US2004191796A1PendingUtilityA1
Fluorescent quenching detection reagents and methods
Est. expiryDec 8, 2019(expired)· nominal 20-yr term from priority
C07F 9/65522C07B 2200/11C07F 9/572C07H 21/00C09B 29/0813C07F 9/6561C07F 9/2408C07D 519/00C07C 245/08C40B 40/00C12Q 1/6818
48
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Claims
Abstract
Oligonucleotide-fluorophore-quencher conjugates wherein the fluorophore moiety has emission wavelengths in the range of about (300) to about (800) nm, and or where the quencher includes a substituted 4-(phenyldiazenyl)phenylamine structure provide improved signal to noise ratios and other advantageous characteristics in hybridization and related assays. The oligonucleotide-fluorophore-quencher conjugates can be synthesized by utilizing novel phosphoramidite reagents that incorporate the quencher moiety based on the substituted 4-(phenyldiazenyl)phenylamine structure, and or novel phosphoramidite reagents that incorporate a fluorophore moiety based on the substituted coumarin, substituted 7-hydroxy-3H-phenoxazin-3-one, or substituted 5,10-dihydro-10 [phenyl]pyrido[2,3-d;6,5-d′]dipyrimidine-2,4,6,8-(1H, 3H, 7H, 9H, 10H)-tetrone structure. Oligonucleotide-fluorophore-quencher-minor groove binder conjugates including a pyrrolo[4,5-e]indolin-7-yl}carbonyl)pyrrolo[4,5-e]indolin-7-yl]carbonyl}pyrrolo[4,5-e]indolin-7-caroxylate (DPI 3 ) moiety as the minor groove binder and the substituted 4-(phenyldiazenyl)phenylamine moiety as the quencher, were synthesized and have substantially improved hybridization and signal to noise ratio properties.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . An oligonucleotide conjugate having the formula
FL-ODN-Q
where ODN is an oligonucleotide or nucleic acid;
FL is a fluorophore moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, and
Q is a quencher moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, the quencher moiety having the structure
where R 0 , R 1 , R 2 , R 3 and R 4 are independently —H, halogen, —O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 where n=0 to 5, —NO 2 , —SO 3 , —N[(CH 2 ) n′ ,CH 3 ] 2 where n′=0 to 5 or —CN, and R 5 =—H or —(CH 2 ) n′ CH 3 where n″=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a.
2 . An oligonucleotide conjugate in accordance with claim 1 where R 0 is H, R 1 is NO 2 in the 4 position of the benzene nucleus, R 2 is H or Cl in the 2 position of the benzene nucleus, and R 3 and R 4 are hydrogen and R 5 is ethyl.
3 . An oligonucleotide conjugate in accordance with claim 1 where the quencher moiety and the linker attaching it to the ODN comprises the structures selected from the moieties shown by the formulas Q-1, Q-2 and Q-3
where q is 1 to 20, X is —O—, —OCH 2 — or —CH 2 —; t and v independently are 1 to 20, r and s independently are 1 to 20, and the conjugated quencher and linker moiety is attached to the ODN through one of the valence bonds designated a or b
4 . An oligonucleotide conjugate in accordance with claim 3 further comprising a minor groove binder moiety attached to the quencher-linker conjugate through one of the valence bonds designated a or b.
5 . An oligonucleotide conjugate in accordance with claim 1 where the quencher moiety and of the linker attaching it to the ODN comprises the structures selected from the moieties shown by the formulas Q-4, and Q-5
where R 6 is —(CH 2 ) n* where n * is 1 to 20, and t and v independently are 1 to 20, and where the quencher moiety is attached to the ODN through the valence bond designated a.
6 . A phosphoramidite reagent for preparing an oligonucleotide-fluorophore-quencher conjugate, the reagent including the moiety
where R 0 , R 1 , R 2 , R 3 and R 4 are independently —H, halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 where n=0 to 5, —NO 2 , —SO 3 , —N[(CH 2 ) n ,CH 3 ] 2 where n′=0 to 5 or —CN, and R 5 =—H or —(CH 2 ) n′ ,CH 3 where n″=0 to 5, and a bis(methylethyl)amino](2-cyanoethoxy)phosphinooxy moiety covalently linked thereto.
