Modified oligonucleotides and applications thereof
View Patent ↗Disclosed, among other things, are primers containing certain modified nucleobases in the 3′ terminal region of the primers that provide reduced formation of primer-dimers during amplification reactions, and various methods of use thereof.
1. A purified single-stranded polynucleotide primer comprising at least one modified pyrimidine nucleobase of the structure
wherein
X is N or C,
R 1 is selected from —H, —F, —Cl, —Br, C 1 -C 6 alkyl, C 1 -C 6 substituted alkyl, C 3 -C 10 aryl, C 3 -C 10 substituted aryl, —CF 3 , —CF 2 H, —CF 2 CH 3 , —CF 2 CF 3 , —CCl 3 , —CN, —CHO, —CO 2 R, —SO 3 R, —PO 3 RR, —C(O)NRR, azido, and —NO 2 where each R is independently —H, C 1 -C 6 alkyl or C 3 -C 10 aryl or alkylaryl, and
R 2 is selected from —H, —F, —Cl, —Br, C 1 -C 6 alkyl, C 1 -C 6 substituted alkyl, C 3 -C 10 aryl, C 3 -C 10 substituted aryl, —CF 3 , —CF 2 H, —CF 2 CH 3 , —CF 2 CF 3 , —CCl 3 , —CN, —CHO, —CO 2 R, —SO 3 R, —PO 3 RR, —C(O)NRR, azido, and —NO 2 where each R is independently H, C 1 -C 6 alkyl or C 3 -C 10 aryl or alkylaryl,
wherein
at least one of R 1 or R 2 is an electron withdrawing substituent or X is N, such that when X is N, R 2 is absent,
at least one said modified pyrimidine nucleobase is no more than 4 nucleotides from the 3′ terminus of the primer, and
said purified single-stranded polynucleotide primer comprising said modified pyrimidine nucleobase now complementary to a target sequence.
2. The polynucleotide primer according to claim 1 , wherein X is C.
3. The polynucleotide primer according to claim 2 , wherein R 2 is —H.
4. The polynucleotide primer according to claim 3 , wherein R 1 is selected from —F, —Cl, —Br, —CO 2 R, —NO 2 , —CHO, —COCH 3 , —CF 3 , —CF 2 H, azido, and —CN.
5. The polynucleotide primer according to claim 4 , wherein R 1 is —F.
6. The polynucleotide primer according to claim 4 , wherein R 1 is —CF 3 .
7. The polynucleotide primer according to claim 4 , wherein R 1 is —CN.
8. The polynucleotide primer according to claim 4 , wherein R 1 is —Cl.
9. The polynucleotide primer according to claim 4 , wherein R 1 is —CO 2 R.
10. The polynucleotide primer according to claim 4 , wherein R 1 is —NO 2 .
11. The polynucleotide primer according to claim 4 , wherein R 1 is —CHO.
12. The polynucleotide primer according to claim 4 , wherein R 1 is —COCH 3 .
13. The polynucleotide primer according to claim 4 , wherein R 1 is —CF 2 H.
14. The polynucleotide primer according to claim 4 , wherein R 1 is —Br.
15. The polynucleotide primer according to claim 1 , wherein R 2 is selected from —F, —Cl, —Br, —CO 2 R, —NO 2 , —CHO, —SO 3 R, —PO 3 RR—C(O)CH 3 , —CF 3 , —CF 2 H, azido, and —CN.
16. The polynucleotide primer of claim 1 , wherein at least one modified nucleotide is the 3′ terminal nucleotide of the polynucleotide.
17. The polynucleotide primer of claim 1 , wherein at least one modified pyrimidine nucleobase is no more than 3 nucleotides from the 3′ terminus of the polynucleotide.
18. The polynucleotide primer of claim 1 , wherein at least one modified pyrimidine nucleobase is no more than 2 nucleotides from the 3′ terminus of the polynucleotide.
19. The polynucleotide primer of claim 1 , wherein the polynucleotide is extendable at the 3′-terminus.