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US 9,435,796 B2Granted
Cyanine dyes
AssigneeBiosearch Technologies, Inc.
InventorsTim Carter, Mark Reddington
Life of this patent
Estimating the term…
Abstract
The invention provides a novel class of cyanine dyes that are functionalized with a linker moiety that facilitates their conjugation to other species. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.
Claims58
A method for detecting a nucleic acid target sequence, said method comprising:
- (a) contacting said target sequence with a detector oligonucleotide comprising a target binding sequence, said detector oligonucleotide having linked thereto,
- i) a cyanine dye, wherein said cyanine dye is covalently attached to said detector oligonucleotide by reaction of a reactive functional group on a cyanine dye reagent and a complementary group on said detector oligonucleotide, said cyanine dye reagent having the formula:
- wherein
- R1 is substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl groups and does not include a carboxylic acid ester moiety;
- R2 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
- R3 is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, or R2 and R3, together with the nitrogen to which they are attached, are optionally joined to form a substituted 5-7-membered heterocycloalkyl;
- wherein one of R2 and R3 comprises said reactive functional group;
- each of L is C(R17)
- wherein each R17 is selected from the group consisting of H, halide, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, and wherein two adjacent R17 groups may join to form a ring;
- n is an integer selected from 1, 2, 3 and 4;
- R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, H, NO2, CN, Z1R19, NR20R21, and C(Z2)R22;
- wherein
- two substituents selected from R4, R5, R6 and R7 on adjacent atoms of the 3H-indolium, together with the carbon atoms to which they are attached, are optionally joined to form an aromatic ring;
- and
- two substituents selected from R8, R9, R10 and R11 on adjacent atoms of the indoline, together with the carbon atoms to which they are attached, are optionally joined to form an aromatic ring;
- Z1 is O or S;
- Z2 is selected from the group consisting of O, S and NH;
- R19 and R20 are independently selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
- R21 is selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl and C(Z3)R22;
- wherein
- Z3 is selected from the group consisting of O, S and NH;
- R22 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR23, and NR24R25,
- wherein
- R23 is selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and C(O)R26
- wherein
- R26 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
- R24 and R25 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- or R20 and R21, together with the nitrogen to which they are attached, are selected from the group consisting of —NHNH2, —N═C═S and —N═C═O;
- R12 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;
- and
- R13, R14, R15 and R16 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- and
- ii) a quencher,
- wherein said detector oligonucleotide is in a conformation allowing donor-acceptor energy transfer between said cyanine dye and said quencher when said cyanine dye is excited;
- (b) hybridizing said target binding sequence to said target sequence, thereby altering said conformation of said detector oligonucleotide, causing a change in a fluorescence parameter;
- and
- (c) detecting said change in said fluorescence parameter, thereby detecting said nucleic acid target sequence.
34 more elements
A method for detecting amplification of a target sequence comprising, in an amplification reaction:
- (a) hybridizing to said target sequence a detector oligonucleotide comprising a single-stranded target binding sequence and an intramolecularly associated secondary structure 5′ to said target binding sequence,
- wherein at least a portion of said detector oligonucleotide is a single stranded tail which is available for hybridization to said target sequence, said detector oligonucleotide having linked thereto,
- i) a cyanine dye, wherein said cyanine dye is covalently attached to said detector oligonucleotide by reaction of a reactive functional group on a cyanine dye reagent and a complementary group on said detector oligonucleotide, said cyanine dye reagent having the formula:
- wherein
- R1 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl groups and does not include a carboxylic acid ester moiety;
- R2 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
- R3 is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, or R2 or R3, together with the nitrogen to which they are attached, are optionally joined to form a substituted 5-7-membered heterocycloalkyl;
- wherein one of R2 and R3 comprises said reactive functional group;
- each of L is C(R17)
- wherein each R17 is a member independently selected from H, halide, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, and wherein two adjacent R17 groups may join to form a ring;
- n is an integer selected from 1, 2, 3 and 4;
- R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, H, NO2, CN, Z1R19, NR20R21, and C(Z2)R22;
- wherein
- two substituents selected from R4, R5, R6 and R7 on adjacent atoms of the 3H-indolium, together with the carbon atoms to which they are attached, are optionally joined to form an aromatic ring;
- and
- two substituents selected from R8, R9, R10 and R11 on adjacent atoms of the indoline, together with the carbon atoms to which they are attached, are optionally joined to form an aromatic ring;
- Z1 is a member selected from O and S;
- Z2 is a member selected from O, S and NH;
- R19 and R20 are independently selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
- R21 is selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl and C(Z3)R22;
- wherein
- Z3 is selected from the group consisting of O, S and NH;
- R22 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR23, and NR24R25,
- wherein
- R23 is selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and C(O)R26
- wherein
- R26 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
- R24 and R25 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- or R20 and R21, together with the nitrogen to which they are attached, are selected from the group consisting of —NHNH2, —N═C═S and —N═C═O;
- R12 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;
- and
- R13, R14, R15 and R16 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- and
- ii) a quencher,
- wherein said detector oligonucleotide is in a conformation allowing donor-acceptor energy transfer between said cyanine dye and said quencher when said cyanine dye is excited;
- (b) extending said hybridized detector oligonucleotide on said target sequence with a polymerase to produce a detector oligonucleotide extension product and separating said detector oligonucleotide extension product from said target sequence;
- (c) hybridizing a primer to said detector oligonucleotide extension product and extending the primer with said polymerase, thereby linearizing said intramolecularly associated secondary structure and producing a change in a fluorescence parameter;
- and
- (d) detecting said change in said fluorescence parameter, thereby detecting said target sequence.
