Enzymes and polymerases for the synthesis of RNA
The invention relates to compositions and methods for the design, evolution, preparation, and/or manufacture of enzymes for use with polynucleotides, primary transcripts and mmRNA molecules.
1. An isolated polynucleotide encoding a chimeric enzyme for synthesizing a capped RNA molecule that comprises at least one chemical modification, the chimeric enzyme comprising (a) at least one capping enzyme that comprises a Vaccinia capping enzyme catalytic subunit (D1) and a Vaccinia capping enzyme stimulation subunit (D12), and (b) at least one T7 RNA polymerase (T7), in one of the following combinations:
(i) a single polypeptide comprising D1 linked to D12 linked to T7;
(ii) a single polypeptide comprising D12 linked to D1 linked to T7;
(iii) a single polypeptide comprising T7 linked to D1 linked to D12; or
(iv) a single polypeptide comprising T7 linked to D12 linked to D1.
2. An expression vector comprising the isolated polynucleotide of claim 1 .
3. The expression vector of claim 2 comprising a pEXpress vector or a pET vector.
4. The isolated polynucleotide of claim 1 , wherein D1, D12 and/or T7 are linked using at least one linker peptide.
5. The isolated polynucleotide of claim 4 , wherein the at least one linker peptide is selected from the group consisting of (GGGGS)n, LGGGGSGGGGSGGGGSAAA (SEQ ID NO: 173), LSGGGGSGGGGSGGGGSGGGGSAAA (SEQ ID NO: 174), and EGKSSGSGSESKST (SEQ ID NO: 175), wherein n refers to any whole integer.
6. The isolated polynucleotide of claim 1 , wherein the T7 RNA polymerase is encoded by the nucleic acid sequence SEQ ID NO: 176.
7. The isolated polynucleotide of claim 1 , wherein the T7 RNA polymerase comprises the amino acid sequence SEQ ID NO: 177.
8. The isolated polynucleotide of claim 1 , wherein the T7 RNA polymerase is a T7 RNA polymerase variant.
9. The isolated polynucleotide of claim 8 , wherein the T7 RNA polymerase variant is produced using continuous evolution.
10. The isolated polynucleotide of claim 9 , wherein the continuous evolution is phage-assisted continuous evolution (PACE).
11. The isolated polynucleotide of claim 8 , wherein the T7 RNA polymerase variant is capable of incorporating at least one chemical modification into RNA during in vitro transcription.
12. The isolated polynucleotide of claim 8 , wherein the T7 RNA polymerase variant is characterized as having an increased transcription efficiency through GC-rich regions as compared to a wild type T7 RNA polymerase.
13. The isolated polynucleotide of claim 8 , wherein the T7 RNA polymerase variant is characterized as having an increased transcription efficiency to produce messenger RNA as compared to a wild type T7 RNA polymerase.
14. The isolated polynucleotide of claim 1 , wherein the chimeric enzyme further comprises an affinity purification tag.
15. The isolated polynucleotide of claim 1 , wherein the capped RNA molecule is a messenger RNA.