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 , B 2 , and B 3 are independently a natural, a modified, or an unnatural nucleoside base; and
R 1 , R 2 , R 3 and R 4 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, a second nucleotide at nucleotide position +2, and a third nucleotide at nucleotide position +3; and
wherein the initiating capped oligonucleotide primer is hybridized to the DNA template at least at nucleotide positions +1, +2, and +3.
2. The complex of claim 1 , wherein R 3 is O-methyl and R 4 is OH.
3. The complex of claim 1 , wherein R 3 and R 4 are O-methyl.
4. The complex of claim 1 , wherein R 4 is O-methyl.
5. The complex of claim 1 , wherein R 1 is OH, R 2 is OH, R 3 is O-methyl, and R 4 is OH.
6. The complex of claim 1 , wherein R 1 is OH, R 2 is OH, R 3 is O-methyl, and R 4 is O-methyl.
7. The complex of claim 1 , wherein at least one of R 1 and R 2 is O-methyl, R 3 is O-methyl, and R 4 is OH.
8. The complex of claim 1 , wherein at least one of R 1 and R 2 is O-methyl, R 3 is O-methyl, and R 4 is O-methyl.
9. The complex of claim 1 , wherein B 1 , B 2 , and B 3 are natural nucleoside bases.
10. The complex of claim 1 , wherein at least one of B 1 , B 2 , and B 3 is a modified or an unnatural base.
11. The complex of claim 1 , wherein at least one of B 1 , B 2 , and B 3 is N6-methyladenine.
12. The complex of claim 1 , wherein B 1 is adenine, cytosine, thymine, or uracil.
13. The complex of claim 1 , wherein B 1 is adenine, B 2 is uracil, and B 3 is adenine.
14. The complex of claim 1 , wherein R 1 and R 2 are OH, R 3 and R 4 are O-methyl, B 1 is adenine, B 2 is uracil, and B 3 is adenine.