7 . A phosphoramidite reagent in accordance with claim 6 having the formula selected from the group consisting of the formulas designated PA-1, PA-2 and PA-3
where R 0 , R 1 , R 2 , R 3 and R4 are independently —H, halogen, —O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 where n=0 to 5, —NO 2 , —SO 3 , —N[(CH 2 ) n′ ,CH 3 ] 2 where n′=0 to 5 or —CN, and R 5 =—H or —(CH 2 ) n″ CH 3 where n″=0 to 5, q is 1 to 20, X is —O— or —CH 2 —; t, v, r and s independently are 1 to 20, and X 2 is H or dimethoxytrityl, methoxytrityl, trityl or an acid labile blocking group.
8 . A phosphoramidite reagent in accordance with claim 7 that has the formula designated PA-1.
9 . A phosphoramidite reagent in accordance with claim 7 that has the formula designated PA-2.
10 . A phosphoramidite reagent in accordance with claim 7 that has the formula designated PA-3.
11 . A phosphoramidite reagent in accordance with claim 7 where R o is H, R 1 is NO 2 in the 4 position of the benzene nucleus, R 2 is Cl in the 2 position of the benzene nucleus, and R 3 and R 4 are hydrogen and R 5 is ethyl.
12 . A covalently linked solid support and quencher conjugate suitable for oligonucleotide synthesis, having the structure
where CPG stands for a polymeric solid support;
LINKER is a moiety having the length of 1 to approximately 30 atoms and linking the diphenylazo moiety to the CPG;
X 2 is OH or, dimethoxytityl, methoxytrityl, trityl or an acid labile blocking group;
R 0 , R 1 , R 2 , R 3 and R4 are independently —H, halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 where n=0 to 5, —NO 2 , —SO 3 , —N[(CH 2 ) n′ CH 3 ] 2 where n′=0 to 5 or —CN, and R 5 =—H or —(CH 2 ) n″ CH 3 where n″=0 to 5.
13 . A covalently linked solid support and quencher conjugate in accordance with claim 12 selected from the structures
where R 6 is —(CH 2 ) n* where n* is 1 to 20, and q, r, t and v independently are 1 to 20.
14 . A covalently linked solid support and quencher conjugate in accordance with claim 13 where R o is H, R 1 is NO 2 in the 4 position of the benzene nucleus, R 2 is Cl in the 2 position of the benzene nucleus, and R 5 is ethyl.
15 . An oligonucleotide conjugate having the formula
FL-ODN-Q-MGB
where ODN is an oligonucleotide or nucleic acid;
FL is a fluorophore covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, and
Q is a quencher moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, the quencher moiety having the structure
where R 0 , R 1 , R 2 , R 3 and R 4 are independently —H, halogen,—O(CH2) n CH 3 , —(CH 2 ) n CH 3 where n=0 to 5, —NO 2 , —SO 3 , —N[(CH 2 ) n′ CH 3 ] 2 where n′=0 to 5 or —CN, and R 5 =—H, —(CH 2 ) n″ CH 3 or —(CH 2 ) n″ — where n″=0 to 5, and
MGB is minor groove binder moiety covalently attached to the ODN moiety or to the quencher moiety through a linker having the length of 0 to approximately 30 atoms.
16 . An oligonucleotide conjugate in accordance with claim 15 where the MGB moiety is attached to the quencher moiety, and the covalently bonded MGB -Q moiety has the structure
where t and v independently are 1 to 20, and the valence bond designated a attaches the MGB-Q moiety to the ODN moiety.
17 . An oligonucleotide conjugate in accordance with claim 16 where R o is H, R 1 is NO 2 in the 4 position of the benzene nucleus, R 2 is H or Cl in the 2 position of the benzene nucleus, and R 3 and R 4 are hydrogen.
18 . A covalently bonded minor groove binder and quencher reagent for oligonucleotide synthesis, having the formula
where R 0 , R 1 , R 2 , R 3 and R 4 are independently —H, halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 where n=0 to 5, —NO 2 , —SO 3 , —N[(CH 2 ) n′ CH 3 ] 2 where n′=0 to 5 or —CN, and t and v independently are 1 to 20;
X 2 is H or dimethoxytrityl, methoxytrityl, trityl or an acid labile blocking group, and
X 3 is pentafluorophenyloxy, or NH-LINKER-CPG or O-LINKER-CPG where CPG is a polymeric solid support and LINKER is a linking moiety having a length of approximately 0 to 30 atoms linking the tricyclic moiety to the CPG.
