FCE mRNA capping enzyme compositions, methods and kits
The present disclosure relates to compositions, kits, and methods of making RNA vaccines having an appropriate cap structure. Systems, apparatus, compositions, and/or methods may include and/or use, in some embodiments, non-naturally occurring single-chain RNA capping enzymes. In some embodiments, an RNA capping enzyme may include an FCE variant having (a) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (b) one or more substitutions relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1.
1. An FCE variant having (a) an amino acid sequence at least 95% identical to positions 1 to 878 of SEQ ID NO: 1, and (b) a substitution relative to SEQ ID NO: 1 at a position corresponding to positions 215, 337, 572, 648, and/or 833 of SEQ ID NO: 1.
2. An FCE variant according to claim 1 , wherein the substitution is at a position corresponding to positions 215, 337, and/or 572 of SEQ ID NO: 1.
3. An FCE variant according to claim 1 further comprising a second substitution at a position (i) other than the position of the first substitution and (ii) corresponding to position 215, 337, 572, 648, or 833 of SEQ ID NO: 1.
4. An FCE variant according to claim 3 further comprising a third substitution at a position (iii) other than the position of the first and second substitutions and (iv) corresponding to position 215, 337, 572, 648, or 833 of SEQ ID NO: 1.
5. An FCE variant according to claim 4 further comprising a fourth substitution at a position (v) other than the position of the first, second and third substitutions and (vi) corresponding to position 215, 337, 572, 648, or 833 of SEQ ID NO: 1.
6. A fusion comprising, in an N-terminal to C-terminal direction, (I) a purification tag or a sorting signal peptide, and (II) the FCE variant according to claim 1 operably linked to (I).
7. A fusion comprising, in an N-terminal to C-terminal direction, (III) the FCE variant according to claim 1 , and (IV) a purification tag or a sorting signal peptide operably linked to (III).
8. A composition comprising the FCE variant according to claim 1 and a polynucleotide.
9. The composition according to claim 8 , wherein the polynucleotide is an uncapped ribonucleic acid.
10. A composition comprising (a) the FCE variant according to claim 1 and (b) a cap, an NTP, a modified NTP, a buffer, S-adenosylmethionine, and/or an RNase inhibitor.
11. The composition according to claim 10 , wherein the NTP is a dNTP or an rNTP.
12. A composition comprising (a) the FCE variant according to claim 1 and (b) one or more additives, salts, reducing agents, chelating agents, detergents, and/or denaturants.
13. A composition comprising (a) the FCE variant according to claim 1 and (b) one or more primers, enzymes other than FCE, or combinations thereof.
14. A composition comprising (a) the FCE variant according to claim 1 and (b) one or more non-ionic, anionic, or zwitterionic surfactants, and/or crowding agents.
15. A glycerol-free, lyophilized composition comprising the FCE variant according to claim 1 .
16. An FCE variant according to claim 1 , wherein the amino acid sequence is at least 98% identical to positions 1 to 878 of SEQ ID NO: 1.