PHOSPHOLINKED DYE ANALOGS WITH AN AMINO ACID LINKER

• US 20120052507A1
• Filed: 08/25/2011
• Published: 03/01/2012
• Est. Priority Date: 08/25/2010
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Shen, Gene
• Granted Patent: US 8,993,737 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C07D 209/14 Radicals substituted by nitrogen atoms, not forming part of a nitro radical
• CPC Class Codes: C07D 401/14 containing three or more hetero rings
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07K 5/0815 with the first amino acid being basic
• CPC Class Codes: C07K 5/1019 with the first amino acid being basic
• CPC Class Codes: C09B 23/0025 the substituent being bound through an oxygen atom
• CPC Class Codes: C09B 23/0033 the substituent being bound through a sulfur atom
• CPC Class Codes: C09B 23/0066 the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 23/105 two >CH- groups
• CPC Class Codes: C09B 23/164 containing one nitrogen atom
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: G01N 2333/9126 DNA-directed DNA polymerase (2.7.7.7)
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

In various embodiments, the present invention provides fluorescent dyes that are linked to another species through an amino acid or peptide linker. In an exemplary embodiment, the dye is linked to a polyphosphate nucleic acid through an amino acid or peptide linker. These conjugates find use in single molecule DNA sequencing and other applications. In various embodiments, the dye moiety is a cyanine dye. Cyanine dyes that are highly charged, such as those including muliple sulfonate, alkylsulfonate, carboxylate and/or alkylcarboxylate moieties are examples of cyanine dyes of use in the compounds of the invention.

Claims

1. A fluorescent dye having the formula:

{R¹-(L¹)_a-(AA)_n}_y-(L²)_b-X

wherein

R¹ is a fluorescent dye moiety;

AA is an amino acid;

n is a selected from the integers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and when n is two or greater, each n amino acid is independently selected;

X is a member selected from a polyvalent moiety, and a moiety having the structure;

2. The fluorescent dye according to claim 1 wherein said polyvalent moiety comprises bonded thereto a member selected from a second fluorescent dye moiety, a moiety having the formula:

3. The fluorescent dye of any preceding claim wherein y and y′

are equal to each other.

4. The fluorescent dye of any preceding claim wherein n is 3.

5. The fluorescent dye of any preceding claim wherein said amino acid is a member selected from lysine, sulfocysteine, glutamic acid, serine, and proline.

6. The fluoresecent dye of any preceding claim wherein each of said n amino acids is the same amino acid.

7. The fluorescent dye of any preceding claim in which said fluorescent dye moiety is a cyanine dye.

8. The fluorescent dye of any preceding claim in which said polyvalent moiety is a residue of a member selected from triazine, perylene, piperidine, phenylalanine, diaminopropanoic acid, aspartic acid, lysine, glutamic acid, serine, aminoadipic acid, 3,5-dihydroxybenzoic acid, 2-amino-4-hydroxy-butyric acid, 4-(1-amino-1-carboxyethyl)-benzoic acid, piperazine-2-carboxylic acid, 4-[4,6-bis-(piperidin-4-ylamino)-[1,3,5]triazin-2-ylamino]-cyclohexanecarboxylic acid and 3-amino-3-[4-(3-amino-prop-1-ynyl)-phenyl]-propionic acid.

9. The fluorescent dye of any preceding claim wherein said fluorescent dye binds to a DNA polymerase with a binding constant greater than an otherwise identical fluorescent dye in which (AA)_n is absent.

10. The fluorescent dye of any of the preceding claims wherein said substituted amino acid side chain comprises a moiety having the formula:

11. A method of monitoring an enzyme reaction, said method comprising:

(a) forming a reaction mixture bycontacting said enzyme with a fluorescent dye according to any preceding claim, wherein said dye is a substrate for said enzyme under conditions sufficient for said enzyme and said dye to react; and

(b) monitoring fluorescence of said reaction mixture.

12. The method according to claim 11 wherein said enzyme is a DNA polymerase and said dye comprises a nucleic acid moiety which is said substrate for said enzyme.

13. The method according to claim 11 or 12 wherein said enzyme reaction is template directed DNA synthesis.

14. The method according to claim 13 wherein said reaction is a component of a single molecule DNA sequencing analysis.