19 . A covalently bonded minor groove binder and quencher reagent in accordance with claim 18 wherein X 3 is pentafluorophenyloxy.
20 . A covalently bonded minor groove binder and quencher reagent in accordance with claim 18 wherein X 3 is NH-LINKER-CPG or O-LINKER-CPG.
21 . A covalently bonded minor groove binder and quencher reagent in accordance with claim 18 where R o is H, R 1 is NO 2 in the 4 position of the benzene nucleus, R 2 is H or Cl in the 2 position of the benzene nucleus, R 3 and R 4 are hydrogen and v=t=3.
22 . An oligonucleotide conjugate having the formula
FL-ODN-Q
where ODN is an oligonucleotide or nucleic acid;
Q is a quencher moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, and
FL is a fluorophore covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, said fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3,
wherein R 8 and R 9 independently are H, halogen, —NO 2 , —SO 3 , —C(═O)NH 2 , or —CN;—OR nn , —SR nn , —OR nn , —NHR nn ,—N[R nn ]2 where R nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;
R 10 , and R 11 independently are H, —CN , —OR 12 , —N(R 12 ) 2 , halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 , —NO 2 , —SO 3 , —C(═O)NH 2 , —N[(CH 2 ) n CH 3 ] 2 where n=0 to 5, or R 12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons;
R 15 is H or alkyl of 1 to 10 carbons;
R 16 is alkyl of 1 to 10 carbons, and
the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.
23 . An oligonucleotide conjugate in accordance with claim 22 where the fluorophore has the formula designated FL-1.
24 . An oligonucleotide conjugate in accordance with claim 23 where R 8 is OC(O)CH(CH 3 ) 2 and R 9 is H.
25 . An oligonucleotide conjugate in accordance with claim 22 where the fluorophore has the formula designated FL-2.
26 . An oligonucleotide conjugate in accordance with claim 25 where R 10 is OC(O)CH(CH 3 ) 2 and R 11 is H.
27 . An oligonucleotide conjugate in accordance with claim 22 where the fluorophore has the formula designated FL-3.
28 . An oligonucleotide conjugate in accordance with claim 28 where R 15 is methyl and R 16 is n-propyl.
29 . An oligonucleotide conjugate in accordance with claim 22 where the quencher moiety comprises the structure
where R 0 , R 1 , R 2 , R 3 and R 4 are independently —H, halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 where n=0 to 5, —NO 2 , —SO 3 , —N[(CH 2 ) n′ CH 3 ] 2 where n′=0 to 5 or —CN, and R 5 =−H or —(CH 2 ) n″ CH 3 where n″=0 to 5.
30 . An oligonucleotide conjugate in accordance with claim 22 comprising an additional minor groove binder moiety (MGB) attached to the quencher moiety through a linker having the length of 0 to approximately 30 atoms, whereby the oligonucleotide conjugate has the formula
FL-ODN-Q-MGB.
31 . An oligonucleotide conjugate of the formula
wherein R 0 , R 1 , R 2 , R 3 and R 4 are independently —H, halogen,—O(CH 2 ) n* CH 3 , —(CH 2 ) n* CH 3 where n*=0 to 5, —NO 2 , —SO 3 , —N[(CH 2 ) n′ CH 3 ] 2 where n′=0 to 5 or —CN;
FL is a fluorophore moiety with emission wavelengths in the range of about 300 to about 800 nm;
K is a linker containing 1 to approximately 30 atoms selected from the group consisting of C, O, N, S, P and H;
[A-B] n symbolizes an ODN, DNA, RNA or PNA or any combination thereof, where A is the sugar phosphate backbone where the sugar and the phosphate may independently be modified; B is a heterocyclic base, where B is independently selected from purine, pyrimidine, pyrazolo[3,4-d]pyrimidine, 7-substituted pyrazolo[3,4-d]pyrimidine- , 7-deazapurine, 7-substiuted 7-deazapurine, and modified purine- and pyrimidine-bases, and where the DNA, RNA, PNA or ODN can include any combinations of these bases, and
and n is the number of nucleotide units in said DNA, RNA, PNA or ODN;
W is a linker of a length of 0 to approximately 30 atoms, selected from the group consisting of C, O, N, S, P and H, and
m is an integer having the values of 1 to 20.
