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 method for synthesizing a fully templated RNA molecule comprising the steps of:
introducing an initiating capped oligonucleotide primer, or a salt thereof of the structure:
wherein:
each of B 1 through B 10 is independently a natural, modified or unnatural nucleoside base;
M is 0 or 1;
L is 0 or 1;
q 1 is 1 and each of q 2 through q 9 is independently 0 or 1;
R 1 is H or methyl;
R 2 and R 3 are each OH;
each of X 1 through X 13 is independently O or S;
each of Y 1 through Y 13 is independently OH, SH, BH 3 , aryl, alkyl, O-alkyl or O-aryl;
each of Z 0 through Z 22 is independently O, S, NH, CH 2 , C(halogen) 2 or CH(halogen); and
each of R 4 through R 12 is independently H, OH, O-methyl or a detectable marker into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template, wherein B 1 in the initiating capped oligonucleotide primer is complementary to the base at position +1 in the promoter sequence of the polynucleotide template; and
incubating said mixture for a time sufficient to allow for transcription of said template.
2. The method of claim 1 , wherein B2 in the initiating capped oligonucleotide primer is complementary to the base at position +2 in the promoter sequence of the polynucleotide template.
3. The method of claim 1 , wherein each of B 1 through B 10 are each independently adenine, N 6 -methyladenine, guanine, cytosine, or uracil.
4. The method of claim 1 , wherein each of q 2 through q 9 is 0.
5. The method of claim 4 , wherein B 1 is adenine, N 6 -methyladenine, or guanine.
6. The method of claim 4 , wherein B 10 is guanine or uracil.
7. The method of claim 4 , wherein B 1 is adenine and B 10 is guanine.
8. The method of claim 4 , wherein B 1 is adenine and B 10 is uracil.
9. The method of claim 4 , wherein B 1 is guanine and B 10 is guanine.
10. The method of claim 4 , wherein B 1 is cytosine and B 10 is guanine.
11. The method of claim 4 , wherein R 1 is H.
12. The method of claim 4 , wherein R 4 is O-methyl.
13. The method of claim 1 , wherein q 2 is 1 and each of q 3 through q 9 is 0.
14. The method of claim 13 , wherein B 2 and B 10 are guanine.
15. The method of claim 13 , wherein B 1 is adenine or N 6 -methyladenine.
16. The method of claim 13 , wherein R 1 is H.
17. The method of claim 13 , wherein R 4 and R 5 are O-methyl.
18. A method for synthesizing a fully templated RNA molecule comprising the steps of:
introducing an initiating capped oligonucleotide primer, or a salt thereof, of the structure:
wherein:
B 1 is adenine or N 6 -methyladenine;
B 10 is uracil;
R 1 is H or methyl;
R 2 and R 3 are each OH; and
R 4 is H, OH, O-methyl or a detectable marker,
into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template; and
incubating said mixture for a time sufficient to allow for transcription of said template.
19. The method of claim 18 , wherein B 1 is adenine.
20. The method of claim 18 , wherein R 1 is H.
21. The method of claim 18 , wherein R 4 is O-methyl.
22. A method for synthesizing a fully templated RNA molecule comprising the steps of:
introducing an initiating capped oligonucleotide primer, or a salt thereof, of the structure:
wherein:
B 1 is guanine;
B 10 is a natural, modified, or unnatural nucleoside base;
R 1 is H or methyl;
R 2 and R 3 are each OH; and
R 4 is H, OH, O-methyl or a detectable marker,
into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template; and
incubating said mixture for a time sufficient to allow for transcription of said template.
23. The method of claim 22 , wherein B 10 is adenine, N 6 -methyladenine, guanine, cytosine, or uracil.
24. The method of claim 22 , wherein B 10 is guanine.
25. The method of claim 22 , wherein R 1 is H.
26. The method of claim 22 , wherein R 4 is O-methyl.
27. A method for synthesizing a fully templated RNA molecule comprising the steps of:
introducing an initiating capped oligonucleotide primer, or a salt thereof, of the structure:
wherein:
B 1 is cytosine;
B 10 is guanine;
R 1 is H or methyl;
R 2 and R 3 are each OH; and
R 4 is H, OH, O-methyl, or a detectable marker,
into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template; and
incubating said mixture for a time sufficient to allow for transcription of said template.
28. The method of claim 27 , wherein R 1 is H.
29. The method of claim 27 , wherein R 4 is O-methyl.
30. A method for synthesizing a fully templated RNA molecule comprising the steps of:
introducing an initiating capped oligonucleotide primer, or a salt thereof, of the structure:
wherein:
B 1 , B 2 , and B 10 are independently a natural, modified or unnatural nucleoside base;
R 1 is H or methyl;
R 2 and R 3 are each OH; and
R 4 and R 5 are independently H, OH, O-methyl, or a detectable marker, into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template; and
incubating said mixture for a time sufficient to allow for transcription of said template.
31. The method of claim 30 , wherein B 1 , B 2 , and B 10 are each independently adenine, N 6 -methyladenine, guanine, cytosine, or uracil.
32. The method of claim 30 , wherein B 2 and B 10 are guanine.
33. The method of claim 30 , wherein B 1 is adenine or N 6 -methyladenine.
34. The method of claim 30 , wherein R 1 is H.
35. The method of claim 30 , wherein R 4 and R 5 are O-methyl.