Compositions and methods for preparing capped mRNA

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 R1 and R2 are each CH3 or H;
• and B1 and B2 are each A, U, G, or C,
• wherein the promoter comprises the sequence of
• TAATACGACTCACTATAX1X2X3 (SEQ ID NO:
• 16),
• wherein A at position 17 is −1 nucleotide and X1 at position 18 is +1 nucleotide,
• when X1 is G, X2 and X3 are each A, T, G, or C, then B1 is A and B2 is G,
• when X1 is C, X2 and X3 are each A, T, G, or C, then B1 is A and B2 is C, and
• when X1 is T, X2 and X3 are each A, T, G, or C, then B1 is A and B2 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.