32 . An oligonucleotide conjugate in accordance with claim 31 where R o is H, R 1 is NO 2 in the 4 position of the benzene nucleus, R 2 is H or Cl in the 2 position of the benzene nucleus, and R 3 and R 4 are hydrogen.
33 . An oligonucleotide conjugate in accordance with claim 31 where said fluorophore moiety has the structure selected from the group designated FL-1, FL-2 and FL-3,
where R 8 is OH or O-alkanoyl where the alkanoyl group has 1 to 10 carbons;
R 9 is H or alkyl of 1 to 10 carbons;
R 10 and R 11 independently are H, —OR 12 , —NHR 13 , halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 , —NO 2 , —SO 3 , —C(═O)NH 2 , —N[(CH 2 ) n CH 3 ] 2 or —CN where n=0 to 5;
R 15 is H or alkyl of 1 to 10 carbons;
R 16 is alkyl of 1 to 10 carbons, and
the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker K.
34 . A phosphoramidite reagent for preparing an oligonucleotide-fluorophore-quencher conjugate, the reagent selected from the group consisting of the structures designated PA-4, PA-5 and PA-6,
wherein R 8 and R 9 independently are H, halogen, —NO 2 , —SO 3 , —C(═O)NH 2 , or —CN; —OR nn , —SR nn , —OR nn, —NHR nn ,—N[R nn ] 2 where R nn is independently H, a blocking group compatible with oligomer synthesis removable under acid or alkaline conditions; or an alkyl or alkanoyl group having 1 to 10 carbon atoms;
j and k independently are 1 to 10,
R 10 and R 11 independently are H —OR 12 , —NHR 13 , halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 , —NO 2 , —SO 3 , —C(═O)NH 2 , —N[(CH 2 ) n CH 3 ] 2 , O-alkyl or O-alkanoyl where the alkanoyl gro has 1 to 10 carbons, or —CN where n=0 to 5; h=1 to 20; and R 12 and R 13 are blocking groups compatible with ODN synthesis;
R 15 is H or alkyl of 1 to 10 carbons;
R 16 is alkyl of 1 to 10 carbons.
35 . A phosphoramidite reagent in accordance with claim 34 that has the formula designated PA-4.
36 . A phosphoramidite reagent in accordance with claim 35 where R 8 is —OC(O)CH(CH 3 ) 2 , R 9 is H, j is 2 and k is 6.
37 . A phosphoramidite reagent in accordance with claim 34 that has the formula designated PA-5.
38 . A phosphoramidite reagent in accordance with claim 37 where R 10 is OC(O)CH(CH 3 ) 2 , R 11 is H and h is 3.
39 . A phosphoramidite reagent in accordance with claim 34 that has the formula designated PA-6.
40 . A phosphoramidite reagent in accordance with claim 39 where R 15 is methyl and R 16 is n-propyl.
41 . A phosphoramidite reagent for preparing an oligonucleotide-fluorophore-quencher conjugate, the reagent having the formula
wherein R 0 , R 1 , R 2 , R 3 and R 4 are independently —H, halogen,—O(CH 2 ) n* CH 3 , —(CH 2 ) n* CH 3 where n*=0 to 5, —NO 2 , —SO 3 , —N[(CH 2 ) n′ CH 3 ] 2 where n′=0 to 5 or —CN, and R 5 =—H or —(CH 2 ) n″ CH 3 where n″=0 to 5;
n is 1 to 10;
q is 1 to 20, and
X 2 is H or dimethoxytrityl, methoxytrityl, trityl or an acid labile blocking group.
42 . A phosphoramidite reagent in accordance with claim 41 where R o is H, R 1 is NO 2 in the 4 position of the benzene nucleus, R 2 is Cl in the 2 position of the benzene nucleus, and R 3 and R 4 are hydrogen, R 5 is ethyl, n is 1 and q is 2.
43 . An oligonucleotide conjugate having the formula
FL-ODN-MGB
where ODN is an oligonucleotide or nucleic acid;
MGB is minor groove binder moiety covalently attached to the ODN moiety or to the quencher moiety through a linker having the length of 0 to approximately 30 atoms;
FL is a fluorophore covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, said fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3,
wherein R 8 and R 9 independently are H, halogen, —NO 2 , —SO 3 , —C(═O)NH 2 , or —CN;—OR nn , —SR nn , —OR nn ,—NHR nn,—N[R nn ] 2 where R nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;
R 10 and R 11 independently are H, —CN, —OR 12 , —N(R 12 ) 2 , halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 , —NO 2 , —SO 3 , —C(═O)NH 2 , —N[(CH 2 ) n CH 3 ] 2 where n=0 to 5, or R 12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,
R 15 is H or alkyl of 1 to 10 carbons;
R 16 is alkyl of 1 to 10 carbons, and
the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.
