Compositions and methods for synthesizing 5′-Capped RNAs
Provided herein are methods and compositions for synthesizing 5′Capped RNAs wherein the initiating capped oligonucleotide primers have the general form m7 Gppp[N 2′Ome ] n [N] m wherein m7 G is N7-methylated guanosine or any guanosine analog, N is any natural, modified or unnatural nucleoside, “n” can be any integer from 0 to 4 and “m” can be an integer from 1 to 9.
1. A complex comprising an initiating capped oligonucleotide primer and a DNA template,
wherein the initiating capped oligonucleotide primer comprises a structure:
wherein
B 1 and B 2 are independently a natural, a modified, or an unnatural nucleoside base; and
R 1 , R 2 , and R 3 are independently OH or O-methyl;
wherein the DNA template comprises a promoter region comprising a transcriptional start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; and
wherein the initiating capped oligonucleotide primer is hybridized to the DNA template at least at nucleotide positions +1 and +2.
2. The complex of claim 1 , wherein B 1 is at nucleotide position +1 and B 2 is at nucleotide position +2.
3. The complex of claim 1 , wherein B 1 and B 2 are natural nucleoside bases.
4. The complex of claim 1 , wherein at least one of B 1 and B 2 is a modified or an unnatural base.
5. The complex of claim 1 , wherein at least one of B 1 and B 2 is N6-methyladenine.
6. The complex of claim 1 , wherein B 1 is adenine and B 2 is guanine.
7. The complex of claim 1 , wherein B 1 is guanine and B 2 is guanine.
8. The complex of claim 1 , wherein B 1 is adenine and B 2 is uracil.
9. The complex of claim 1 , wherein R 3 is OH.
10. The complex of claim 1 , wherein R 3 is O-methyl.
11. The complex of claim 1 , wherein at least one of R 1 and R 2 is O-methyl, B 1 is adenine, and B 2 is guanine.
12. The complex of claim 1 , wherein at least one of R 1 and R 2 is O-methyl, and B 1 and B 2 are guanine.
13. The complex of claim 1 , wherein at least one of R 1 and R 2 is O-methyl, B 1 is adenine, and B 2 is uracil.
14. The complex of claim 1 , wherein B 1 is N6-methyladenine and B 2 is a natural, a modified, or an unnatural base.
15. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
16. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
17. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
18. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
19. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
20. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
21. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
22. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
23. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
24. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
25. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
26. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
27. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
28. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
29. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
30. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure:
31. The complex of claim 1 , wherein the initiating capped oligonucleotide primer comprises the structure: