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 transcript comprising an RNA encoding an amino acid sequence having (a) at least 90% 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 833 of SEQ ID NO: 1, and (c) optionally, a cap.
2. The FCE variant transcript according to claim 1 , wherein the substitution is at a position corresponding to positions 215, 337, and 572 of SEQ ID NO: 1.
3. The FCE variant transcript according to claim 1 , wherein the encoded amino acid sequence further comprises 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. The FCE variant transcript according to claim 3 , wherein the encoded amino acid sequence further comprises 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. The FCE variant transcript according to claim 4 , wherein the encoded amino acid sequence further comprises 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 transcript comprising, in a 5′ to 3′ direction, (I) a nucleotide sequence encoding a purification tag or a sorting signal peptide, and (II) the FCE variant transcript according to claim 1 , wherein the purification tag or sorting signal peptide is operably linked to the FCE variant encoded by (II).
7. A fusion transcript comprising, in a 5′ to 3′ direction, (I) the FCE variant transcript according to claim 1 , and (II) a nucleotide sequence encoding a purification tag or a sorting signal peptide, wherein the purification tag or sorting signal peptide is operably linked to the FCE variant encoded by (I).
8. The FCE variant transcript according to claim 1 , further comprising (c) a cap.
9. A cell comprising the FCE variant transcript according to claim 2 .
10. The cell according to claim 9 , wherein the cell is a yeast cell.
11. A method of capping a target RNA, the method comprising contacting (a) an FCE variant having (i) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and (ii) a substitution at a position corresponding to positions 215, 337, 572, 648, and 833 of SEQ ID NO: 1, (b) the target RNA, and (c) one or more of a cap, an NTP, and a modified NTP, and optionally (d) a buffer, S-adenosylmethionine, and/or an RNase inhibitor, to form a capped target RNA.
12. The method according to claim 11 , wherein the contacting further comprises contacting at a temperature in the range of 37° C.-60° C. and/or for a time in the range of seconds to hours.
13. The method according to claim 11 , wherein the target RNA is uncapped.
14. The method according to claim 11 , further comprising contacting the target RNA with a decapping enzyme prior to contacting the target RNA with the FCE variant.
15. The method according to claim 11 , further comprising contacting the capped target RNA with one or more pharmaceutically acceptable additives.