# US 9,435,796 B2 · Cyanine dyes

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 9,435,796 B2
- Document ID: US9435796B2
- Status: Granted
- Kind: B2
- Application number: 13/179,345
- Filed: 2011-07-08
- Published: 2012-05-10
- Granted: 2016-09-06
- Earliest claimed: 2004-02-04
- Assignees: Biosearch Technologies, Inc.
- Applicants: Tim Carter; Mark Reddington
- Inventors: Tim Carter; Mark Reddington
- Web page: https://patentgrep.com/patent/US9435796B2

## CPC

- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- C12Q 1/37: involving peptidase or proteinase
- C12Q 2337/00: N-linked chromogens for determinations of peptidases and proteinases
- G01N 2333/95: Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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.

## Claims

### Claim 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
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.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said fluorescence parameter is detected in real time.

### Claim 3 (depends on 1)

The method according to claim 1, wherein R2 comprises said reactive functional group, and said reactive functional group is an oxygen-containing reactive functional group.

### Claim 4 (depends on 3)

The method according to claim 3, wherein said oxygen-containing reactive functional group is selected from the group consisting of hydroxyl and phosphoramidite.

### Claim 5 (depends on 1)

The method according to claim 1,
wherein
R1 is unsubstituted alkyl;
R3 is H;
R4, R5, R6, R7, R8, R9, R10, and R11 are each H;
R12 is unsubstituted alkyl;
R13, R14, R15 and R16 are each unsubstituted alkyl; and
R17 is H.

### Claim 6 (depends on 5)

The method according to claim 5, wherein n is 1 or 2.

### Claim 7 (depends on 6)

The method according to claim 6,
wherein
R1 is pentylene;
R12 is ethyl; and
R13, R14, R15 and R16 are each methyl.

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 1)

The method according to claim 1, wherein said detector oligonucleotide is not required to adopt a secondary structure for said detector oligonucleotide to function.

### Claim 10 (depends on 1)

The method according to claim 1, 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.

### Claim 11 (depends on 10)

The method according to claim 10, wherein said aromatic ring is phenyl.

### Claim 12 (depends on 1)

The method according to claim 1, wherein one of R2 and R3 is attached to said detector oligonucleotide through a phosphodiester moiety.

### Claim 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
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.

### Claim 14 (depends on 13)

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.

### Claim 15 (depends on 13)

The method according to claim 13, wherein said secondary structure further comprises a partially or entirely single-stranded restriction endonuclease site.

### Claim 16 (depends on 13)

The method according to claim 13, wherein a change in fluorescence intensity is detected.

### Claim 17 (depends on 16)

The method according to claim 16, wherein said change in fluorescence intensity is detected in real-time.

### Claim 18 (depends on 13)

The method according to claim 13, wherein said intramolecularly associated secondary structure comprises a portion of said target binding sequence.

### Claim 19 (depends on 13)

The method according to claim 13, wherein R2 comprises said reactive functional group, and said reactive functional group is an oxygen-containing reactive functional group.

### Claim 20 (depends on 19)

The method according to claim 19, wherein said oxygen-containing reactive functional group is selected from the group consisting of hydroxyl and phosphoramidite.

### Claim 21 (depends on 13)

The method according to claim 13,
wherein
R1 is unsubstituted alkyl;
R3 is H;
R4, R5, R6, R7, R8, R9, R10, and R11 are each H;
R12 is unsubstituted alkyl;
R13, R14, R15 and R16 are each unsubstituted alkyl; and
R17 is H.

### Claim 22 (depends on 21)

The method according to claim 21, wherein n is 1 or 2.

### Claim 23 (depends on 22)

The method according to claim 22,
wherein
R1 is pentylene;
R12 is ethyl; and
R13, R14, R15 and R16 are each methyl.

### Claim 24 (depends on 13)

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.