36 more elements
A method for detecting a nucleic acid target sequence, said method comprising:
- (a) contacting said target sequence with a detector oligonucleotide comprising a target binding sequence, said detector oligonucleotide having linked thereto,
- i) a cyanine dye, wherein said cyanine dye is covalently attached to said detector oligonucleotide by reaction of a reactive functional group on a cyanine dye reagent and a complementary group on said detector oligonucleotide, said cyanine dye reagent having the formula:
- wherein
- R1 is a member selected from substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl groups and does not include a carboxylic acid ester moiety;
- R2 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- R3 is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, or R2 and R3, together with the nitrogen to which they are attached, are optionally joined to form a substituted 5-7-membered heterocycloalkyl;
- wherein one of R2 and R3 comprises said reactive functional group;
- -(L=L)n-L= has a structure which is selected from the group consisting of:
- wherein
- Z4 is H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl;
- R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, H, NO2, CN, Z1R19, NR20R21, and C(Z2)R22;
- wherein
- Z1 is a member selected from O and S;
- Z2 is selected from the group consisting of O, S and NH;
- R19 and R20 are independently selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
- R21 is selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl and C(Z3)R22;
- wherein
- Z3 is selected from the group consisting of O, S and NH;
- R22 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR23, and NR24R25,
- wherein
- R23 is selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and C(O)R26
- wherein
- R26 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
- R24 and R25 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- or R20 and R21, together with the nitrogen to which they are attached, are selected from the group consisting of —NHNH2, —N═C═S and —N═C═O;
- R12 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;
- and
- R13, R14, R15 and R16 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- and
- ii) a quencher,
- wherein said detector oligonucleotide is in a conformation allowing donor-acceptor energy transfer between said cyanine dye and said quencher when said cyanine dye is excited;
- (b) hybridizing said target binding sequence to said target sequence, thereby altering said conformation of said detector oligonucleotide, causing a change in a fluorescence parameter;
- and
- (c) detecting said change in said fluorescence parameter, thereby detecting said nucleic acid target sequence.
31 more elements
A method for detecting amplification of a target sequence comprising, in an amplification reaction:
- (a) hybridizing to said target sequence a detector oligonucleotide comprising a single-stranded target binding sequence and an intramolecularly associated secondary structure 5′ to said target binding sequence,
- wherein at least a portion of said detector oligonucleotide is a single stranded tail which is available for hybridization to said target sequence, said detector oligonucleotide having linked thereto,
- i) a cyanine dye, wherein said cyanine dye is covalently attached to said detector oligonucleotide by reaction of a reactive functional group on a cyanine dye reagent and a complementary group on said detector oligonucleotide, said cyanine dye reagent having the formula:
- wherein
- R1 is a member selected from substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl groups and does not comprise a carboxylic acid ester moiety;
- R2 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- R3 is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, or R2 and R3, together with the nitrogen to which they are attached, are optionally joined to form a substituted 5-7-membered heterocycloalkyl;
- wherein one of R2 and R3 comprises said reactive functional group;
- -(L=L)n-L= has a structure which is a member selected from:
- wherein
- Z4 is H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl;
- R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, H, NO2, CN, Z1R19, NR20R21, and C(Z2)R22;
- wherein
- Z1 is a member selected from O and S;
- Z2 is selected from the group consisting of O, S and NH;
- R19 and R20 are independently selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
- R21 is a member selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl and C(Z3)R22;
- wherein
- Z3 is selected from the group consisting of O, S and NH;
- R22 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR23, and NR24R25,
- wherein
- R23 is selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and C(O)R26
- wherein
- R26 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
- R24 and R25 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- or R20 and R21, together with the nitrogen to which they are attached, are selected from the group consisting of —NHNH2, —N═C═S and —N═C═O;
- R12 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;
- and
- R13, R14, R15 and R16 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- and
- ii) a quencher,
- wherein said detector oligonucleotide is in a conformation allowing donor-acceptor energy transfer between said cyanine dye and said quencher when said cyanine dye is excited;
- (b) extending said hybridized detector oligonucleotide on said target sequence with a polymerase to produce a detector oligonucleotide extension product and separating said detector oligonucleotide extension product from said target sequence;
- (c) hybridizing a primer to said detector oligonucleotide extension product and extending the primer with said polymerase, thereby linearizing said intramolecularly associated secondary structure and producing a change in a fluorescence parameter;
- and
- (d) detecting said change in said fluorescence parameter, thereby detecting said target sequence.
