Methods of producing rna
AssigneeModernaTX, Inc.
InventorsMargaret Franklin, Amy E. Rabideau, Ruchi Jain, Mihir Metkar
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Abstract
Aspects of the disclosure relate to invitro transcription reactions comprising an initiator oligonucleotide, a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA) polymerase, and nucleoside triphosphates (NTPs), wherein the initiator oligonucleotide comprises at least four contiguous nucleotides. In some embodiments, the disclosure relates to new and improved compositions of initiator oligonucleotides.
Claims31
An in vitro transcription reaction comprising:
- an initiator oligonucleotide, a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA) polymerase, and nucleoside triphosphates (NTPs), wherein the initiator oligonucleotide comprises at least four contiguous nucleotides.
The in vitro transcription reaction of any one of the preceding claims, wherein the initiator oligonucleotide comprises at least five, at least six, or at least seven contiguous nucleotides.
The in vitro transcription reaction of any one of the preceding claims, wherein the initiator oligonucleotide consist of no more than ten nucleotides.
The in vitro transcription reaction of any one of the preceding claims, wherein the initiator oligonucleotide comprises modified nucleotides.
The in vitro transcription reaction of any one of the preceding claims, wherein the initiator oligonucleotide comprises 2′-O-methyl nucleotides and/or phosphorothioate bonds.
The in vitro transcription reaction of any one of the preceding claims, wherein the NTPs comprise unmodified adenosine triphosphate (ATP), unmodified cytidine triphosphate (CTP), unmodified uridine triphosphate (UTP), and/or unmodified guanosine triphosphate (GTP).
The in vitro transcription reaction of any one of the preceding claims, wherein the NTPs comprise modified ATP, modified CTP, modified UTP, optionally 1-methylpseudo-UTP, and/or modified GTP.
The in vitro transcription reaction of any one of the preceding claims, wherein the reaction mixture comprises an equimolar concentration of NTPs.
The in vitro transcription reaction of any one of the preceding claims, wherein the initiator oligonucleotide comprises an adenine-guanine (AG) dinucleotide.
The in vitro transcription reaction of any one of the preceding claims, wherein the initiator oligonucleotide comprises a nucleotide sequence selected from GCAG, GCAAG, GGCAG, GCGAG, and GCAGG.
The in vitro transcription reaction of any one of the preceding claims further comprising water, buffer, and/or pyrophosphatase.
The in vitro transcription reaction of any one of the preceding claims, wherein the RNA polymerase is a T7 RNA polymerase.
The in vitro transcription reaction of any one of the preceding claims, wherein the RNA polymerase is a naturally occurring RNA polymerase.
The in vitro transcription reaction of any one of the preceding claims further comprising a ribonucleic acid (RNA) encoded by the DNA.
Dependencies are inferred from the claim text.