Phospholinked dye analogs with an amino acid linker

• US 9,499,862 B2
• Filed: 03/25/2015
• Issued: 11/22/2016
• 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
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/667,869
• Publication Number:
  US 20150197794A1
• Time in Patent Office: 608 Days
• Field of Search: 536/4.1, 530/322, 435/6.11
• US Class Current: 1/1
• 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 multiple 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:

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 nucleic acid and a combination thereof.

3. The fluorescent dye according to claim 2, wherein said first fluorescent dye moiety and said second fluorescent dye moiety form a FRET pair.

4. The fluorescent dye of claim 1, wherein said amino acid linker includes amino acids selected from hydrophobic amino acids, cationic amino acids, and a combination thereof.

5. The fluorescent dye of claim 4, wherein said amino acid linker further comprises an anionic amino acid.

6. The fluorescent dye of claim 1, wherein n is 1, 2, 3, 4, 5, or 6.

7. The fluorescent dye of claim 1, wherein the amino acids forming (AA)_n are the same amino acid or a different amino acid.

8. The fluorescent dye of claim 1, wherein said amino acid linker is other than Pro, Pro₂, Pro₃, Pro₄, Pro₅ or Pro₆.

9. The fluorescent dye of claim 1, wherein said amino acid linker is other than Lys, Lys₄, Lys₅ or Lys₆.

10. The fluorescent dye of claim 5, wherein said anionic amino acid is a member selected from, sulfocysteine, glutamic acid, and O-phosphoserine.

11. The fluorescent dye of claim 1, wherein L¹ comprises an adaptor selected from an alkyl amine and a nitrogen-containing heterocyclic moiety.

12. The fluorescent dye of claim 11, wherein said alkyl amine is:

—

NH(CH₂)_gC(O)NH(CH₂)_h—

,

wherein

g and h are independently selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or higher.

13. The fluorescent dye of claim 1, wherein each of said n amino acids is the same amino acid.

14. The fluorescent dye of claim 1, wherein said fluorescent dye moiety is a cyanine dye.

15. The fluorescent dye of claim 14, wherein said cyanine is selected from Cy3 and Cy5 cyanine dyes.

16. The fluorescent dye of claim 1, wherein 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.

17. The fluorescent dye of claim 1, 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.

18. The fluorescent dye of claim 1 wherein said amino acid comprises a side chain comprising a moiety having the formula:

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

(a) forming a reaction mixture by contacting said enzyme with a fluorescent dye according to claim 1, 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.

20. The method according to claim 19, wherein said enzyme is a DNA polymerase and said dye comprises a nucleic acid moiety which is said substrate for said enzyme.

21. The method according to claim 19 wherein said enzyme reaction is template directed DNA synthesis.

22. The method according to claim 21, wherein said reaction is a component of a single molecule DNA sequencing analysis.