44 . An oligonucleotide conjugate in accordance with claim 43 where the fluorophore has the formula designated FL-1.
45 . An oligonucleotide conjugate in accordance with claim 44 where R 8 is —OC(O)CH(CH 3 ) 2 and R 9 is H.
46 . An oligonucleotide conjugate in accordance with claim 43 where the fluorophore has the formula designated FL-2.
47 . An oligonucleotide conjugate in accordance with claim 46 where R 10 is OC(O)CH(CH 3 ) 2 and R 11 is H.
48 . An oligonucleotide conjugate in accordance with claim 43 where the fluorophore has the formula designated FL-3.
49 . An oligonucleotide conjugate in accordance with claim 49 where R 15 is methyl and R 16 is n-propyl.
50 . An oligonucleotide conjugate having the formula
FL-ODN
where ODN is an oligonucleotide or nucleic acid;
FL is a fluorophore covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, said fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3,
wherein R 8 and R 9 independently are H, halogen, —NO 2 , —SO 3 , —C(═O)NH 2 , or —CN; —OR nn , —SR nn , —OR nn , —NHR nn ,—N[R nn ] 2 where R nn , is independently H, an alkyl group of 1 to 10 carbons or an- alkanoyl group of 1 to 10 carbons;
R 10 and R 11 independently are H, —CN, —OR 12 , —N(R 12 ) 2 , halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 , —NO 2 , —SO 3 , —C(=O)NH 2 , —N[(CH 2 ) n CH 3 ] 2 where n=0 to 5,or R 12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,;
R 15 is H or alkyl of 1 to 10 carbons;
R 16 is alkyl of 1 to 10 carbons, and
the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.
51 . An oligonucleotide conjugate in accordance with claim 50 where the fluorophore has the formula designated FL-1.
52 . An oligonucleotide conjugate in accordance with claim 51 where R 8 is OC(O)CH(CH 3 ) 2 and R 9 is H.
53 . An oligonucleotide conjugate in accordance with claim 50 where the fluorophore has the formula designated FL-2.
54 . An oligonucleotide conjugate in accordance with claim 53 where R 10 is OC(O)CH(CH 3 ) 2 and R 11 is H.
55 . An oligonucleotide conjugate in accordance with claim 50 where the fluorophore has the formula designated FL-3.
56 . An oligonucleotide conjugate in accordance with claim 55 where R 15 is methyl and R 16 is n-propyl.
57 . A method for hybridizing nucleic acids, comprising the steps of:
(a) providing a first nucleic acid and a second nucleic acid, (b) incubating the nucleic acids under hybridization conditions, and (c) identifying hybridized nucleic acids; wherein at least one of the nucleic acids comprises a FL-nucleic-acid-Q conjugate where FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and Q is a quencher moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, the quencher moiety having the structure where R 0 , R 1 , R 2 , R 3 and R 4 are independently —H, halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 where n=0 to 5, —NO 2 , —SO 3 , —N[(CH 2 ) n′ CH 3 ] 2 where n′=0 to 5 or —CN, and R 5 =—H or —(CH 2 ) n″ CH 3 where n″=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a.
58 . A method in accordance with claim 57 where in the formula Q of the quencher moiety R o is H, R 1 is NO 2 in the 4 position of the benzene nucleus, R 2 is Cl in the 2 position of the benzene nucleus, and R 3 and R4 are hydrogen and R 5 is ethyl.
59 . A method for hybridizing nucleic acids, comprising the steps of:
(a) providing a first nucleic acid and a second nucleic acid, (b) incubating the nucleic acids under hybridization conditions, and (c) identifying hybridized nucleic acids; wherein at least one of the nucleic acids comprises a FL-nucleic-acid-Q conjugate where Q is a quencher moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and wherein FL is a fluorophore covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, said fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3, wherein R 8 and R 9 independently are H, halogen, —NO 2 , —SO 3 , —C(═O)NH 2 , or —CN;—OR nn , —SR nn , —OR nn , —NHR nn ,—N[R nn ] 2 where R nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons; R 10 and R 11 independently are H, —CN, —OR 12 , —N(R 12 ) 2 , halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 , —NO 2 , —SO 3 , —C(═O)NH 2 , —N[(CH 2 ) n CH 3 ] 2 where n=0 to 5,or R 12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,; R 15 is H or alkyl of 1 to 10 carbons; R 16 is alkyl of 1 to 10 carbons, and the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.
