Energy transfer dyes with enhanced fluorescence

• US 7,169,939 B2
• Filed: 02/26/2004
• Issued: 01/30/2007
• Est. Priority Date: 05/03/1996
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lee, Linda G., Spurgeon, Sandra L., Rosenblum, Barnett
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/788,836
• Publication Number:
  US 20050069912A1
• Time in Patent Office: 1,069 Days
• Field of Search: 549/223, 435/6, 536/24.33, 536/25.3, 536/26.6
• US Class Current: 549/223
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 311/90 with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C09B 11/04 derived from triarylmethanes , i.e. central C-atom is substituted by amino, cyano, alkyl
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 69/00 Dyes not provided for by a single group of this subclass
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R₂₁Z₁C(O)R₂₂R₂₈ where R₂₁ is a C_1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z₁ is either NH, sulfur or oxygen, R₂₂ is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R₂₈ includes a functional group which attaches the linker to the acceptor dye.

Claims

1. A fluorescently-labeled reagent comprising a reagent and an energy transfer dye, wherein the energy transfer dye comprises:

a xanthene donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response thereto;

a 4,7-dichlororhodamine acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response thereto; and

a non-nucleosidic linker linking the 5- or 6-ring position of the donor dye to the 5- or 6-ring position of the acceptor dye,

and wherein the energy transfer dye is covalently linked to the reagent.

2. The fluorescently-labeled reagent of claim 1 in which the reagent is selected from the group consisting of proteins, polypeptides, polysaccharides, nucleosides/tides, oligonucleotides, oligonucleotide analogs, lipids, solid supports and organic and inorganic polymers.

3. The fluorescently-labeled reagent of claim 2 in which the reagent is a nucleoside/tide.

4. The fluorescently labeled reagent of claim 3 in which the energy transfer dye is covalently linked to the base of the nucleoside/tide at the 4′

-position of the donor or acceptor dye.

5. The fluorescently labeled reagent of claim 4 in which the energy transfer dye is covalently linked to the base of the nucleoside/tide by way of an acetylenic amido or alkenic amido linkage.

6. The fluorescently labeled reagent of claim 5 in which the acetylenic amido or alkenic amido linkage is selected from —

C≡

C—

CH₂—

NH—

C(O)—

, 3-amino-1-propyn-1-yl, —

C≡

C—

CH₂—

NH—

C(O)—

(CH₂)₅—

C(O)—

, —

C═

CH—

C(O)—

NH—

(CH₂)₅—

NH—

C(O)— and

—

C≡

CH₂—

O—

CH₂—

CH₂—

NR—

, where R is hydrogen, a protecting group or alkyl.

7. The fluorescently-labeled reagent of claim 3 in which the donor dye is a fluorescein.

8. The fluorescently-labeled reagent of claim 3 in which the linker has a backbone that is less than 9 atoms in length.

9. The fluorescently-labeled reagent of claim 3 in which the linker comprises a functional group selected from an alkene, a diene, an alkyne, a five membered ring having at least one unsaturated bond, a six membered ring having at least one unsaturated bond and a fused ring structure.

10. The fluorescently-labeled reagent of claim 3 in which the energy transfer dye comprises the structure:

11. The fluorescently labeled reagent of claim 10 in which the nucleoside/tide is a 2′

-deoxyribonucleoside.

12. The fluorescently labeled reagent of claim 10 in which the nucleoside/tide is a 2′

-deoxyribonucleotide.

13. The fluorescently labeled reagent of claim 12 in which the 2′

-deoxyribonucleotide is a 2′

-deoxyribonucleoside-5′

-triphosphate.

14. The fluorescently labeled reagent of claim 10 in which the nucleoside/tide is a terminating nucleotide.

15. The fluorescently labeled nucleoside/tide of claim 14 in which the terminating nucleotide is a 2′

,3′

-dideoxynucleoside-5′

-triphosphate.

16. A fluorescently-labeled reagent of claim 2 in which the reagent is an oligonucleotide or oligonucleotide analog.

17. The fluorescently-labeled reagent of claim 16 in which the energy transfer dye is attached to the 5′

-terminus of the oligonucleotide or analog.

18. The fluorescently-labeled reagent of claim 16 in which the energy transfer dye is attached to the 3′

-terminus of the oligonucleotide or analog.

19. The fluorescently-labeled reagent of claim 16 in which the energy transfer dye is attached to a nucleobase of the oligonucleotide or analog.

20. The fluorescently-labeled reagent of claim 16 in which the energy transfer dye comprises the structure: