Compositions and methods for synthesizing 5′-capped RNAs

1. An initiating capped oligonucleotide primer comprising the following 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 independently H, OH, alkyl, O-alkyl, halogen, a linker or a detectable marker;

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 are independently H, OH, OMe or a detectable marker.

2. An initiating capped oligonucleotide primer according to claim 1 wherein:

q 2 is 1; and

each of q 3 through q 9 is 0.

3. An initiating capped oligonucleotide primer according to claim 1 , wherein:

B 1 is completely complementary to nucleoside base on the nucleic acid template at transcription template position +1,

B 2 through B 9 , if present, are completely complementary to respective nucleoside bases on nucleic acid template at transcription start site from position +2 and on, and

B 10 is completely complementary to the last hybridized transcription template nucleotide.

4. An initiating capped oligonucleotide primer according to claim 3 wherein:

each of q 2 through q 9 is 0, and

B 10 hybridizes to template nucleotide 2.

5. An initiating capped oligonucleotide primer according to claim 4 wherein:

B 1 is adenine or N6-methyladenine.

6. An initiating capped oligonucleotide primer according to claim 1 wherein:

q 2 is 1;

q 3 is 1; and

each of q 4 through q 9 is 0.

7. An initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 4 is 1, and

each of q 5 through q 9 is 0.

8. An initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 5 is 1, and

each of q 6 through q 9 is 0.

9. An initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 6 is 1, and

each of q 7 through q 9 is 0.

10. An initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 7 is 1, and

each of q 8 through q 9 is 0.

11. An initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 8 is 1, and

q 9 is 0.

12. An initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 9 is 1.

13. An initiating capped oligonucleotide primer according to claim 1 wherein:

at least one of R 2 and R 3 is H, alkyl, O-alkyl, halogen, a linker or a detectable marker.

14. An RNA molecule comprising the initiating capped oligonucleotide primer according to claim 1 .

15. An isolated cell containing an RNA molecule comprising the initiating capped oligonucleotide primer according to claim 1 .

16. An isolated cell containing a protein or a peptide translated from an RNA molecule comprising the initiating capped oligonucleotide primer according to claim 1 .

17. A pharmaceutical composition comprising an RNA molecule comprising the initiating capped oligonucleotide primer according to claim 1 and a pharmaceutical acceptable carrier.

18. A method for synthesizing a fully templated RNA molecule comprising the steps of:

introducing the initiating capped oligonucleotide primer with 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 independently H, OH, alkyl, O-alkyl, halogen, a linker or a detectable marker;

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 are independently H, OH, OMe or a detectable marker into a mixture comprising 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 initiating capped oligonucleotide primer according to claim 1 wherein:

at least one of X 1 through X 13 is S.

20. The initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 9 is 0, and

at least one of X 1 through X 4 is S.

21. The initiating capped oligonucleotide primer according to claim 20 , wherein:

B 1 is adenine or N6-methyladenine and B 10 is guanine.

22. The initiating capped oligonucleotide primer according to claim 1 wherein:

at least one of Y 1 through Y 12 is SH, BH 3 , aryl, alkyl, O-alkyl or O-aryl.

23. The initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 9 is 0, and

at least one of Y 1 through Y 4 is SH, BH 3 , aryl, alkyl, O-alkyl or O-aryl.

24. The initiating capped oligonucleotide primer according to claim 23 , wherein:

B 1 is adenine or N6-methyladenine and B 10 is guanine.

25. The initiating capped oligonucleotide primer according to claim 1 wherein:

at least one of Z 1 through Z 22 is S, NH, CH 2 , C(halogen) 2 or CH(halogen).

26. The initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 9 is 0, and

at least one of Z 1 through Z 22 is S, NH, CH 2 , C(halogen) 2 or CH(halogen).

27. The initiating capped oligonucleotide primer according to claim 26 , wherein:

B 1 is adenine or N6-methyladenine and B 10 is guanine.

28. The initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 9 is 0, and

B 1 is adenine or N6-methyladenine and B 10 is uracil.

