Cyanine dyes
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.
1. 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
R 1 is substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl groups and does not include a carboxylic acid ester moiety;
R 2 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
R 3 is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, or R 2 and R 3 , together with the nitrogen to which they are attached, are optionally joined to form a substituted 5-7-membered heterocycloalkyl;
wherein one of R 2 and R 3 comprises said reactive functional group;
each of L is C(R 17 )
wherein each R 17 is selected from the group consisting of H, halide, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, and wherein two adjacent R 17 groups may join to form a ring;
n is an integer selected from 1, 2, 3 and 4;
R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 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, NO 2 , CN, Z 1 R 19 , NR 20 R 21 , and C(Z 2 )R 22 ;
wherein
two substituents selected from R 4 , R 5 , R 6 and R 7 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 R 8 , R 9 , R 10 and R 11 on adjacent atoms of the indoline, together with the carbon atoms to which they are attached, are optionally joined to form an aromatic ring;
Z 1 is O or S;
Z 2 is selected from the group consisting of O, S and NH;
R 19 and R 20 are independently selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
R 21 is selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl and C(Z 3 )R 22 ;
wherein
Z 3 is selected from the group consisting of O, S and NH;
R 22 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR 23 , and NR 24 R 25 ,
wherein
R 23 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)R 26
wherein
R 26 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
R 24 and R 25 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
or R 20 and R 21 , together with the nitrogen to which they are attached, are selected from the group consisting of —NHNH 2 , —N═C═S and —N═C═O;
R 12 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
R 13 , R 14 , R 15 and R 16 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.
2. The method according to claim 1 , wherein said fluorescence parameter is detected in real time.
3. The method according to claim 1 , wherein R 2 comprises said reactive functional group, and said reactive functional group is an oxygen-containing reactive functional group.
4. The method according to claim 3 , wherein said oxygen-containing reactive functional group is selected from the group consisting of hydroxyl and phosphoramidite.
5. The method according to claim 1 ,
wherein
R 1 is unsubstituted alkyl;
R 3 is H;
R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are each H;
R 12 is unsubstituted alkyl;
R 13 , R 14 , R 15 and R 16 are each unsubstituted alkyl; and
R 17 is H.
6. The method according to claim 5 , wherein n is 1 or 2.
7. The method according to claim 6 ,
wherein
R 1 is pentylene;
R 12 is ethyl; and
R 13 , R 14 , R 15 and R 16 are each methyl.
8. The method according to claim 1 , wherein the 3′-terminal nucleotide of said detector oligonucleotide is blocked or rendered incapable of extension by a nucleic acid polymerase.
9. The method according to claim 1 , wherein said detector oligonucleotide is not required to adopt a secondary structure for said detector oligonucleotide to function.
10. The method according to claim 1 , wherein
two substituents selected from R 4 , R 5 , R 6 and R 7 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 R 8 , R 9 , R 10 and R 11 on adjacent atoms of the indoline, together with the carbon atoms to which they are attached, are joined to form an aromatic ring.
11. The method according to claim 10 , wherein said aromatic ring is phenyl.
12. The method according to claim 1 , wherein one of R 2 and R 3 is attached to said detector oligonucleotide through a phosphodiester moiety.
13. 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
R 1 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl groups and does not include a carboxylic acid ester moiety;
R 2 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
R 3 is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, or R 2 or R 3 , together with the nitrogen to which they are attached, are optionally joined to form a substituted 5-7-membered heterocycloalkyl;
wherein one of R 2 and R 3 comprises said reactive functional group;
each of L is C(R 17 )
wherein each R 17 is a member independently selected from H, halide, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, and wherein two adjacent R 17 groups may join to form a ring;
n is an integer selected from 1, 2, 3 and 4;
R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 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, NO 2 , CN, Z 1 R 19 , NR 20 R 21 , and C(Z 2 )R 22 ;
wherein
two substituents selected from R 4 , R 5 , R 6 and R 7 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 R 8 , R 9 , R 10 and R 11 on adjacent atoms of the indoline, together with the carbon atoms to which they are attached, are optionally joined to form an aromatic ring;
Z 1 is a member selected from O and S;
Z 2 is a member selected from O, S and NH;
R 19 and R 20 are independently selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
R 21 is selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl and C(Z 3 )R 22 ;
wherein
Z 3 is selected from the group consisting of O, S and NH;
R 22 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR 23 , and NR 24 R 25 ,
wherein
R 23 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)R 26
wherein
R 26 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
R 24 and R 25 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
or R 20 and R 21 , together with the nitrogen to which they are attached, are selected from the group consisting of —NHNH 2 , —N═C═S and —N═C═O;
R 12 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
R 13 , R 14 , R 15 and R 16 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.
