Universal spacer/energy transfer dyes

• US 5,728,528 A
• Filed: 09/20/1995
• Issued: 03/17/1998
• Est. Priority Date: 09/20/1995
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Ju, Jingyue, Mathies, Richard A., Glazer, Alexander N.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): TRAN, PAUL
• Application Number:
  US08/531,132
• Time in Patent Office: 909 Days
• Field of Search: 435/6, 435/810, 435/91.1, 536/25.32, 536/6.2, 536/22.1, 536/18.4
• US Class Current: 435/6.12
• 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: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Fluorescent labels are provided employing energy absorber/donor components and energy acceptor/fluorescer components joined together by a spacer which comprises sugar phosphate monomeric units, particularly ribosyl phosphate monomeric units, where the sugar groups are free of active hydroxyl groups. Particularly, an energy transfer component is substituted at the 5'"'"' position of the spacer chain, while the other energy transfer component is substituted at the 1'"'"' position of the 3'"'"' terminal ribosyl group of the label forming an ET cassette for linking to a nucleic acid sequence with any compositions. By employing combinations of ET components, with a common energy absorber/donor and different fluorescers, one can provide for families of labels which can be tagged to any target molecules and which can be excited at a single wavelength and fluoresce at different wavelengths with large Stokes shifts. The compositions find particular application in sequencing.

Claims

1. In a method for identifying different nucleic acid sequences using a combination of fluorescent labels in an electrophoresis separation, said fluorescent labels fluorescing at different wavelengths and each different label joined to a different nucleic acid sequence, the improvement which comprises:

said labels comprising an energy donor component absorbing at a common wavelength, an acceptor component absorbing energy transferred by said energy donor component, where the acceptor for each label emits at a different wavelength, and said energy donor component and acceptor component linked by a backbone comprising sugar phosphate ester monomer linkages with the 1'"'"'-hydroxyl group being replaced with a hydrogen or etherified.

2. A method according to claim 1, wherein said sugar phosphate ester linkages are 1'"'"',2'"'"'-dideoxyribosyl-3'"'"', 5'"'"'-phosphate linkages.

3. A method according to claim 2, wherein said backbone comprises 3 to 15 phosphate ester monomers.

4. A method according to claim 3, wherein said backbone has a 5'"'"' hydroxyl terminus and said energy donor component is linked at said 5'"'"' terminus.

5. A method according to claim 3, wherein said backbone has a 3'"'"' terminus comprising a linking functionality at the 1'"'"'-position of said 1'"'"', 2'"'"'-dideoxyribosyl group to which said acceptor is covalently bonded.

6. In a method for identifying different nucleic acid sequences using a combination of fluorescent labels in an electrophoresis separation, said fluorescent labels linked to the appropriate primers and fluorescing at different wavelengths and each different label joined to a different nucleic acid sequence, the improvement which comprises:

said labels comprising an energy donor component absorbing at a common wavelength, an acceptor component absorbing energy transferred by said donor component, where the acceptor for each label emits at a different wavelength, and said energy donor component and acceptor component are linked by a backbone comprising from 3 to 15 1'"'"',2'"'"'-dideoxyribosyl 3'"'"',5'"'"'-phosphate ester monomer linkages, said energy donor component covalently linked at the 5'"'"' terminus of said backbone and said acceptor component linked to the 1'"'"'-position of the 3'"'"'-terminal 1'"'"',2'"'"'-dideoxyribose of said backbone.

7. A method according to claim 6, wherein said backbone comprises from 5 to 8 1'"'"', 2'"'"'-dideoxyribosyl 3'"'"', 5'"'"'-phosphate ester monomer linkages.

8. A method according to claim 6, wherein said donor component is a fluorescein.

9. A method according to claim 6, wherein said acceptor is linked through a uridine.

10. A method according to claim 6, wherein said acceptor is linked through a carboxy group bonded to the 1'"'"'-position at the 3'"'"'-terminus of the sugar monomer.

11. A method according to claim 6, wherein at least one fluorescer is a rhodamine.

12. In a method for identifying different nucleic acid sequences using a combination of fluorescent labels in an electrophoresis separation for determining a nucleic acid sequence, where each of said labels is associated with the presence of a different nucleotide, said fluorescent labels fluorescing at different wavelengths and each different label joined to a different nucleic acid sequence, the improvement which comprises:

said labels comprising an energy donor component absorbing at a common wavelength, an acceptor component absorbing energy from said energy donor component, where the acceptor for each label emits at a different wavelength, and said energy donor component and acceptor component linked by a backbone comprising from 3 to 9 1'"'"', 2'"'"'-dideoxyribosyl 3'"'"', 5'"'"'-phosphate ester monomer linkages, said energy donor component covalently linked at the 5'"'"' terminus of said backbone and said acceptor component linked to the 1'"'"'-position of the 3'"'"'-terminal 1'"'"', 2'"'"'-dideoxyribose of said backbone.

13. A compound comprising an energy donor component and an acceptor component absorbing energy transferred by said energy donor component, said components linked by a backbone comprising sugar phosphate ester monomer linkages with the 1'"'"'-hydroxyl group being replaced by a hydrogen or etherified.

14. A compound according to claim 13, wherein said backbone comprises from 3 to 15 1'"'"', 2'"'"'-dideoxyribosyl 3'"'"', 5'"'"'-phosphate ester monomer linkages.

15. A compound according to claim 14, wherein said energy donor component is covalently bonded at the 5'"'"' backbone terminus and said acceptor is covalently bonded to the backbone 3'"'"'-terminal of 1'"'"', 2'"'"'-dideoxyribosyl 3'"'"', 5'"'"'-phosphate ester at the 1'"'"'-position through a linking group.

16. A compound according to claim 15, wherein the 3'"'"'-terminus of said backbone is an hydroxyl, protected hydroxyl, phosphoramidite, phosphite, nucleotide or oligonucleotide.

20. A kit comprising at least two compounds according to claim 13 differing in said acceptor and having the same energy donor.

21. A kit comprising at least two compounds according to claim 15 differing in said acceptor and having the same energy donor.

17. A compound of the formula 5'"'"'-fluorescein substituted poly-1'"'"', 2'"'"'-dideoxyribose-3'"'"', 5'"'"'-phosphate, having from 3 to 15 1'"'"', 2'"'"'-dideoxyribose-3'"'"', 5'"'"'-phosphate monomeric units, and substituted through a linking group at the 1'"'"'-position of the 3'"'"'-terminal 1'"'"', 2'"'"'-dideoxyribose-3'"'"', 5'"'"' -phosphate monomeric unit with a fluorescein or a rhodamine.

18. A compound according to claim 17, wherein the 3'"'"'-terminus of said backbone is an hydroxyl, protected hydroxyl, phosphoramidite, phosphite, nucleotide or oligonucleotide.

19. A compound according to claim 17, wherein said linking group is a carboxyl or uridyl group.