29. The initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 9 is 0, and

B 1 is guanine and B 10 is guanine.

30. The initiating capped oligonucleotide primer according to claim 1 wherein:

R 1 is methyl.

31. The initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 9 is 0, and

B 1 is guanine, cytosine, or uracil.

32. The initiating capped oligonucleotide primer according to claim 1 wherein:

each of q 2 through q 9 is 0, and

B 10 is adenine or N6-methyladenine, cytosine, or uracil.

33. The initiating capped oligonucleotide primer according to claim 1 wherein:

the initiating capped primer is selected from the group consisting of m7 GpppApA, m7 GpppApC, m7 GpppApU, m7 GpppCpA, m7 GpppCpC, m7 GpppCpG, m7 GpppCpU, m7 GpppGpA, m7 GpppGpC, m7 GpppGpG, m7 GpppGpU, m7 GpppUpA, m7 GpppUpC, m7 GpppUpG, m7 GpppUpU, m7 G 3′Ome pppApA, m7 G 3′Ome pppApC, m7 G3′OmepppApG, m7 G 3′Ome pppApU, m7 G 3′Ome pppCpA, m7 G 3′Ome pppCpC, m7 G 3′Ome pppCpG, m7 G 3′Ome pppCpU, m7 G 3′Ome pppGpA, m7 G 3′Ome pppGpC, m7 G 3′Ome pppGpG, m7 G 3′Ome pppGpU, m7 G 3′Ome pppUpA, m7 G 3′Ome pppUpC, m7 G 3′Ome pppUpG, m7 G 3′Ome pppUpU, m7 G 3′Ome pppA 2′Ome pA, m7 G 3′Ome pppA 2′Ome pC, m7 G 3′Ome pppA 2′Ome pG, m7 G 3′Ome pppA 2′Ome pU, m7 G 3′Ome pppC 2′Ome pA, m7 G 3′Ome pppC 2′Ome pC, m7 G 3′Ome pppC 2′Ome pG, m7 G 3′Ome pppC 2′Ome pU, m7 G 3′Ome pppG 2′Ome pA, m7 G 3′Ome pppG 2′Ome pC, m7 G 3′Ome pppG 2′Ome pG, m7 G 3′Ome pppG 2′Ome pU, m7 G 3′Ome pppU 2′Ome pA, m7 G 3′Ome pppU 2′Ome pC, m7 G 3′Ome pppU 2′Ome pG, m7 G 3′Ome pppU 2′Ome pU, m7 GpppA 2′Ome pA, m7 GpppA 2′Ome pC, m7 GpppA 2′Ome pU, m7 GpppC 2′Ome pA, m7 GpppC 2′Ome pC, m7 GpppC 2′Ome pG, m7 GpppC 2′Ome pU, m7 GpppG 2′Ome pA, m7 GpppG 2′Ome pC, m7 GpppG 2′Ome pG, m7 GpppG 2′Ome pU, m7 GpppU 2′Ome pA, m7 GpppU 2′Ome pC, m7 GpppU 2′Ome pG, and m7 GpppU 2′Ome pU.

34. The initiating capped oligonucleotide primer according to claim 1 wherein:

the initiating capped primer is selected from the group consisting of m7 G 2′Ome pppA 2′Ome pA, m7 G 2′Ome pppA 2′Ome pC, m7 G 2′Ome pppA 2′Ome pG, m7 G 2′Ome pppA 2′Ome pU, m7 G 2′Ome pppC 2′Ome pA, m7 G 2′Ome pppC 2′Ome pC, m7 G 2′Ome pppC 2′Ome pG, m7 G 2′Ome pppC 2′Ome pU, m7 G 2′Ome pppG 2′Ome pA, m7 G 2′Ome pppG 2′Ome pC, m7 G 2′Ome pppG 2′Ome pG, m7 G 2′Ome pppG 2′Ome pU, m7 G 2′Ome pppU 2′Ome pA, m7 G 2′Ome pppU 2′Ome pC, m7 G 2′Ome pppU 2′Ome pG, and m7 G 2′Ome pppU 2′Ome pU, m7 G 2′Ome pppApA, m7 G 2′Ome pppApC, m7 G 2′Ome pppApG, m7 G 2′Ome pppApU, m7 G 2′Ome pppCpA, m7 G 2′Ome pppCpC, m7 G 2′Ome pppCpG, m7 G 2′Ome pppCpU, m7 G 2′Ome pppGpA, m7 G 2′Ome pppGpC, m7 G 2′Ome pppGpG, m7 G 2′Ome pppGpU, m7 G 2′Ome pppUpA, m7 G 2′Ome pppUpC, m7 G 2′Ome pppUpG, and m7 G 2′Ome pppUpU.

