Fluorescence energy transfer probes with stabilized conformations
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.
1. A probe nucleic acid having the formula:
wherein,
CHOL is a cholesterol derivative;
R 1 , R 2 , R 3 and R 4 are linker moieties independently selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
Nu 1 and Nu 2 are members independently selected from the group consisting of nucleotide residues and nucleoside residues;
NA is a nucleic acid sequence;
D is a donor of light energy; and
Q is a quencher of light energy,
wherein the CHOL moieties interact to bring D and Q into position to enable transfer of energy from D to Q, and
wherein said probe nucleic acid sequence is not hybridized to a target nucleic acid.
2. The probe nucleic acid according to claim 1 , wherein R 2 —CHOL and R 3 —CHOL are independently selected and have structures according to the formula:
wherein,
R 11 is a member selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
PEG is polyethylene glycol;
Y 3 is an organic functional group adjoining said PEG to said CHOL.
3. The probe nucleic acid according to claim 2 , wherein said PEG has from about 2 to about 20 ethylene glycol subunits.
4. The probe nucleic acid according to claim 2 in which R 11 is substituted or unsubstituted alkyl.
5. The probe nucleic acid according to claim 3 , wherein R 11 is C 1 –C 6 substituted or unsubstituted alkyl.
6. The probe nucleic acid according to claim 2 , wherein Y 3 —CHOL has the structure:
7. The probe nucleic acid according to claim 1 , 2 wherein Nu 1 and Nu 2 are nucleotides having an exocyclic amine group to which —R 1 —D and —R 4 Q are attached, respectively.
8. A probe nucleic acid having the formula:
wherein,
NA is a nucleic acid sequence;
Nu 1 and Nu 2 are members independently selected from the group consisting of nucleotide residues and nucleoside residues;
Y 1 and Y 2 are linking groups independently selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
R 5 and R 6 are linking groups independently selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
D is a donor of light energy; and
Q is a quencher of light energy,
wherein the CHOL moieties interact to bring D and Q into position to enable transfer of energy from D to Q, and
wherein said probe nucleic acid sequence is not hybridized to a target nucleic acid.
9. The probe nucleic acid according to claim 8 , wherein Y 1 and Y 2 are members independently selected from substituted or unsubstituted heteroalkyl.
10. The probe nucleic acid according to claim 9 , wherein Y 1 and Y 2 are polyethylene glycol.
11. The probe nucleic acid according to claim 10 , wherein said polyethylene glycol has from about 2 to about 20 ethylene glycol subunits.
12. The probe nucleic acid according to claim 8 , wherein Y 1 —CHOL and Y 2 —CHOL have the structure: