture;
wherein said nucleic acid stain normally exhibits a luminescence when associated noncovalently with nucleic acids, and the luminescence of said nucleic acid stain that is associated with said quenching primer or a dimer of said quenching primer is detectably quenched; and
d) illuminating said amplification product mixture to yield a luminescence response. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 61)
-
-
20. A method of elongating an oligonucleotide comprising:
-
a) preparing a mixture comprising an elongation enzyme and a quenching primer;
wherein said quenching primer is an oligonucleotide having 6-60 nucleotides that is covalently bound to a quenching moiety;
b) incubating said mixture under conditions such that the quenching primer is extended to produce an elongation product;
c) adding a nucleic acid stain before, during, or after said incubating step, such that the nucleic acid stain associates noncovalently with the nucleic acids present in said mixture;
wherein said nucleic acid stain normally exhibits a luminescence when associated noncovalently with nucleic acids, and said luminescence due to nucleic acid stain associated with said primer or a dimer of said primer is detectably quenched; and
d) illuminating said mixture to yield a luminescence response. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 62)
-
-
28. A method of assaying for a protein, comprising:
-
a) preparing a mixture comprising i) a quenching oligonucleotide, wherein said quenching oligonucleotide is an oligonucleotide having 6-60 nucleotides that is covalently bound to a quenching moiety; and
ii) a nucleic acid stain, wherein said nucleic acid stain normally exhibits a luminescence when associated noncovalently with nucleic acids;
such that said nucleic acid stain associates noncovalently with the oligonucleotide, and the luminescence due to the nucleic acid stain associated with said oligonucleotide is detectably quenched;
c) adding a sample that contains or is thought to contain said protein to said mixture;
d) incubating the mixture for a time sufficient for said protein to interact with said oligonucleotide;
e) illuminating said mixture to yield a luminescence response; and
f) correlating said luminescence response with the presence or activity of said protein. - View Dependent Claims (29, 30, 31, 63)
-
-
32. A composition, comprising
an oligonucleotide having 6-60 bases; -
a quenching moiety that is covalently bound to said oligonucleotide, wherein said quenching moiety is essentially nonfluorescent; and
a plurality of nucleic acid stain molecules, which may be the same or different, that are associated non-covalently with said oligonucleotide;
wherein said nucleic acid stain molecules normally exhibit a luminescence when associated noncovalently with nucleic acids;
wherein at least one of said nucleic acid stain molecules undergoes energy transfer of said luminescence to said quenching moiety. - View Dependent Claims (33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49)
wherein R1 is H, or R1 taken in combination with R2 is a fuised six-membered aromatic ring; R2 and R5 are independently H, F, Cl, Br, I, CN;
or C1-C18 alkyl, or C1-C18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
or —
SO3X where X is H or a counterion;
or R2 taken in combination with R1 is a fused six-membered aromatic ring;
or R5 taken in combination with R6 is a fused six-membered aromatic ring;
R3 and R4 are independently C1-C6 alkoxy, the alkyl portions of which are linear or branched, saturated or unsaturated;
R6 is H, or R6 taken in combination with R5 is a fused six-membered aromatic ring;
R10 is H, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
or R10 is a saturated or unsaturated C1-C18 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alcohol, —
SO3X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;
or R10 has the formula
where R12, R13, R14, R15 and R16 are independently H, F, Cl, Br, I, —
SO3X, a carboxylic acid, a salt of carboxylic acid, CN, hydroxy, amino, hydrazino;
or C1-C18 alkyl, C1-C18 alkoxy, C1-C18 alkylthio, C1-C18 alkanoylamino, C1-C18 alkylaminocarbonyl, C2-C36 dialkylaminocarbonyl, C1-C18 alkyloxycarbonyl, or C6-C18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alcohol, —
SO3X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;
or one pair of adjacent substituents R13 and R14, R14 and R15 or R15 and R16, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid;
or one of R12, R13, R14, R15 and R16 is a covalent linkage L;
provided that at least one of R10, R12, R13, R14, R15, R16, R18, or R19 is a covalent linkage to said oligonucleotide, L, wherein L is a single covalent bond, or a linear or branched, cyclic or heterocyclic, saturated or unsaturated covalent linkage having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;
or single, double, triple or aromatic carbon-carbon bonds;
or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen-nitrogen or nitrogen-oxygen bonds;
or aromatic or heteroaromatic bonds.
