Fluorescent quenching detection reagents and methods
First Claim
1. An oligonucleotide conjugate having the formula FL-ODN-Qwhere 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 R0, R1, R2, R3 and R4 are independently —
H, halogen, —
O(CH2)nCH3, —
(CH2)nCH3 where n=0 to 5, —
NO2, —
SO3, —
N[(CH2)n′
,CH3]2 where n′
=0 to 5 or —
CN, and R5=—
H or —
(CH2)n′
CH3 where n″
=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a.
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Accused Products
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 (DPI3) 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.
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Citations
78 Claims
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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 R0, R1, R2, R3 and R4 are independently —
H, halogen, —
O(CH2)nCH3, —
(CH2)nCH3 where n=0 to 5, —
NO2, —
SO3, —
N[(CH2)n′
,CH3]2 where n′
=0 to 5 or —
CN, and R5=—
H or —
(CH2)n′
CH3 where n″
=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a. - View Dependent Claims (2, 3, 4, 5)
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- 6. A phosphoramidite reagent for preparing an oligonucleotide-fluorophore-quencher conjugate, the reagent including the moiety
- 12. A covalently linked solid support and quencher conjugate suitable for oligonucleotide synthesis, having the structure
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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 R0, R1, R2, R3 and R4 are independently —
H, halogen,—
O(CH2)nCH3, —
(CH2)nCH3 where n=0 to 5, —
NO2, —
SO3, —
N[(CH2)n′
CH3]2 where n′
=0 to 5 or —
CN, and R5=—
H, —
(CH2)n″
CH3 or —
(CH2)n″
—
where n″
=0 to 5, andMGB 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. - View Dependent Claims (16, 17)
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- 18. A covalently bonded minor groove binder and quencher reagent for oligonucleotide synthesis, having the formula
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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 R8and R9 independently are H, halogen, —
NO2, —
SO3, —
C(═
O)NH2, or —
CN;
—
ORnn, —
SRnn, —
ORnn, —
NHRnn,—
N[Rnn]2 where Rnn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;
R10, and R11 independently are H, —
CN , —
OR12, —
N(R12)2, halogen,—
O(CH2)nCH3, —
(CH2)nCH3, —
NO2, —
SO3, —
C(═
O)NH2, —
N[(CH2)nCH3]2 where n=0 to 5, or R12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons;
R15 is H or alkyl of 1 to 10 carbons;
R16 is alkyl of 1 to 10 carbons, and the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker. - View Dependent Claims (23, 24, 25, 26, 27, 29, 30)
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28. An oligonucleotide conjugate in accordance with claim 28 where R15 is methyl and R16 is n-propyl.
- 31. An oligonucleotide conjugate of the formula
- 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,
- 41. A phosphoramidite reagent for preparing an oligonucleotide-fluorophore-quencher conjugate, the reagent having the formula
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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 R8 and R9 independently are H, halogen, —
NO2, —
SO3, —
C(═
O)NH2, or —
CN;
—
ORnn, —
SRnn, —
ORnn,—
NHRnn,—
N[Rnn]2 where Rnn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;
R10 and R11 independently are H, —
CN, —
OR12, —
N(R12)2, halogen,—
O(CH2)nCH3, —
(CH2)nCH3, —
NO2, —
SO3, —
C(═
O)NH2, —
N[(CH2)nCH3]2 where n=0 to 5, or R12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,R15 is H or alkyl of 1 to 10 carbons;
R16 is alkyl of 1 to 10 carbons, and the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker. - View Dependent Claims (44, 45, 46, 47, 48)
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49. An oligonucleotide conjugate in accordance with claim 49where R15 is methyl and R16 is n-propyl.