35. The initiating capped oligonucleotide primer according to claim 1 wherein:

the initiating capped primer is selected from the group consisting of m7 Gppp(N6-methyladenine)pA, m7 Gppp(N6-methyladenine)pC, m7 Gppp(N6-methyladenine)pU, m7 G 3′Ome ppp(N6-methyladenine)pA, m7 G 3′Ome PPP(N6-methyladenine)pC, m7 G 3′Ome ppp(N6-methyladenine)pG, m7 G 3′Ome ppp(N6-methyladenine)pU, m7 G 2′Ome ppp(N6-methyladenine)pA, m7 G 2′Ome ppp(N6-methyladenine)pC, m7 G 2′Ome ppp(N6-methyladenine)pG, m7 G 2′Ome ppp(N6-methyladenine)pU, m7 Gppp(N6-methyladenine) 2′Ome pC, m7 Gppp(N6-methyladenine) 2′Ome pG, and m7 Gppp(N6-methyladenine) 2′Ome pU, m7 G 2′Ome ppp(N6-methyladenine) 2′Ome pA, m7 G 2′Ome ppp(N6-methyladenine) 2′Ome pC, m7 G 2′Ome ppp(N6-methyladenine) 2′Ome pG, m7 G 2′Ome ppp(N6-methyladenine) 2′Ome pU m7 G 3′Ome ppp(N6-methyladenine) 2′Ome pA, m7 G 3′Ome ppp(N6-methyladenine) 2′Ome pC, m7 G 3′Ome ppp(N6-methyladenine) 2′Ome pG, and m7 G 3′Ome ppp(N6-methyladenine) 2′Ome pU.

36. The initiating capped oligonucleotide primer according to claim 1 , wherein

the initiating capped oligonucleotide primer comprises the structure:

37. The initiating capped oligonucleotide primer according to claim 1 , wherein

the initiating capped oligonucleotide primer comprises the structure:

38. A complex comprising a DNA template and an initiating capped oligonucleotide primer comprising the following 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 independently H, OH, alkyl, O-alkyl, halogen, a linker or a detectable marker;

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 are independently H, OH, OMe or a detectable marker; and

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.

39. The initiating capped oligonucleotide primer according to claim 38 wherein:

each of q 2 through q 9 is 0.

40. The initiating capped oligonucleotide primer according to claim 39 wherein:

B 1 is adenine or N6-methyladenine and B 10 is guanine.

41. The complex according to claim 39 , wherein:

B 1 is adenine or N6-methyladenine and B 10 is uracil.

42. The complex according to claim 38 , wherein:

R 1 is Methyl.

43. The complex according to claim 38 , wherein:

each of q 2 through q 9 is 0, and

at least one of X 1 through X 13 is S.

44. The complex according to claim 38 , wherein:

at least one of Y 1 through Y 12 is SH, BH 3 , aryl, alkyl, O-alkyl or O-aryl.

45. The complex according to claim 38 , wherein:

at least one of Z 1 through Z 22 is S, NH, CH 2 , C(halogen) 2 or CH(halogen).

46. An initiating capped oligonucleotide primer comprising the following structure:

wherein:

B 1 and B 2 are independently a natural, modified or unnatural nucleoside base;

R 1 is H or methyl; and

R 2 and R 3 are independently H, OH, alkyl, or O-alkyl.