Compositions and methods for preparing capped mRNA
A method for in vitro transcription of a DNA template into RNA includes providing a mixture containing a buffer substance, ribonucleoside triphosphates (NTPs), one or more magnesium salts in a concentration of from about 2 mM to about 60 mM, the DNA template, and a recombinant RNA polymerase, and incubating the reaction mixture at from about 25° C. to about 40° C. for from about 1 hour to about 12 hours thereby producing the RNA. A method for in vitro transcription includes providing a DNA template and a cap analogue that binds to −1 and/or +1 nucleotides of promoter for in vitro transcription, thus producing more full length mRNAs, allowing for more flexibility on the choice of first mRNA base, and providing +2 position open for custom sequence.
1. A method for in vitro transcription of a DNA template into RNA, comprising
providing (1) a DNA template comprises a promoter operably linked to a nucleic acid comprising a 5′ untranslated region (5′ UTR), an open reading frame (ORF) encoding the RNA of interest, a 3′ UTR, and a poly A region, and
(2) a cap analogue comprises the structure of
wherein R 1 and R 2 are each CH 3 or H; and B 1 and B 2 are each A, U, G, or C,
wherein the promoter comprises the sequence of
TAATACGACTCACTATAX 1 X 2 X 3 (SEQ ID NO: 16),
wherein A at position 17 is −1 nucleotide and X 1 at position 18 is +1 nucleotide,
when X 1 is G, X 2 and X 3 are each A, T, G, or C, then B 1 is A and B 2 is G,
when X 1 is C, X 2 and X 3 are each A, T, G, or C, then B 1 is A and B 2 is C, and
when X 1 is T, X 2 and X 3 are each A, T, G, or C, then B 1 is A and B 2 is U,
wherein the cap analogue binds to −1 and +1 nucleotides of the promoter, and
incubating the DNA template and the cap analogue in a reaction mixture, wherein the incubating comprises incubating the reaction mixture at from about 15° C. to about 35° C. for from about 1 hour to about 12 hours, thereby producing the RNA, wherein
the 5′ UTR consists of SEQ ID NO: 1 and the 3′ UTR consists of SEQ ID NO: 4, or
the 5′ UTR consists of SEQ ID NO: 1 and the 3′ UTR consists of SEQ ID NO: 6, or
the 5′ UTR consists of SEQ ID NO: 3 and the 3′ UTR consists of SEQ ID NO: 4, or
the 5′ UTR consists of SEQ ID NO: 3 and the 3′ UTR consists of SEQ ID NO: 6, or
the 5′ UTR consists of SEQ ID NO: 3 and the 3′ UTR consists of SEQ ID NO: 8, or
the 5′ UTR consists of SEQ ID NO: 5 and the 3′ UTR consists of SEQ ID NO: 4, or
the 5′ UTR consists of SEQ ID NO: 5 and the 3′ UTR consists of SEQ ID NO: 6, or
the 5′ UTR consists of SEQ ID NO: 7 and the 3′ UTR consists of SEQ ID NO: 4, or
the 5′ UTR consists of SEQ ID NO: 7 and the 3′ UTR consists of SEQ ID NO: 6, or
the 5′ UTR consists of SEQ ID NO: 7 and the 3′ UTR consists of SEQ ID NO: 8, or
the 5′ UTR consists of SEQ ID NO: 9 and the 3′ UTR consists of SEQ ID NO: 4, or
the 5′ UTR consists of SEQ ID NO: 9 and the 3′ UTR consists of SEQ ID NO: 6.
2. The method of claim 1 , wherein the promoter comprises a sequence selected from SEQ ID NO: 10, 11, 13, and 14.
3. The method of claim 1 , wherein the 5′ UTR consists of SEQ ID NO: 1 and the 3′ UTR consists of SEQ ID NO: 4.
4. The method of claim 1 , wherein the cap analogue is selected from the group consisting of m 7 GpppApC, m 7 GpppApG, m 7 GpppApU, m 7 G 3′Ome pppApC, m 7 G 3′Ome pppApG, m 7 G 3′Ome pppApU, m 7 G 3′Ome pppA 2′Ome pC, m 7 G 3′Ome pppA 2′Ome pG, m 7 G 3′Ome pppA 2′Ome pU, m 7 GpppA 2′Ome pC, m 7 GpppA 2′Ome pG, and m 7 GpppA 2′Ome pU.