60 . A method in accordance with claim 59 where the fluorophore has the formula designated FL-1.
61 . A method in accordance with claim 60 where R 8 is OC(O)CH(CH 3 ) 2 and R 9 is H.
62 . A method in accordance with claim 59 where the fluorophore has the formula designated FL-2.
63 . A method in accordance with claim 62 where R 10 is OC(O)CH(CH 3 ) 2 and R 11 is H.
64 . A method in accordance with claim 59 where the fluorophore has the formula designated FL-3.
65 . A method in accordance with claim 64 where R 15 is methyl and R 16 is n-propyl.
66 . A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps:
(a) providing a polynucleotide comprising a target sequence, (b) providing at least two FL-ODN-Q conjugates, wherein ODN represents an oligonucleotide moiety, one of the at least two FL-ODN-Q conjugates has a sequence that is perfectly complementary to the target sequence and at least one other of the FL-ODN-Q conjugates has a single-nucleotide mismatch with the target sequence; (c) separately incubating each of the FL-ODN-Q conjugates with the polynucleotide under hybridization conditions; and (d) determining the hybridization strength between each of the FL-ODN-Q and the polynucleotide, wherein FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and Q is a quencher moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, the quencher moiety having the structure where R 0 , R 1 , R 2 , R 3 and R are independently —H, halogen, —O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 where n=0 to 5, —NO 2 , —SO 3 , —N[(CH 2 ) n′ CH 3 ] 2 where n′=0 to 5 or —CN, and R 5 =—H or —(CH 2 ) n″ CH 3 where n″=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a.
67 . A method in accordance with claim 66 where in the formula of the quencher moiety Q R o is H, R 1 is NO 2 in the 4 position of the benzene nucleus, R 2 is Cl in the 2 position of the benzene nucleus, and R 3 and R 4 are hydrogen and R 5 is ethyl.
68 . A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps:
(a) providing a polynucleotide comprising a target sequence, (b) providing at least two FL-ODN-Q conjugates, wherein ODN represents an oligonucleotide moiety, one of the at least two FL-ODN-Q conjugates has a sequence that is perfectly complementary to the target sequence and at least one other of the FL-ODN-Q conjugates has a single-nucleotide mismatch with the target sequence; (c) separately incubating each of the FL-ODN-Q conjugates with the polynucleotide under hybridization conditions; and (d) determining the hybridization strength between each of the FL-ODN-Q and the polynucleotide, wherein Q is a quencher moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and the fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3, wherein R 8 and R 9 independently are H, halogen, —NO 2 , —SO 3 , —C(═O)NH 2 , or —CN;—OR nn , —SR nn , —OR nn , —NHR nn ,—N[R nn ] 2 where R nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons; R 10 and R 11 independently are H, —CN, —OR 12 , —N(R 12 ) 2 , halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 , —NO 2 , —SO 3 , —C(═O)NH 2 , —N[(CH 2 ) n CH 3 ] 2 where n=0 to 5,or R 12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons; R 15 is H or alkyl of 1 to 10 carbons; R 16 is alkyl of 1 to 10 carbons, and the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.
69 . A method in accordance with claim 68 where the fluorophore has the formula designated FL-1.
70 . A method in accordance with claim 69 where R 8 is OC(O)CH(CH 3 ) 2 and R 9 is H.
71 . A method in accordance with claim 68 where the fluorophore has the formula designated FL-2.
72 . A method in accordance with claim 71 where R 10 is OC(O)CH(CH 3 ) 2 and R 11 is H.
73 . A method in accordance with claim 68 where the fluorophore has the formula designated FL-3.
74 . A method in accordance with claim 73 where R 15 is methyl and R 16 is n-propyl.