-
-
37. A composition, as claimed in claim 35, wherein the covalently bound quenching moiety is a triarylmethane dye having the formula
wherein R1, R2, R3, R4, R5, R6, R7, and R8 are independently H, F, Cl, Br, I, CN; - or C1-C18 alkyl, or C1-C18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
or —
SO3X where X is H or a counterion;
or R1 and R2, R3 and R4, R5 and R6, or R7 and R8, taken in combination form a fused six-membered aromatic ring;
A is OH or NR14R15;
wherein R14 and R15 are independently H, C1-C6 alkyl, C1-C6 carboxyalkyl, C1-C6 sulfoalkyl, a salt of C1-C6 carboxyalkyl, or a salt of C1-C6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alkyl;
or a covalent linkage L;
or R14 taken in combination with R15 forms a saturated 5- or 6-membered heterocycle that is optionally further substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alkyl, or a covalent linkage L;
or R14 in combination with R2, or R15 in combination with R3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C1-C6 alkyls or —
CH2SO3X;
B is O or N+R16R17;
wherein R16 and R17 are independently H, C1-C6 alkyl, C1-C6 carboxyalkyl, C1-C6 sulfoalkyl, a salt of C1-C6 carboxyalkyl, or a salt of C1-C6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alkyl;
or a covalent linkage L;
or R16 taken in combination with R17 forms a saturated 5- or 6-membered heterocycle that is optionally further substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alkyl;
or a covalent linkage L;
or R16 in combination with R6, or R17 in combination with R7, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C1-C6 alkyls or —
CH2SO3X;
where R9, R10, R11, R12 and R13 are independently H, F, Cl, Br, I, —
SO3X, a carboxylic acid, a salt of carboxylic acid, CN, hydroxy, amino, hydrazino;
or C1-C18 alkyl, C1-C18 alkoxy, C1-C18 alkylthio, C1-C18 alkanoylamino, C1-C18 alkylaminocarbonyl, C2-C36 dialkylaminocarbonyl, C1-C18 alkyloxycarbonyl, or C6-C18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alcohol, —
SO3X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;
or one pair of adjacent substituents R10 and R11, R11 and R12 or R12 and R13, when taken in combination, form a fuised 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid;
or one of R9, R10R11, R12, R13 and R14 is a covalent linkage L;
provided that at least one of R14, R15, R16, R17, R9, R10, R11, R12, R13, R14, or R15 is a covalent linkage to said oligonucleotide, L, wherein L is a single covalent bond, or a linear or branched, cyclic or heterocyclic, saturated or unsaturated covalent linkage having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;
or single, double, triple or aromatic carbon-carbon bonds;
or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen-nitrogen or nitrogen-oxygen bonds;
or aromatic or heteroaromatic bonds.
- or C1-C18 alkyl, or C1-C18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
-
38. A composition, as claimed in claim 34, wherein the covalently bound quenching moiety has the formula
wherein R1 is H, or R1 taken in combination with R2 is a fused six-membered aromatic ring; -
R2, R3, R4 and R5 are independently H, F, Cl, Br, I, CN;
or C1-C18 alkyl, or C1-C18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
or —
SO3X where X is H or a counterion;
or R2 taken in combination with R1 is a fused six-membered aromatic ring;
or R5 taken in combination with R6 is a fused six-membered aromatic ring;
R6 is H, or R6 taken in combination with R5 is a fused six-membered aromatic ring;
R8 and R9 are independently H, C1-C6 alkyl, C1-C6 carboxyalkyl, C1-C6 sulfoalkyl, a salt of C1-C6 carboxyalkyl, or a salt of C1-C6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alkyl;
or a covalent linkage L;
or one or more of R8 and R9 is a Q moiety;
or R8 taken in combination with R9 forms a saturated 5- or 6-membered heterocycle that is optionally fused to a Q moiety and optionally further substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alkyl, or a covalent linkage L;
or R8 in combination with R2, or R9 in combination with R3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C1-C6 alkyls or —
CH2SO3X;
wherein each Q moiety is 1-4 fused aromatic or heteroaromatic rings that is optionally substituted by halogen, cyano, sulfo, alkali or ammonium salt of sulfo, carboxy, alkali or ammonium salt of carboxy, nitro, alkyl, perfluoroalkyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino;
alkylamidol;
or a covalent linkage L;
wherein each heteroaromatic ring in Q is a 5- or 6-membered aromatic heterocycle having 1 to 3 heteroatoms selected from the group consisting of O, N or S in any combination; and
