Large-scale synthesis of messenger RNA
The present invention provides methods for large-scale production of a composition enriched for full-length mRNA molecules using an SP6 RNA polymerase and compositions produced using such methods and uses thereof.
1. A method for large-scale production of a composition enriched for full-length mRNA molecules comprising synthesizing in vitro mRNA using a recombinant SP6 RNA polymerase having 90% identity to SEQ ID NO: 1, wherein the mRNA is synthesized in about 1 gram to 250 gram quantities in a single batch in a reaction mixture comprising NTPs at a concentration ranging from 1-10 mM each NTP, a DNA template at a concentration ranging from 0.01-0.5 mg/ml, the recombinant SP6 RNA polymerase at a concentration ranging from 0.01-0.1 mg/ml, and a salt or buffering agent, at pH 6.5-8.0, and wherein the reaction is incubated for 60-120 minutes at between 37° C.−42° C.
2. The method of claim 1 , wherein at least 80% of the synthesized mRNA molecules are full-length.
3. The method of claim 1 , wherein at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the synthesized mRNA molecules are full-length.
4. The method of claim 1 , wherein the composition comprises less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of abortive transcripts.
5. The method of claim 3 or 4 , wherein the full-length or abortive transcripts of mRNA are detected by agarose gel electrophoresis.
6. The method of claim 5 , wherein the mRNA is denatured by glyoxal before agarose gel electrophoresis (“glyoxal agarose gel electrophoresis”).
7. The method of claim 3 or 4 , wherein the full-length or abortive transcripts of mRNA are detected by capillary electrophoresis.
8. The method of claim 7 , wherein the capillary electrophoresis is coupled with a fluorescence-based detection.
9. The method of claim 7 , wherein the capillary electrophoresis is coupled with UV absorption spectroscopy detection.
10. The method of claim 1 , wherein the composition is substantially free of abortive transcripts.
11. The method of claim 1 , wherein the method further comprises a step of capping and/or tailing the synthesized mRNA.
12. The method of claim 1 , wherein the full-length mRNA molecule is at least 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb in length.
13. The method of claim 1 , wherein the method further comprises a step of purifying synthesized mRNA to remove abortive transcripts.
14. The method of claim 1 , wherein the recombinant SP6 RNA polymerase has the amino acid sequence of SEQ ID NO: 1.
15. The method of claim 14 , wherein the recombinant SP6 RNA polymerase further comprises a tag.
16. The method of claim 1 , wherein the DNA template is sequence optimized.
17. The method of claim 1 , wherein the NTPs comprise one or more modified NTPs.
18. The method of claim 1 , wherein the mRNA is codon optimized.