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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 R8 and R9 independently are H, halogen, —
NO2, —
SO3, —
C(═
O)NH2, or —
CN;
—
ORnn, —
SRnn, —
ORnn, —
NHRnn,—
N[Rnn]2 where Rnn, is independently H, an alkyl group of 1 to 10 carbons or an- alkanoyl group of 1 to 10 carbons;
R10 and R11 independently are H, —
CN, —
OR12, —
N(R12)2, halogen,—
O(CH2)nCH3, —
(CH2)nCH3, —
NO2, —
SO3, —
C(=O)NH2, —
N[(CH2)nCH3]2 where n=0 to 5,or R12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,;
R15 is H or alkyl of 1 to 10 carbons;
R16 is alkyl of 1 to 10 carbons, and the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker. - View Dependent Claims (51, 52, 53, 54, 55, 56)
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57. A method for hybridizing nucleic acids, comprising the steps of:
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(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 structurewhere R0, R1, R2, R3 and R4 are independently —
H, halogen,—
O(CH2)nCH3, —
(CH2)nCH3 where n=0 to 5, —
NO2, —
SO3, —
N[(CH2)n′
CH3]2 where n′
=0 to 5 or —
CN, and R5=—
H or —
(CH2)n″
CH3 where n″
=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a. - View Dependent Claims (58)
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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, andwherein 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 R8 and R9 independently are H, halogen, —
NO2, —
SO3, —
C(═
O)NH2, or —
CN;
—
ORnn, —
SRnn, —
ORnn, —
NHRnn,—
N[Rnn]2 where Rnn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;
R10 and R11 independently are H, —
CN, —
OR12, —
N(R12)2, halogen,—
O(CH2)nCH3, —
(CH2)nCH3, —
NO2, —
SO3, —
C(═
O)NH2, —
N[(CH2)nCH3]2 where n=0 to 5,or R12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,;
R15 is H or alkyl of 1 to 10 carbons;
R16 is alkyl of 1 to 10 carbons, and the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker. - View Dependent Claims (60, 61, 62, 63, 64, 65)
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66. A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps:
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(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 R0, R1, R2, R3 and R are independently —
H, halogen, —
O(CH2)nCH3, —
(CH2)nCH3 where n=0 to 5, —
NO2, —
SO3, —
N[(CH2)n′
CH3]2 where n′
=0 to 5 or —
CN, and R5=—
H or —
(CH2)n″
CH3 where n″
=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a. - View Dependent Claims (67)
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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 R8and R9 independently are H, halogen, —
NO2, —
SO3, —
C(═
O)NH2, or —
CN;
—
ORnn, —
SRnn, —
ORnn, —
NHRnn,—
N[Rnn]2 where Rnn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;
R10 and R11 independently are H, —
CN, —
OR12, —
N(R12)2, halogen,—
O(CH2)nCH3, —
(CH2)nCH3, —
NO2, —
SO3, —
C(═
O)NH2, —
N[(CH2)nCH3]2 where n=0 to 5,or R12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons;
R15 is H or alkyl of 1 to 10 carbons;
R16 is alkyl of 1 to 10 carbons, and the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker. - View Dependent Claims (69, 70, 71, 72, 73, 74)
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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 R0, R1, R2, R3 and R4 are independently —
H, halogen,—
O(CH2)nCH3, —
(CH2)nCH3 where n=0 to 5, —
NO2, —
SO3, —
N[(CH2)n′
CH3]2 where n′
=0 to 5 or —
CN, and R5=—
H or —
(CH2)n″
CH3 where n″
=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a.
-
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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 R8 and R9 independently are H, halogen, —
NO2, —
SO3, —
C(═
O)NH2, or —
CN;
—
ORnn, —
SRn, —
ORnn,—
NHRnn,—
N[Rnn]2 where Rnn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;
R10 and R11 independently are H, —
CN, —
OR12, —
N(R12)2, halogen,—
O(CH2)nCH3, —
(CH2)nCH3, —
NO2, —
SO3, —
C(═
O)NH2, —
N[(CH2)nCH3]2 where n=0 to 5,or R12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,R15 is H or alkyl of 1 to 10 carbons;
R16 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.
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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 R8 and R9 independently are H, halogen, —
NO2, —
SO3, —
C(═
O)NH2, or —
CN;
—
ORnn, —
SRnn, —
ORnn,—
NHRnn,—
N[Rnn]2 where Rnn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;
R10 and R11 independently are H, —
CN, —
OR12, —
N(R12)2, halogen,—
O(CH2)nCH3, —
(CH2)nCH3, —
NO2, —
SO3, —
C(═
O)NH2, —
N[(CH2)nCH3]2 where n=0 to 5 or R12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,R15 is H or alkyl of 1 to 10 carbons;
R16 is alkyl of 1 to 10 carbons, and the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.
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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 R8 and R9 independently are H, halogen, —
NO2, —
SO3, —
C(═
O)NH2, or —
CN;
—
ORnn, —
SRnn, —
ORnn, —
NHRnn,—
N[Rnn2 where Rnn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;
R10 and R11 independently are H, —
CN, —
OR12, —
N(R12)2, halogen,—
O(CH2)nCH3, —
(CH2)nCH3, —
NO2, —
SO3, —
C(═
O)NH2, —
N[(CH2)nCH3]2 where n=0 to 5, or R12 is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,R15 is H or alkyl of 1 to 10 carbons;
R16 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.
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Specification