Gamma-PNA miniprobes for fluorescent labeling

Patent metadata

Field	Value
Publication number	US-9926592-B2
Application number	US-201214357874-A
Country	US
Kind code	B2
Filing date	Nov 14, 2012
Priority date	Nov 14, 2011
Publication date	Mar 27, 2018
Grant date	Mar 27, 2018

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• Univ Carnegie Mellon
• Univ Of Pittsburgh—Of The Commonwealth System Of Higher Education

Inventors

• Armitage Bruce A
• Opresko Patricia L
• Ly Danith
• Shank Nathaniel

Classifications

• C12N2310/3181

  Peptide nucleic acid, PNA · CPC title

• C12Q1/6825 Primary

  Nucleic acid detection involving sensors · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12Q2525/107

  incorporating a peptide nucleic acid · CPC title

• G01N33/533 Primary

  with fluorescent label · CPC title