75 . A method for hybridizing nucleic acids, comprising the steps of:
(a) providing a first nucleic acid and a second nucleic acid, (b) incubating the nucleic acids under hybridization conditions, and (c) identifying hybridized nucleic acids; wherein at least one of the nucleic acids comprises a FL-nucleic-acid-Q-MGB conjugate where FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, MGB is minor groove binder moiety covalently attached to the ODN moiety or to the quencher moiety through a linker having the length of 0 to approximately 30 atoms and Q is a quencher moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, the quencher moiety having the structure where R 0 , R 1 , R 2 , R 3 and R 4 are independently —H, halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 where n=0 to 5, —NO 2 , —SO 3 , —N[(CH 2 ) n′ CH 3 ] 2 where n′=0 to 5 or —CN, and R 5 =—H or —(CH 2 ) n″ CH 3 where n″=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a.
76 . A method for hybridizing nucleic acids, comprising the steps of:
(a) providing a first nucleic acid and a second nucleic acid, (b) incubating the nucleic acids under hybridization conditions, and (c) identifying hybridized nucleic acids; wherein at least one of the nucleic acids comprises a FL-ODN-Q-MGB conjugate where ODN is a nucleic acid or modified nucleic acid, MGB is minor groove binder moiety covalently attached to the ODN moiety or to the quencher moiety through a linker having the length of 0 to approximately 30 atoms, Q is a quencher moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, and FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and the fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3, wherein R 8 and R 9 independently are H, halogen, —NO 2 , —SO 3 , —C(═O)NH 2 , or —CN;—OR nn , —SR n , —OR nn ,—NHR nn ,—N[R nn ] 2 where R nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons; R 10 and R 11 independently are H, —CN, —OR 12 , —N(R 12 ) 2 , halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 , —NO 2 , —SO 3 , —C(═O)NH 2 , —N[(CH 2 ) n CH 3 ] 2 where n=0 to 5,or R 12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons, R 15 is H or alkyl of 1 to 10 carbons; R 16 is alkyl of 1 to 10 carbons, and the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker; and Q comprises a diazo moiety having the formula: wherein covalent attachment to the linker is through the nitrogen atom designated as b.
77 . A method for hybridizing nucleic acids, comprising the steps of:
(a) providing a first nucleic acid and a second nucleic acid, (b) incubating the nucleic acids under hybridization conditions, and (c) identifying hybridized nucleic acids; wherein at least one of the nucleic acids comprises a FL-ODN-Q-MGB conjugate where ODN is a nucleic acid or modified nucleic acid, MGB is minor groove binder moiety covalently attached to the ODN moiety or to the quencher moiety through a linker having the length of 0 to approximately 30 atoms, Q is a quencher moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, and FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms,, and the fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3, wherein R 8 and R 9 independently are H, halogen, —NO 2 , —SO 3 , —C(═O)NH 2 , or —CN;—OR nn , —SR nn , —OR nn ,—NHR nn ,—N[R nn ] 2 where R nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons; R 10 and R 11 independently are H, —CN, —OR 12 , —N(R 12 ) 2 , halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 , —NO 2 , —SO 3 , —C(═O)NH 2 , —N[(CH 2 ) n CH 3 ] 2 where n=0 to 5 or R 12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons, R 15 is H or alkyl of 1 to 10 carbons; R 16 is alkyl of 1 to 10 carbons, and the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.
78 . A method for hybridizing nucleic acids, comprising the steps of:
(a) providing a first nucleic acid and a second nucleic acid, (b) incubating the nucleic acids under hybridization conditions, and (c) identifying hybridized nucleic acids; wherein at least one of the nucleic acids comprises a FL-ODN-Q conjugate where ODN is a nucleic acid or modified nucleic acid, Q is a quencher moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, and FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and the fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3 , wherein R 8 and R 9 independently are H, halogen, —NO 2 , —SO 3 , —C(═O)NH 2 , or —CN;—OR nn , —SR nn , —OR nn , —NHR nn ,—N[R nn 2 where R nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons; R 10 and R 11 independently are H, —CN, —OR 12 , —N(R 12 ) 2 , halogen,—O(CH 2 ) n CH 3 , —(CH 2 ) n CH 3 , —NO 2 , —SO 3 , —C(═O)NH 2 , —N[(CH 2 ) n CH 3 ] 2 where n=0 to 5, or R 12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons, R 15 is H or alkyl of 1 to 10 carbons; R 16 is alkyl of 1 to 10 carbons, and the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker; and Q comprises a diazo moiety having the formula: wherein covalent attachment to the linker is through the nitrogen atom designated as b.Cited by (0)
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