14. The method according to claim 13 , wherein said target sequence is amplified by a method selected from Strand Displacement Amplification, Polymerase Chain reaction, Self Sustained Sequence Replication, Transcription Mediated Amplification, and Nucleic Acid Sequence Based Amplification.
15. The method according to claim 13 , wherein said secondary structure further comprises a partially or entirely single-stranded restriction endonuclease site.
16. The method according to claim 13 , wherein a change in fluorescence intensity is detected.
17. The method according to claim 16 , wherein said change in fluorescence intensity is detected in real-time.
18. The method according to claim 13 , wherein said intramolecularly associated secondary structure comprises a portion of said target binding sequence.
19. The method according to claim 13 , wherein R 2 comprises said reactive functional group, and said reactive functional group is an oxygen-containing reactive functional group.
20. The method according to claim 19 , wherein said oxygen-containing reactive functional group is selected from the group consisting of hydroxyl and phosphoramidite.
21. The method according to claim 13 ,
wherein
R 1 is unsubstituted alkyl;
R 3 is H;
R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are each H;
R 12 is unsubstituted alkyl;
R 13 , R 14 , R 15 and R 16 are each unsubstituted alkyl; and
R 17 is H.
22. The method according to claim 21 , wherein n is 1 or 2.
23. The method according to claim 22 ,
wherein
R 1 is pentylene;
R 12 is ethyl; and
R 13 , R 14 , R 15 and R 16 are each methyl.
24. The method according to claim 13 , wherein the 3′-terminal nucleotide of said detector oligonucleotide is blocked or rendered incapable of extension by a nucleic acid polymerase.
25. The method according to claim 13 , wherein said detector oligonucleotide is not required to adopt a secondary structure for said detector oligonucleotide to function.
26. The method according to claim 13 , wherein
two substituents selected from R 4 , R 5 , R 6 and R 7 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 R 8 , R 9 , R 10 and R 11 on adjacent atoms of the indoline, together with the carbon atoms to which they are attached, are joined to form an aromatic ring.
27. The method according to claim 26 , wherein said aromatic ring is phenyl.
28. The method according to claim 13 , wherein said intramolecularly associated secondary structure is selected from the group consisting of hairpins, stem-loop structures, pseudoknots, and triple helices.
29. The method according to claim 13 , wherein one of R 2 and R 3 is attached to said detector oligonucleotide through a phosphodiester moiety.
30. 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
R 1 is a member selected from substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl groups and does not include a carboxylic acid ester moiety;
R 2 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
R 3 is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, or R 2 and R 3 , together with the nitrogen to which they are attached, are optionally joined to form a substituted 5-7-membered heterocycloalkyl;
wherein one of R 2 and R 3 comprises said reactive functional group;
-(L=L) n -L= has a structure which is selected from the group consisting of:
wherein
Z 4 is H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl;
R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 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, NO 2 , CN, Z 1 R 19 , NR 20 R 21 , and C(Z 2 )R 22 ;
wherein
Z 1 is a member selected from O and S;
Z 2 is selected from the group consisting of O, S and NH;
R 19 and R 20 are independently selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
R 21 is selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl and C(Z 3 )R 22 ;
wherein
Z 3 is selected from the group consisting of O, S and NH;
R 22 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR 23 , and NR 24 R 25 ,
wherein
R 23 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)R 26
wherein
R 26 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
R 24 and R 25 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
or R 20 and R 21 , together with the nitrogen to which they are attached, are selected from the group consisting of —NHNH 2 , —N═C═S and —N═C═O;
R 12 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
R 13 , R 14 , R 15 and R 16 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. The method according to claim 30 , wherein R 2 comprises said reactive functional group, and said reactive functional group is an oxygen-containing reactive functional group.
