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US 12,624,391 B2Granted
Cyanine dyes
AssigneePacific Biosciences of California, Inc.
InventorsStephen Yue, Gene Shen, Wei-Chuan (David) Sun
Life of this patent
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Abstract
The invention provides a novel class of cyanine dyes that are functionalized with sulfonic acid groups and a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.
Claims13
A method of synthesizing a first polynucleotide having a first nucleic acid sequence, the method comprising:
- under conditions of a template dependent polymerization reaction, contacting a nucleic acid polymerase complexed with a second polynucleotide having a second nucleic acid sequence complementary to the first nucleic acid sequence with a cyanine compound of Formula X:
- wherein
- Rf, Rg, Rh, and Ri are members independently selected from halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heteroalkyl;
- Rc, and Rd are independently selected from alkyl and heteroalkyl, substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, and phosphoric acid;
- a and e are independently selected from selected from the integers 0, 1, 2, 3 and 4, with the proviso that at least one member selected from a and e is 2 or greater, such that when a is 2 or greater, each Ra is independently selected, and when e is 2 or greater, each Re is independently selected;
- wherein when a is 2 or greater, ring A is selected from phenyl substituted at least at the 4- and 6-positions with (Ra) a, and substituted or unsubstituted napthyl;
- and when e is 2, or more ring E is selected from phenyl substituted at least at the 4- and 6-positions with (Re)e, and substituted or unsubstituted naphthyl;
- Q is a member selected from
- wherein
- n is selected from the integers from 1 to 3;
- each Ra and Re, is independently selected from C(O)R9, OR12, NR12R13, CR12C(O)R13, NR12C(O)2R13, SO3H, and C(O) NR12R13, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, a bond to M, and a ring structure formed by joining a member selected from two Ra moieties together with the atoms they are attached, two Re moieties together with the atoms they are attached, and a combination thereof, to form said ring structure, which is a member selected from a substituted or unsubstituted aryl and a substituted or unsubstituted heteroaryl,
- wherein
- R9 is a member selected from OR10, and a bond to M wherein
- R10 is a member selected from H and substituted or unsubstituted alkyl;
- R11 is a nucleic acid moiety, which is a substrate for the polymerase;
- and
- R12 and R13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl, and a bond to M,
- wherein
- M comprises a linker bound to a nucleic acid carrier, which is a substrate for the polymerase,
- and at least one of Ra, Rc, Rd, Re, Rf, Rg, Rh, Ri, R9, R12 and R13 is a bond to M, thereby synthesizing the first polynucleotide.
17 more elements
A method of sequencing a first polynucleotide having a first nucleic sequence, the method of sequencing comprising:
- under conditions of a template dependent polymerization reaction, contacting a nucleic acid polymerase complexed with a second polynucleotide having a second nucleic acid sequence complementary to the first nucleic acid sequence with a cyanine compound of Formula X:
- wherein
- Rf, Rg, Rh, and Ri are members independently selected from halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heteroalkyl;
- Rc, and Rd are independently selected from alkyl and heteroalkyl, substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, and phosphoric acid;
- a and e are independently selected from selected from the integers 0, 1, 2, 3 and 4, with the proviso that at least one member selected from a and e is 2 or greater, such that when a is 2 or greater, each Ra is independently selected, and when e is 2 or greater, each Re is independently selected;
- wherein when a is 2 or greater, ring A is selected from phenyl substituted at least at the 4- and 6-positions with (Ra)a, and substituted or unsubstituted napthyl;
- and when e is 2, or more ring E is selected from phenyl substituted at least at the 4- and 6-positions with (Re)e, and substituted or unsubstituted naphthyl;
- Q is a member selected from
- wherein
- n is selected from the integers from 1 to 3;
- Ra and Re, are independently selected from C(O)R9, OR12, NR12R13, CR12C(O)R13, NR12C(O)2R13, SO3H, and C(O) NR12R13, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and a ring structure formed by joining a member selected from two Ra moieties together with the atoms they are attached, two Re moieties together with the atoms they are attached, and a combination thereof, to form said ring structure, which is a member selected from a substituted or unsubstituted aryl and a substituted or unsubstituted heteroaryl
- wherein
- R9 is a member selected from OR10, and a bond to M wherein
- R10 is a member selected from H and substituted or unsubstituted alkyl;
- R11 is a nucleic acid moiety, which is a substrate for the polymerase;
- and
- R12 and R13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl, and a bond to M,
- wherein
- M comprises a linker bound to a nucleic acid carrier, which is a substrate for the polymerase,
- and at least one of Ra, Rc, Rd, Re, Rf, Rg, Rh, Ri, R9, R12 and R13 is a bond to M,
- such that under the conditions of the template dependent polymerization reaction the cyanine compound is released from the nucleic acid moiety which is a substrate for the polymerase, and is detected, thereby sequencing the first polynucleotide.
18 more elements
Dependencies are inferred from the claim text.