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 . A composition comprising:
(a) an FCE variant having (a) an amino acid sequence 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
(b) a polynucleotide, a dNTP, an rNTP, a primer, an enzyme other than the FCE variant, a buffering agent, or a combination thereof.
2 . A composition according to claim 1 , wherein the composition comprises the enzyme other than the FCE variant.
3 . A composition according to claim 2 , wherein the enzyme other than the FCE variant is a polymerase.
4 . A composition according to claim 3 , wherein the FCE variant and the polymerase together form a fusion protein.
5 . A composition according to claim 1 , wherein the substitution is at a position corresponding to positions 215, 337, and 572 of SEQ ID NO: 1.
6 . A composition according to claim 1 , wherein the FCE variant 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.
7 . A composition according to claim 6 , wherein the FCE variant 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.
8 . A composition according to claim 7 , wherein the FCE variant 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.
9 . A composition according to claim 1 further comprising a purification tag or a sorting signal peptide, wherein the FCE variant is operably linked to the purification tag or the sorting signal peptide.
10 . A composition according to claim 1 , wherein the polynucleotide is an uncapped ribonucleic acid.
11 . A composition according to claim 1 further comprising a cap, S-adenosylmethionine, and/or an RNase inhibitor.
12 . A composition according to claim 1 comprising the dNTP or the rNTP.
13 . A composition according to claim 1 further comprising one or more additives, salts, reducing agents, chelating agents, detergents, and/or denaturants.
14 . A composition according to claim 1 further comprising one or more non-ionic, anionic, or zwitterionic surfactants, and/or crowding agents.
15 . A composition according to claim 1 , wherein the composition is a glycerol-free, lyophilized composition.
16 . A composition according to claim 1 , wherein the enzyme other than the FCE variant is a decapping enzyme.
17 . A method comprising:
(a) contacting a polymerase with one or more of a polynucleotide template encoding a target RNA, rNTPs and/or modified rNTPs, and a buffer to form a transcription product comprising the target RNA; and
(b) contacting an FCE variant with one or more of the transcription products, an NTP, a modified NTP, a cap, a methyl donor, and a buffering agent to form a capped target RNA,
wherein the FCE variant has (i) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and (ii) 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
wherein the FCE variant and the polymerase together form a fusion protein.
18 . A method according to claim 17 , wherein the contacting in (b) 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.
19 . A method according to claim 17 further comprising (c) contacting the capped target RNA with a pharmaceutically acceptable additive, binder, buffer, coating, color, controlled release agent, delivery agent, diluent, disintegrant, dye, excipient, filler, lipid, lubricant, salt, sorbant, stabilizer, or combinations thereof.
20 . A method according to claim 17 , wherein the capped product comprises a cap 0 product.
21 . A method according to claim 17 , wherein (b) further comprises contacting the capped RNA with 2′—O-methyltransferase to form a second capped product, wherein the second capped product comprises a cap 1 product.