32. The method according to claim 31 , wherein said oxygen-containing reactive functional group is selected from the group consisting of hydroxyl and phosphoramidite.
33. The method according to claim 30 , wherein the 3′-terminal nucleotide of said detector oligonucleotide is blocked or rendered incapable of extension by a nucleic acid polymerase.
34. The method according to claim 30 , wherein said detector oligonucleotide is not required to adopt a secondary structure for said detector oligonucleotide to function.
35. The method according to claim 30 , wherein one of R 2 and R 3 is attached to said detector oligonucleotide through a phosphodiester moiety.
36. 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
R 1 is a member selected from substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl groups and does not comprise a carboxylic acid ester moiety;
R 2 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
R 3 is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, or R 2 and R 3 , together with the nitrogen to which they are attached, are optionally joined to form a substituted 5-7-membered heterocycloalkyl;
wherein one of R 2 and R 3 comprises said reactive functional group;
-(L=L) n -L= has a structure which is a member selected from:
wherein
Z 4 is H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl;
R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 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, NO 2 , CN, Z 1 R 19 , NR 20 R 21 , and C(Z 2 )R 22 ;
wherein
Z 1 is a member selected from O and S;
Z 2 is selected from the group consisting of O, S and NH;
R 19 and R 20 are independently selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
R 21 is a member selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl and C(Z 3 )R 22 ;
wherein
Z 3 is selected from the group consisting of O, S and NH;
R 22 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR 23 , and NR 24 R 25 ,
wherein
R 23 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)R 26
wherein
R 26 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
R 24 and R 25 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
or R 20 and R 21 , together with the nitrogen to which they are attached, are selected from the group consisting of —NHNH 2 , —N═C═S and —N═C═O;
R 12 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
R 13 , R 14 , R 15 and R 16 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.
37. The method according to claim 36 , wherein R 2 comprises said reactive functional group, and said reactive functional group is an oxygen-containing reactive functional group.
38. The method according to claim 37 , wherein said oxygen-containing reactive functional group is selected from the group consisting of hydroxyl and phosphoramidite.
39. The method according to claim 36 , wherein the 3′-terminal nucleotide of said detector oligonucleotide is blocked or rendered incapable of extension by a nucleic acid polymerase.
40. The method according to claim 36 , wherein said detector oligonucleotide is not required to adopt a secondary structure for said detector oligonucleotide to function.
41. The method according to claim 36 , wherein said intramolecularly associated secondary structure is selected from the group consisting of hairpins, stem-loop structures, pseudoknots, and triple helices.
42. The method according to claim 36 , wherein one of R 2 and R 3 is attached to said detector oligonucleotide through a phosphodiester moiety.
43. 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
R 1 is a member selected from substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl groups and does not include a carboxylic acid ester moiety;
R 2 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
R 3 is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, or R 2 and R 3 , together with the nitrogen to which they are attached, are optionally joined to form a substituted 5-7-membered heterocycloalkyl;
wherein one of R 2 and R 3 comprises said reactive functional group;
each of L is C(R 17 )
wherein each R 17 is a member independently selected from H, halide, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, and wherein two adjacent R 17 groups may join to form a ring;
n is an integer selected from 1, 2, 3 and 4;
R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 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, NO 2 , CN, Z 1 R 19 , NR 20 R 21 , and C(Z 2 )R 22 ;
wherein
two substituents selected from R 4 , R 5 , R 6 and R 7 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 R 8 , R 9 , R 10 and R 11 on adjacent atoms of the indoline, together with the carbon atoms to which they are attached, are joined to form an aromatic ring
Z 1 is a member selected from O and S;
Z 2 is selected from the group consisting of O, S and NH;
R 19 and R 20 are independently selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
R 21 is selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl and C(Z 3 )R 22 ;
wherein
Z 3 is selected from the group consisting of O, S and NH;
R 22 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR 23 , and NR 24 R 25 ,
wherein
R 23 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)R 26
wherein
R 26 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
R 24 and R 25 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
or R 20 and R 21 , together with the nitrogen to which they are attached, are selected from the group consisting of —NHNH 2 , —N═C═S and —N═C═O;
R 12 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
R 13 , R 14 , R 15 and R 16 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.