### Claim 25 (depends on 13)

The method according to claim 13, wherein said detector oligonucleotide is not required to adopt a secondary structure for said detector oligonucleotide to function.

### Claim 26 (depends on 13)

The method according to claim 13, 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.

### Claim 27 (depends on 26)

The method according to claim 26, wherein said aromatic ring is phenyl.

### Claim 28 (depends on 13)

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.

### Claim 29 (depends on 13)

The method according to claim 13, wherein one of R2 and R3 is attached to said detector oligonucleotide through a phosphodiester moiety.

### Claim 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
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.

### Claim 31 (depends on 30)

The method according to claim 30, wherein R2 comprises said reactive functional group, and said reactive functional group is an oxygen-containing reactive functional group.

### Claim 32 (depends on 31)

The method according to claim 31, wherein said oxygen-containing reactive functional group is selected from the group consisting of hydroxyl and phosphoramidite.

### Claim 33 (depends on 30)

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.

### Claim 34 (depends on 30)

The method according to claim 30, wherein said detector oligonucleotide is not required to adopt a secondary structure for said detector oligonucleotide to function.

### Claim 35 (depends on 30)

The method according to claim 30, wherein one of R2 and R3 is attached to said detector oligonucleotide through a phosphodiester moiety.

### Claim 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
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.

### Claim 37 (depends on 36)

The method according to claim 36, wherein R2 comprises said reactive functional group, and said reactive functional group is an oxygen-containing reactive functional group.

### Claim 38 (depends on 37)

The method according to claim 37, wherein said oxygen-containing reactive functional group is selected from the group consisting of hydroxyl and phosphoramidite.

### Claim 39 (depends on 36)

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.

### Claim 40 (depends on 36)

The method according to claim 36, wherein said detector oligonucleotide is not required to adopt a secondary structure for said detector oligonucleotide to function.

### Claim 41 (depends on 36)

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.

### Claim 42 (depends on 36)

The method according to claim 36, wherein one of R2 and R3 is attached to said detector oligonucleotide through a phosphodiester moiety.

### Claim 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
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.

### Claim 44 (depends on 43)

The method according to claim 43, wherein R2 comprises said reactive functional group, and said reactive functional group is an oxygen-containing reactive functional group.

### Claim 45 (depends on 44)

The method according to claim 44, wherein said oxygen-containing reactive functional group is selected from the group consisting of hydroxyl and phosphoramidite.

### Claim 46 (depends on 43)

The method according to claim 43,
wherein
R1 is unsubstituted alkyl;
R3 is H;
R4, R5, R6, R7, R8, R9, R10, and R11 are each H;
R12 is unsubstituted alkyl;
R13, R14, R15 and R16 are each unsubstituted alkyl; and
R17 is H.

### Claim 47 (depends on 46)

The method according to claim 46, wherein n is 1 or 2.

### Claim 48 (depends on 47)

The method according to claim 47,
wherein
R1 is pentylene;
R12 is ethyl; and
R13, R14, R15 and R16 are each methyl.

### Claim 49 (depends on 43)

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.

### Claim 50 (depends on 43)

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.

### Claim 51 (depends on 43)

The method according to claim 43, wherein said aromatic ring is phenyl.

### Claim 52 (depends on 43)

The method according to claim 43, wherein one of R2 and R3 is attached to said first carrier molecule through a phosphodiester moiety.

### Claim 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
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.

### Claim 54 (depends on 53)

The method according to claim 53, wherein R2 comprises said reactive functional group, and said reactive functional group is an oxygen-containing reactive functional group.

### Claim 55 (depends on 54)

The method according to claim 54, wherein said oxygen-containing reactive functional group is selected from the group consisting of hydroxyl and phosphoramidite.

### Claim 56 (depends on 53)

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.

### Claim 57 (depends on 53)

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.

### Claim 58 (depends on 53)

The method according to claim 53, wherein one of R2 and R3 is attached to said first carrier molecule through a phosphodiester moiety.