K is O or N+R18R19;
wherein R18 and R19 are independently H, C1-C6 alkyl, C1-C6 carboxyalkyl, C1-C6 sulfoalkyl, a salt of C1-C6 carboxyalkyl, or a salt of C1-C6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a w carboxylic acid ester of a C1-C6 alkyl;
or a covalent linkage L;
or one or more of R18 and R19 is a Q moiety;
or R18 taken in combination with R19 forms a saturated 5- or 6-membered heterocycle that is a piperidine, or a pyrrolidine that is optionally fused to a Q moiety, and optionally further substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alkyl;
or a covalent linkage L;
or R18 in combination with R4, or R19 in combination with R5, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C1-C6 alkyls or —
CH2SO3X moieties;
R10 is H, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
or R10 is a saturated or unsaturated C1-C18 alkyl that is optionally substituted one more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester a C1-C6 alcohol, —
SO3X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;
or R10 has the formula
where R12, R13, R14, R15 and R16 are independently H, F, Cl, Br, I, —
SO3X;
a carboxylic acid, a salt of carboxylic acid, CN, hydroxy, amino, hydrazino;
or C1-C18 alkyl, C1-C18 alkoxy, C1-C18 alkylthio, C1-C18 alkanoylamino, C1-C18 alkylaminocarbonyl, C2-C36 dialkylaminocarbonyl, C1-C18 alkyloxycarbonyl, or C6-C18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alcohol, —
SO3X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;
or one pair of adjacent substituents R13 and R14, R14 and R15 or R15 and R16, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid;
or one of R12, R13, R14, R15 and R16 is a covalent linkage L;
provided that at least one of R8, R9, R18, and R19 is, or is fused to, a Q moiety; and
further provided that at least one of R8, R9, R12, R13, R14, R15, R16, R18, or R19 is a covalent linkage to said oligonucleotide, L, wherein L is a single covalent bond, or a linear or branched, cyclic or heterocyclic, saturated or unsaturated covalent linkage having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;
or single, double, triple or aromatic carbon-carbon bonds;
or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen-nitrogen or nitrogen-oxygen bonds;
or aromatic or heteroaromatic bonds.
-
-
39. A composition, as claimed in claim 38, wherein for said covalently bound quenching moiety:
-
each Q moiety is a substituted or unsubstituted phenyl, naphthyl, anthracenyl, benzothiazole, benzoxazole, or benzimidazole;
K is N+R18R19;
R8═
R19 and R9═
R18; and
R10 has the formula
wherein one of R12-R16 is the covalent linkage L.
-
-
40. A composition, as claimed in claim 39, wherein each Q moiety is a phenyl or substituted phenyl;
- and R12 is the covalent linkage L;
wherein L is bound to the oligonucleotide at the 5′
-terminus, a base, a sugar, or a phosphate.
- and R12 is the covalent linkage L;
-
41. A composition, as claimed in claim 40, wherein the covalent linkage L is bound to the oligonucleotide at one or more purine or pyrimidine bases through an amide, ester, ether or thioether bond, or a transition metal complex.
-
42. A composition, as claimed in claim 40, wherein the covalent linkage L is bound to the oligonucleotide at the 5′
- -terminus.
-
43. A composition, as claimed in claim 32, further comprising a second oligonucleotide that is hybridized to said oligonucleotide.
-
44. A composition, as claimed in claim 32, wherein the nucleic acid stain is a monomeric cyanine dye, a dimeric cyanine dye, a phenanthridinium dye, an acridine dye, an indole dye, or a Hoechst dye.
-
45. A composition, as claimed in claim 42, wherein the nucleic acid stain is Hoechst 33258, Hoechst 33342, Hoechst 34580, ethidium, ethidium dimer, propidium, aminoactinomycin, DAPI, or acridine orange.
-
46. A composition, as claimed in claim 42, wherein the nucleic acid stain is an unsymmetrical cyanine dye or a dimer of an unsymmetrical cyanine dye.
-
47. A composition, as claimed in claim 44, wherein the nucleic acid stain exhibits a maximum absorption at from about 350 nm to about 520 nm when associated noncovalently with a nucleic acid.
-
48. A composition, as claimed in claim 32, further comprising additional nucleotides covalently attached at the 3′
- -terminus of said oligonucleotide.
-
49. A composition, as claimed in claim 32, wherein said oligonucleotide is covalently bound to one or more additional quenching moieties, that may be the same or different.