44. The method according to claim 43 , wherein R 2 comprises said reactive functional group, and said reactive functional group is an oxygen-containing reactive functional group.
45. The method according to claim 44 , wherein said oxygen-containing reactive functional group is selected from the group consisting of hydroxyl and phosphoramidite.
46. The method according to claim 43 ,
wherein
R 1 is unsubstituted alkyl;
R 3 is H;
R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are each H;
R 12 is unsubstituted alkyl;
R 13 , R 14 , R 15 and R 16 are each unsubstituted alkyl; and
R 17 is H.
47. The method according to claim 46 , wherein n is 1 or 2.
48. The method according to claim 47 ,
wherein
R 1 is pentylene;
R 12 is ethyl; and
R 13 , R 14 , R 15 and R 16 are each methyl.
49. The method according to claim 43 , wherein said first carrier molecule is a nucleic acid probe, wherein the 3′-terminal nucleotide of said nucleic acid probe is blocked or rendered incapable of extension by a nucleic acid polymerase.
50. The method according to claim 43 , wherein said first carrier molecule is a nucleic acid probe, wherein said nucleic acid probe does not require said first carrier molecule to adopt a secondary structure for said nucleic acid probe to function.
51. The method according to claim 43 , wherein said aromatic ring is phenyl.
52. The method according to claim 43 , wherein one of R 2 and R 3 is attached to said first carrier molecule through a phosphodiester moiety.
53. 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
R 1 is a member selected from substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl groups and does not include a carboxylic acid ester moiety;
R 2 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
R 3 is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, or R 2 and R 3 , together with the nitrogen to which they are attached, are optionally joined to form a substituted 5-7-membered heterocycloalkyl;
wherein one of R 2 and R 3 comprises said reactive functional group;
-(L=L) n -L= has a structure which is a member selected from:
wherein
Z 4 is H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl;
R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 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, NO 2 , CN, Z 1 R 19 , NR 20 R 21 , and C(Z 2 )R 22 ;
wherein
Z 1 is a member selected from O and S;
Z 2 is selected from the group consisting of O, S and NH;
R 19 and R 20 are independently selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;
R 21 is a member selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl and C(Z 3 )R 22 ;
wherein
Z 3 is elected from the group consisting of O S and NH;
R 22 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR 23 , and NR 24 R 25 ,
wherein
R 23 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)R 26
wherein
R 26 is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
R 24 and R 25 are independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
or R 20 and R 21 , together with the nitrogen to which they are attached, are selected from the group consisting of —NHNH 2 , —N═C═S and —N═C═O;
R 12 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
R 13 , R 14 , R 15 and R 16 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.
54. The method according to claim 53 , wherein R 2 comprises said reactive functional group, and said reactive functional group is an oxygen-containing reactive functional group.
55. The method according to claim 54 , wherein said oxygen-containing reactive functional group is selected from the group consisting of hydroxyl and phosphoramidite.
56. The method according to claim 53 , wherein said first carrier molecule is a nucleic acid probe, wherein the 3′-terminal nucleotide of said nucleic acid probe is blocked or rendered incapable of extension by a nucleic acid polymerase.
57. The method according to claim 53 , wherein said first carrier molecule is a nucleic acid probe, wherein said nucleic acid probe does not require said first carrier molecule to adopt a secondary structure for said nucleic acid probe to function.
58. The method according to claim 53 , wherein one of R 2 and R 3 is attached to said first carrier molecule through a phosphodiester moiety.