High fidelity in vitro transcription
The present disclosure provides methods and compositions for high fidelity in vitro transcription reactions.
1. A method of producing a messenger RNA (mRNA), the method comprising
(a) identifying a non-canonical terminator motif NNATCTGTTNN (SEQ ID NO: 50) in a DNA encoding a mRNA that encodes a polypeptide, wherein N at each position of the non-canonical terminator motif is any nucleotide selected from A, T, C, and G;
(b) producing a modified DNA comprising at least one codon substitution in the non-canonical terminator motif that preserves the amino acid sequence of the polypeptide; and
(c) producing a modified mRNA in an in vitro transcription reaction that comprises the modified DNA, nucleoside triphosphates, and a T7 RNA polymerase.
2. The method of claim 1 , wherein:
the polypeptide comprises an amino acid sequence X1-Ser-Val, and X1 is selected from the group consisting of Ile, Leu, Val, Ala, Gly, Pro, Thr, Ser, Gln, Glu, Lys, and Arg;
the polypeptide comprises an amino acid sequence X2-Leu-Phe, and X2 is selected from the group consisting of Tyr, His, Asn, and Asp;
the polypeptide comprises an amino acid sequence X2-Leu-Leu, and X2 is selected from the group consisting of Tyr, His, Asn, and Asp;
the polypeptide comprises an amino acid sequence Ile-Cys-X3, and X3 is selected from the group consisting of Leu, Phe, Cys, Ser, Tyr, and Trp.
3. The method of claim 1 , wherein the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1.
4. The method of claim 1 , wherein the T7 RNA polymerase comprises an amino acid substitution at position 47, relative to a T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.
5. The method of claim 4 , wherein the amino acid substitution at position 47 is G47A.
6. The method of claim 5 , wherein the amino acid modification comprises an additional C-terminal amino acid, relative to the T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.
7. The method of claim 6 , wherein the additional C-terminal amino acid is selected from glycine, threonine, serine, alanine, and proline.
8. The method of claim 1 , wherein the T7 RNA polymerase comprises an amino acid substitution at a position selected from positions 350, 351, 387, 394, 425, 427, 437, 441, 632, 811, and 880, relative to T7 RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.
9. The method of claim 8 , wherein the amino acid substitution is at position 350, and the amino acid substitution at position 350 is selected from E350K, E350N, E350A, and E350W.
10. A method of producing a modified DNA encoding a messenger RNA (mRNA), the method comprising
(a) identifying a non-canonical terminator motif NNATCTGTTNN (SEQ ID NO: 50) in a DNA encoding a mRNA that encodes a polypeptide, wherein N at each position of the non-canonical terminator motif is any nucleotide selected from A, T, C, and G; and
(b) producing a modified DNA encoding the mRNA, wherein the modified DNA comprises at least one codon substitution in the non-canonical terminator motif that preserves the amino acid sequence of the polypeptide encoded by the mRNA.
11. The method of claim 8 , wherein the amino acid substitution is at position 351, and the amino acid substitution at position 351 is D351V.
12. The method of claim 8 , wherein the amino acid substitution is at position 387, and the amino acid substitution at position 387 is K387H, K387N, and K387S.
13. The method of claim 8 , wherein the amino acid substitution is at position 437, and the amino acid substitution at position 437 is N437T, N437I, N437Y and N437F.
14. The method of claim 8 , wherein the amino acid substitution is at position 441, and the amino acid substitution at position 441 is K441R.
15. The method of claim 8 , wherein the amino acid substitution is at position 880, and the amino acid substitution at position 880 is F880Y.