Gamma-PNA miniprobes for fluorescent labeling
A category of γPNA miniprobes and chimeric γPNA probes is especially useful for detecting RNA and telomeric DNA in a cell sample. In particular, the probes can be used to deliver fluorescent dyes to the telomeres, allowing direct visualization of telomeres in cells.
1. A kit for detecting a telomeric DNA in a cell sample, comprising (A) a receptacle that contains a mini-PEG modified γPNA miniprobe that hybridizes with the telomeric DNA, and (B) a receptacle that contains a diluent suitable for hybridization,
wherein the mini-PEG modified γPNA miniprobe comprises at least one PNA monomer that is a mini-PEG modified γPNA monomer having the structure
wherein Base is a nucleobase.
2. The kit according to claim 1 , wherein the mini-PEG modified γPNA miniprobe consists of six, nine or twelve PNA monomers.
3. The kit according to claim 1 , wherein the mini-PEG modified γPNA miniprobe comprises at least one fluorescent dye as a detectable label.
4. The kit according to claim 3 , wherein the fluorescent dye is selected from the group consisting of Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, coumarin, acridine derivatives, eosin derivatives and fluorescein.
5. The kit according to claim 3 , wherein the detectable label is attached to a N-terminal or a C-terminal PNA monomer or a N-terminal or a C-terminal γPNA monomer of the mini-PEG modified γPNA miniprobe.
6. The kit according to claim 3 , wherein the detectable label is attached to an internal PNA monomer or an internal γPNA monomer of the mini-PEG modified γPNA miniprobe.
7. The kit according to claim 3 , wherein the detectable label is attached to a uracil or a cytosine nucleobase of a PNA monomer.
8. The kit according to claim 6 , wherein the detectable label is attached to a γ-carbon of the γPNA monomer.
9. The kit according to claim 8 , wherein the detectable label is attached to the γ-carbon of the γPNA monomer via a linker CH 2 —(O—CH 2 —CH 2 ) n —X, wherein X is selected from the group consisting of —NH 2 —, —CH═CH—, —COOH and —N 3 and n is an integer from 1 to 10.
10. The kit according to claim 1 , wherein the mini-PEG modified γPNA miniprobe comprises two fluorescent dyes.
11. The kit according to claim 1 , wherein the nucleobase sequence of the mini-PEG modified γPNA miniprobe consists of:
(A) CCCTAA (nucleobases 13-18 of SEQ ID NO: 2), wherein at least one C is replaced with a G-clamp nucleobase;
(B) CCCTAACCC (nucleobases 10-18 of SEQ ID NO: 2); or
(C) CCCTAACCCTAA (nucleobases 7-18 of SEQ ID NO: 2).
12. A chimeric mini-PEG modified γPNA miniprobe, comprising:
(i) at least one PNA monomer that is a mini-PEG modified γPNA monomer having the structure
wherein Base is a nucleobase;
(ii) a first, right-handed, domain comprising right-handed PNA monomers, and
(iii) a second, left-handed, domain comprising left-handed PNA monomers,
wherein the first and second domains are attached to each other end-to-end, optionally via a linker.
13. The chimeric mini-PEG modified γPNA miniprobe according to claim 12 , wherein (A) the first domain comprises from 4 to 10 right-handed PNA monomers; and (B) the second domain comprises from 4 to 7 left handed PNA monomers.
14. The chimeric mini-PEG modified γPNA miniprobe according to claim 12 , wherein the sequence of the first domain is complementary to a telomeric DNA and the second domain does not hybridize with the telomeric DNA.
15. The chimeric mini-PEG modified γPNA miniprobe according to claim 14 , wherein the first domain comprises nine right-handed γPNA monomers and the second domain comprises six left-handed γPNA monomers.
16. The chimeric mini-PEG modified γPNA miniprobe of claim 12 , further comprising at least one detectable label covalently attached to the first domain or the second domain.
17. The chimeric mini-PEG modified γPNA miniprobe of claim 12 , wherein the first domain is attached to the second domain via a linker.
18. The chimeric mini-PEG modified γPNA miniprobe of claim 12 , further comprising at least one detectable label covalently attached to the first domain or the second domain, wherein the first domain is attached to the second domain via a linker.
19. A mini-PEG modified γPNA miniprobe comprising at least one PNA monomer that is a mini-PEG modified γPNA monomer having structure:
wherein Base is a nucleobase;
wherein the nucleobase sequence of the miniprobe consists of:
(A) CCCTAA (nucleobases 18-13 of SEQ ID NO: 2), wherein at least one C is replaced with a G-clamp nucleobase;
(B) CCCTAACCC (nucleobases 18-10 of SEQ ID NO: 2); or
(C) CCCTAACCCTAA (nucleobases 7-18 of SEQ ID NO: 2).
20. The γPNA miniprobe according to claim 19 , wherein the miniprobe comprises at least one detectable label.
21. The γPNA miniprobe according to claim 19 , comprising two or more of said mini-PEG modified γPNA monomers.
22. The γPNA miniprobe according to claim 19 , wherein all PNA monomers of the miniprobe are said mini-PEG modified γPNA monomers.
23. The γ-RNA miniprobe according to claim 19 , wherein at least one mini-PEG modified γPNA monomer comprises a fluorophore.