-
50. A kit, comprising:
-
a) a nucleic acid stain that normally exhibits a luminescence when associated noncovalently with nucleic acids; and
b) an oligonucleotide having 6-60 bases that is covalently bound to a quenching moiety capable of accepting energy transfer of said luminescence from said nucleic acid stain, wherein said quenching moiety is essentially nonfluorescent. - View Dependent Claims (51, 52, 53, 54, 55, 56, 57, 64)
wherein R1 is H, or R1 taken in combination with R2 is a fused six-membered aromatic ring; R2, R3, R4 and R5 are independently H, F, Cl, Br, I, CN;
or C1-C18 alkyl, or C1-C18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
or —
SO3X where X is H or a counterion;
or R2 taken in combination with R1 is a fused six-membered aromatic ring;
or R5 taken in combination with R6 is a fused six-membered aromatic ring;
R6 is H, or R6 taken in combination with R5 is a fused six-membered aromatic ring;
R8 and R9 are independently H, C1-C6 alkyl, C1-C6 carboxyalkyl, C1-C6 sulfoalkyl, a salt of C1-C6 carboxyalkyl, or a salt of C1-C6 sulfoaLkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alkyl;
or a covalent linkage L;
or one or more of R8 and R9 is a Q moiety;
or R8 taken in combination with R9 forms a saturated 5- or 6-membered heterocycle that is optionally fused to a Q moiety and optionally further substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alkyl, or a covalent linkage L;
or R8 in combination with R2, or R9 in combination with R3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C1-C6 alkyls or —
CH2SO3X;
wherein each Q moiety is a substituted or unsubstituted phenyl, naphthyl, anthracenyl, benzothiazole, benzoxazole, or benzimidazole that is optionally substituted by halogen, cyano, sulfo, alkali or ammonium salt of sulfo, carboxy, alkali or ammonium salt of carboxy, nitro, alkyl, perfluoroalkyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino;
alkylamido;
or a covalent linkage L;
wherein each heteroaromatic ring in Q is a 5- or 6-membered aromatic heterocycle having 1 to 3 heteroatoms selected from the group consisting of O, N or S in any combination; and
wherein R18 and R19 are independently H, C1-C6 alkyl, C1-C6 carboxyalkyl, C1-C6 sulfoalkyl, a salt of C1-C6 carboxyalkyl, or a salt of C1-C6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alkyl;
or a covalent linkage L;
or one or more of R18 and R19 is a Q moiety;
or R18 taken in combination with R19 forms a saturated 5- or 6-membered heterocycle that is a piperidine, or a pyrrolidine that is optionally fused to a Q moiety, and optionally further substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alkyl;
or a covalent linkage L;
or R18 in combination with R4, or R19 in combination with R5, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C1-C6 alkyls or —
CH2SO3X moieties;
R12, R13, R14, R15 and R16 are independently H, F, Cl, Br, I, —
SO3X, a carboxylic acid, a salt of carboxylic acid, CN, hydroxy, amino, hydrazino;
or C1-C18 alkyl, C1-C18 alkoxy, C1-C18 alkylthio, C1-C18 alkanoylamino, C1-C18 alkylaminocarbonyl, C2-C36 dialkylaminocarbonyl, C1-C18 alkyloxycarbonyl, or C6-C18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alcohol, —
SO3X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;
or one pair of adjacent substituents R13 and R14, R14 and R15 or R15 and R16, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid;
or one of R12, R13, R14, R15 and R16 is a covalent linkage L;
provided that at least one of R8, R9, R18, and R19 is, or is fused to, a Q moiety; and
further provided that at least one of R8, R9, R12, R13, R14, R15, R16, R18, or R19 is a covalent linkage to said oligonucleotide, L, wherein L is a single covalent bond, or a linear or branched, cyclic or heterocyclic, saturated or unsaturated covalent linkage having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;
or single, double, triple or aromatic carbon-carbon bonds;
or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen-nitrogen or nitrogen-oxygen bonds;
or aromatic or heteroaromatic bonds.
-
-
54. A kit, as claimed in claim 53, wherein each Q moiety is a phenyl or substituted phenyl;
- and R12 is the covalent linkage L;
wherein L is bound to the oligonucleotide at the 5′
-terminus, a base, a sugar, or a phosphate.
- and R12 is the covalent linkage L;
-
55. A kit, as claimed in claim 50, wherein the covalently bound quenching moiety is bound to the oligonucleotide at one or more purine or pyrimidine bases through an amide, ester, ether or thioether bond, or a transition metal complex.
-
56. A kit, as claimed in claim 50, wherein the covalently bound quenching moiety is bound to the oligonucleotide at the 5′
- -terminus.
-
57. A kit, as claimed in claim 50, wherein said nucleic acid stain is an unsymmetrical cyanine dye or a dimer of an unsymmetrical cyanine dye.
-
64. A kit, as claimed in claim 54, wherein said nucleic acid stain is an unsymmetrical cyanine dye or a dimer of an unsymmetrical cyanine dye.
Specification