5. The method of claim 1 , wherein the reaction mixture comprises
a buffer substance in a concentration of from about 45 mM to about 55 mM,
an RNase inhibitor in a concentration of from about 0.01 U/μl to about 0.03 U/μl,
NTPs in a concentration of from about 3 mM to about 5 mM,
the cap analogue in a concentration of from about 6 mM to about 8 mM,
one or more magnesium salts in a concentration of from about 20 mM to about 30 mM,
a polyamine in a concentration of from about 1.5 mM to about 2.5 mM,
the DNA template in a concentration of from about 0.01 μg/μl to about 0.05 μg/μl,
a pyrophosphatase in a concentration of from about 0.1 mU/μl to about 0.5 mU/μl, and
an RNA polymerase in a concentration of from about 0.01 μg/μl to about 0.05 μg/μl.
6. The method of claim 5 , wherein the RNA polymerase is selected from wild type T7 RNA polymerase or a variant thereof.
7. The method of claim 1 , wherein the incubating comprises incubating the reaction mixture at from about 18° C. to about 31° C.
8. The method of claim 7 , wherein the incubating comprises incubating the reaction mixture at about 30° C. for about 4 hours.
9. The method of claim 1 , wherein the DNA template further comprises at least one transcriptional terminator located upstream and/or downstream of the open reading frame (ORF).
10. The method of claim 1 , wherein X 1 is G, X 2 and X 3 are each A, T, G, or C, B 1 is A, and B 2 is G.
11. The method of claim 1 , wherein X 1 is C, X 2 and X 3 are each A, T, G, or C, B 1 is A, and B 2 is C.
12. The method of claim 1 , wherein X 1 is T, X 2 and X 3 are each A, T, G, or C, B 1 is A, and B 2 is U.
13. The method of claim 1 , wherein the cap analogue in a concentration of from about 0.5 mM to about 50 mM.
14. The method of claim 1 , wherein the incubating the reaction mixture is performed at about 25° C. for from about 1 hour to about 5 hours.
15. The method of claim 2 , wherein the promoter comprises the sequence of SEQ ID NO: 10.
16. The method of claim 2 , wherein the promoter comprises the sequence of SEQ ID NO: 11.
17. The method of claim 2 , wherein the promoter comprises the sequence of SEQ ID NO: 13.
18. The method of claim 2 , wherein the promoter comprises the sequence of SEQ ID NO: 14.
19. The method of claim 1 , wherein the 5′ UTR consists of SEQ ID NO: 1 and the 3′ UTR consists of SEQ ID NO: 6.
20. The method of claim 1 , wherein the 5′ UTR consists of SEQ ID NO: 3 and the 3′ UTR consists of SEQ ID NO: 4.
21. The method of claim 1 , wherein the 5′ UTR consists of SEQ ID NO: 3 and the 3′ UTR consists of SEQ ID NO: 6.
22. The method of claim 1 , wherein the 5′ UTR consists of SEQ ID NO: 3 and the 3′ UTR consists of SEQ ID NO: 8.
23. The method of claim 1 , wherein the 5′ UTR consists of SEQ ID NO: 5 and the 3′ UTR consists of SEQ ID NO: 4.
24. The method of claim 1 , wherein the 5′ UTR consists of SEQ ID NO: 5 and the 3′ UTR consists of SEQ ID NO: 6.
25. The method of claim 1 , wherein the 5′ UTR consists of SEQ ID NO: 7 and the 3′ UTR consists of SEQ ID NO: 4.
26. The method of claim 1 , wherein the 5′ UTR consists of SEQ ID NO: 7 and the 3′ UTR consists of SEQ ID NO: 6.
27. The method of claim 1 , wherein the 5′ UTR consists of SEQ ID NO: 7 and the 3′ UTR consists of SEQ ID NO: 8.
28. The method of claim 1 , wherein the 5′ UTR consists of SEQ ID NO: 9 and the 3′ UTR consists of SEQ ID NO: 4.
29. The method of claim 1 , wherein the 5′ UTR consists of SEQ ID NO: 9 and the 3′ UTR consists of SEQ ID NO: 6.