33 more elements
A method for detecting or quantifying a nucleic acid, said method comprising:
- (a) contacting the nucleic acid with a mixture comprising at least a first carrier molecule and a second carrier molecule,
- wherein
- said first carrier molecule has covalently bound thereto a first quencher and a first fluorophore, wherein said first fluorophore is a cyanine dye, wherein said cyanine dye is covalently attached to said first carrier molecule by reaction of a reactive functional group on a cyanine dye reagent and a complementary group on said first carrier molecule, said cyanine dye reagent having the formula:
- wherein
- R1 is a member selected from substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl groups and does not include a carboxylic acid ester moiety;
- R2 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- R3 is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, or R2 and R3, together with the nitrogen to which they are attached, are optionally joined to form a substituted 5-7-membered heterocycloalkyl;
- wherein one of R2 and R3 comprises said reactive functional group;
- each of L is C(R17)
- wherein each R17 is a member independently selected from H, halide, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, and wherein two adjacent R17 groups may join to form a ring;
- n is an integer selected from 1, 2, 3 and 4;
- R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, H, NO2, CN, Z1R19, NR20R21, and C(Z2)R22;
- wherein
- two substituents selected from R4, R5, R6 and R7 on adjacent atoms of the 3H-indolium, together with the carbon atoms to which they are attached, are joined to form an aromatic ring;
- and
- two substituents selected from R8, R9, R10 and R11 on adjacent atoms of the indoline, together with the carbon atoms to which they are attached, are joined to form an aromatic ring
- Z1 is a member selected from O and S;
- Z2 is selected from the group consisting of O, S and NH;
- R19 and R20 are independently selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
- R21 is selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl and C(Z3)R22;
- wherein
- Z3 is selected from the group consisting of O, S and NH;
- R22 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR23, and NR24R25,
- wherein
- R23 is selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and C(O)R26
- wherein
- R26 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- R24 and R25 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- or R20 and R21, together with the nitrogen to which they are attached, are selected from the group consisting of —NHNH2, —N═C═S and —N═C═O;
- R12 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;
- and
- R13, R14, R15 and R16 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- and
- said second carrier molecule has attached thereto a member selected from a second fluorophore, a second quencher and a combination thereof,
- wherein said second fluorophore, said second quencher, or said second fluorophore and said second quencher is different from said first fluorophore and said first quencher, respectively;
- and
- (b) detecting a change in a fluorescent property of one or more component of the mixture, the nucleic acid or a combination thereof, thereby detecting or quantifying the nucleic acid.
34 more elements
A method for detecting or quantifying a nucleic acid, said method comprising:
- (a) contacting the nucleic acid with a mixture comprising at least a first carrier molecule and a second carrier molecule,
- wherein
- said first carrier molecule has covalently bound thereto a first quencher and a first fluorophore, wherein said first fluorophore is a cyanine dye, wherein said cyanine dye is covalently attached to said first carrier molecule by reaction of a reactive functional group on a cyanine dye reagent and a complementary group on said first carrier molecule, said cyanine dye reagent having the formula:
- wherein
- R1 is a member selected from substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl groups and does not include a carboxylic acid ester moiety;
- R2 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- R3 is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, or R2 and R3, together with the nitrogen to which they are attached, are optionally joined to form a substituted 5-7-membered heterocycloalkyl;
- wherein one of R2 and R3 comprises said reactive functional group;
- -(L=L)n-L= has a structure which is a member selected from:
- wherein
- Z4 is H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl;
- R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, H, NO2, CN, Z1R19, NR20R21, and C(Z2)R22;
- wherein
- Z1 is a member selected from O and S;
- Z2 is selected from the group consisting of O, S and NH;
- R19 and R20 are independently selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
- R21 is a member selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl and C(Z3)R22;
- wherein
- Z3 is elected from the group consisting of O S and NH;
- R22 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR23, and NR24R25,
- wherein
- R23 is selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and C(O)R26
- wherein
- R26 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
- R24 and R25 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- or R20 and R21, together with the nitrogen to which they are attached, are selected from the group consisting of —NHNH2, —N═C═S and —N═C═O;
- R12 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;
- and
- R13, R14, R15 and R16 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
- said second carrier molecule has attached thereto a member selected from a second fluorophore, a second quencher and a combination thereof,
- wherein said second fluorophore, said second quencher, or said second fluorophore and said second quencher is different from said first fluorophore and said first quencher, respectively;
- and
- (b) detecting a change in a fluorescent property of one or more component of the mixture, the nucleic acid or a combination thereof, thereby detecting or quantifying the nucleic acid.
30 more elements
Dependencies are inferred from the claim text.