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 modified 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;
each R 1 is independently H or methyl;
R 2 and R 3 are independently H, OH, alkyl, O-alkyl, halogen, a linker or a detectable marker, wherein at least one of R 2 and R 3 is H, 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 is independently H, OH, O-methyl, or a detectable marker.
2 . The modified initiating capped oligonucleotide primer of claim 1 , wherein each R 1 is H.
3 . The modified initiating capped oligonucleotide primer of claim 1 , wherein R 2 is OH or O-methyl.
4 . The modified initiating capped oligonucleotide primer of claim 1 , wherein R 3 is OH or O-methyl.
5 . The modified initiating capped oligonucleotide primer of claim 1 , wherein R 4 is OH or O-methyl.
6 . The modified initiating capped oligonucleotide primer of claim 1 , wherein each of B 1 through B 10 is independently adenine, N 6 -methyladenine, guanine, cytosine, or uracil.
7 . The modified initiating capped oligonucleotide primer of claim 1 , wherein B 1 is adenine.
8 . The modified initiating capped oligonucleotide primer of claim 1 , wherein B 1 is N 6 -methyladenine.
9 . The modified initiating capped oligonucleotide primer of claim 1 , wherein B 1 is guanine.
10 . The modified initiating capped oligonucleotide primer of claim 1 , wherein B 10 is guanine.
11 . The modified initiating capped oligonucleotide primer of claim 1 , wherein B 10 is uracil.
12 . The modified initiating capped oligonucleotide primer of claim 1 , wherein each of q 2 through q 9 is 0.
13 . The modified initiating capped oligonucleotide primer of claim 12 , wherein B 1 is adenine, N 6 -methyladenine, or guanine.
14 . The modified initiating capped oligonucleotide primer of claim 12 , wherein B 10 is guanine or uracil.
15 . The modified initiating capped oligonucleotide primer of claim 12 , wherein B 1 is adenine and B 10 is guanine.
16 . The modified initiating capped oligonucleotide primer of claim 12 , wherein B 1 is N 6 -methyladenine and B 10 is guanine.
17 . The modified initiating capped oligonucleotide primer of claim 12 , wherein B 1 is adenine and B 10 is uracil.
18 . A modified RNA molecule comprising the modified initiating capped oligonucleotide primer according to claim 1 .
19 . A cell containing a modified RNA molecule comprising the modified initiating capped oligonucleotide primer according to claim 1 .
20 . A pharmaceutical composition comprising a modified RNA molecule comprising the modified initiating capped oligonucleotide primer according to claim 1 and a pharmaceutical acceptable carrier.
21 . A complex comprising an initiating capped oligonucleotide primer and a DNA template,
wherein the initiating capped oligonucleotide primer comprises a structure:
wherein:
B 1 is N 6 -methyladenine;
B 10 is guanine;
R 1 is H or methyl;
R 2 and R 3 are OH; and
R 4 is 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.