Fluorescence energy transfer probes with stabilized conformations

• US 20070059752A1
• Filed: 09/13/2006
• Published: 03/15/2007
• Est. Priority Date: 06/09/1999
• Status: Abandoned Application

• Current Assignee: HSB Corporate Trust Company (UK) Limited
• Original Assignee: Biosearch Technologies Incorporated
• Inventors: Cook, Ronald
• Application Number:
  US11/520,152
• Publication Number:
  US 20070059752A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07J 51/00 Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

The present invention provides a class of Conformationally Assisted Probes comprising (a) a nucleic acid moiety; (b) an energy donor moiety; (c) an energy acceptor moiety; and (d) one or more stabilizing moieties.

Claims

1. A compound having a structure selected from:

X—

R-A-Q-(Y)_n, R—

X-A-(Y)_n-Q, R—

X-A-Q-(Y)_n, and
X—

R-A-(Y)_n-Q

wherein,

A is a nucleic acid chain comprising nucleic acid monomers selected from the group consisting of natural nucleic acids, modified nucleic acids and combinations thereof;

R is a molecular energy transfer donor;

Q is a molecular energy acceptor; and

X and Y are the same or different and are non-nucleic acid stabilizing moieties that interact to bring R and Q into operative proximity, thereby enabling transfer of energy from R to Q; and

n is 0 or 1.

2. The compound according to claim 1, wherein said molecular energy donor is a fluorophore.

3. The compound according to claim 1, wherein said molecular energy acceptor is a fluorescence quencher.

4. The compound according to claim 1, wherein X and Y are both hydrophobic moieties.

5. The compound according to claim 4, wherein X and Y are members independently selected from the group consisting of saturated hydrocarbons, unsaturated hydrocarbons, steroids, fatty acids, fatty alcohols and hydrophobic peptides.

6. The compound according to claim 1, wherein natural nucleic acids are members selected from the group consisting of deoxyribonucleotides, ribonucleotides and combinations thereof.

7. The compound according to claim 6, wherein said modified nucleic acids are peptide nucleic acids.

8. The compound according to claim 1, wherein said nucleic acid monomers are joined by linkages that are members independently selected from the group consisting phosphodiesters and modified phosphodiesters.

9. The compound according to claim 8, wherein said modified phosphodiesters are members selected from the group consisting of phosphorothioates and phosphoramidates.

10. The compound according to claim 1, wherein said nucleic acid sequence further comprises a hybridization enhancing moiety.

11. The compound according to claim 10, wherein said hybridization enhancing moiety is a member selected from the group consisting of intercalating agents, minor groove binders and modified exocyclic bases.

12. The compound according to claim 1 wherein X and Y are independently attached to members selected from the group consisting of a natural base of said nucleic acid chain, a modified base of said nucleic acid chain, a 3′

-hydroxyl group of said nucleic acid chain, a 5′

-hydroxyl group of said nucleic acid chain, a 2′

-hydroxyl group of said nucleic acid chain, and a linkage joining nucleic acid groups in said nucleic acid chain.

13. The compound according to claim 1, wherein said compound is immobilized on a solid surface.

14. A method for amplifying a polynucleotide, wherein a compound according to claim 1 is a primer in said method.

15. The method according to claim 14, wherein said method comprises a member selected from the group consisting of polymerase chain reaction (PCR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA) and combinations thereof.

16. A method for analyzing or quantitating DNA, wherein the compound according to claim 1 is used as a probe.

17. The method according to claim 16, wherein said method comprises a member selected from the group consisting of 5′

-nuclease assay, rolling circle amplification and combinations thereof.

18. A kit for quantitating nucleic acid, said kit comprising a compound according to claim 1.

19. A compound having the formula:

20. The compound according to claim 19, wherein R¹ and R² are independently selected and have structures according to the formula:

21. The compound according to claim 20, wherein said PEG has from about 2 to about 20 ethylene glycol subunits.

22. The compound according to claim 20 in which R¹¹ is substituted or unsubstituted alkyl.

23. The compound according to claim 22, wherein R¹¹ is C₁-C₆ substituted or unsubstituted alkyl.

24. The compound according to claim 20, wherein Y³—

CHOL has the structure;

25. The compound according to claim 19, wherein Nu¹ and Nu² are nucleotides having an exocyclic amine group to which —

R¹-D and —

R⁴Q are attached, respectively.

31. The compound according to claim 19, wherein Nu¹ and Nu² are nucleotides having an exocyclic amine group to which —

R⁵-D and —

R⁶Q are attached, respectively.

26. A compound having the structure:

27. The compound according to claim 26, wherein Y¹ and Y² are members independently selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

28. The compound according to claim 27, wherein Y¹ and Y² are polyethylene glycol.

29. The compound according to claim 28, wherein said PEG has from about 2 to about 20 ethylene glycol subunits.

30. The compound according to claim 26, wherein Y¹—

X and Y²—

Y have the structure;