Methods and compositions for increasing capping efficiency of transcribed RNA
Methods and compositions for capping RNA in an in vitro transcription mixture are provided that include a thermostable RNA polymerase variant and a cap analog such that when a DNA template is added to the mixture, and the mixture is then incubated under conditions for in vitro transcription, capped RNA is produced.
1. A method for capping an RNA with a cap analog, comprising:
(a) combining in a mixture,
ribonucleotide triphosphates (rNTPs) and/or modifications thereof,
a cap analog, and
an RNA polymerase, wherein the RNA polymerase: (i) has at least 90% sequence identity to SEQ ID NO: 1; and (ii) comprises an amino acid substitution at the position corresponding to position 388 and at the position corresponding to position 567 of SEQ ID NO: 1,
(b) co-transcriptionally capping the RNA to produce capped RNA with an efficiency of co-transcriptional capping of at least 95% as measured using mass spectrometry and comparing the capped RNA to uncapped RNA having a 5′ppp.
2. The method according to claim 1 , wherein the RNA polymerase further comprises an amino acid substitution of at least one position corresponding to positions selected from the group consisting of: 109, 205, 534, and 618 of SEQ ID NO:1.
3. The method according to claim 1 , wherein the RNA polymerase further comprises an amino acid substitution of at least two positions corresponding to positions selected from the group consisting of: 109, 205, 534, and 618 of SEQ ID NO:1.
4. The method according to claim 1 , wherein the RNA polymerase further comprises an amino acid substitution at four positions corresponding to positions 109, 205, 534, and 618 of SEQ ID NO:1.
5. The method according to claim 1 , wherein the amino acid substitutions at positions corresponding to positions 388 and 567 of SEQ ID NO:1 are D388E and V567P.
6. The method according to claim 1 , wherein the RNA polymerase further comprises an amino acid substitution at one or more positions corresponding to positions selected from the group consisting of: 75, 83, 108, 206, 227, 281, 297, 312, 323, 327, 333, 340, 354, 362, 375, 428, 446, 454, 461, 495, 510, 584, 591, 642, 711, 724, 740, 788, 832, 834, 835, 843, 847, 849, 856, 863, 866 and 877 of SEQ ID NO:1.
7. The method according to claim 1 , wherein the RNA polymerase further comprises at least 10 of the following amino acid substitutions corresponding to positions selected from the group consisting of: 75, 83, 108, 206, 227, 281, 297, 312, 323, 327, 333, 340, 354, 362, 375, 428, 446, 454, 461, 495, 510, 584, 591, 642, 711, 724, 740, 788, 832, 834, 835, 843, 847, 849, 856, 863, 866 and 877 of SEQ ID NO:1.
8. The method according to claim 1 , wherein the RNA polymerase further comprises one or more of the following amino acid substitutions selected from the group consisting of: T75Q, A83K, E108L, K206P, V227I, I281P, V297I, Y312D, A323I, A327P, K333P, V340E, A354Q, M362P, T375K, T375N, A428P, L446F, K454P, K461R, S495N, C510Q, A584K, D591E, K642R, K711R, A724P, K740R, G788A, M832F, D834E, T835L, A843Q, D847E, F849V, S856T, A863P, A866K and E877R, wherein the amino acid substitutions are at positions that correspond to positions in SEQ ID NO:1.
9. The method according to claim 1 , wherein the RNA polymerase further comprises at least 10 of the following acid substitutions selected from the group consisting of: T75Q, A83K, E108L, K206P, V227I, I281P, V297I, Y312D, A323I, A327P, K333P, V340E, A354Q, M362P, T375K, T375N, A428P, L446F, K454P, K461R, S495N, C510Q, A584K, D591E, K642R, K711R, A724P, K740R, G788A, M832F, D834E, T835L, A843Q, D847E, F849V, S856T, A863P, A866K and E877R wherein the amino acid substitutions are at positions that correspond to positions in SEQ ID NO:1.
10. The method according to claim 1 , wherein the RNA polymerase comprises a fusion to an exogenous DNA binding domain.
11. The method according to claim 1 , wherein the cap analog comprises Formula 1, wherein Formula 1, comprises:
R 1 and/or R 2 =O-alkyl, halogen, a linker, hydrogen or a hydroxyl;
R 3 =guanine, adenine, cytosine, uridine, guanine analog, adenine analog, cytosine analog, or uridine analog;
R 4 =N 1 (p−Nx) where N is a nucleoside or a modified nucleoside where the nucleoside is selected from adenine, uridine, guanine, or cytidine or analogs of adenine, uridine, guanine, or cytidine, and the modified nucleoside is selected from N 6 -methyladenine, N 1 -methyladenine,N 6 -2′-O-dimethyladenosine, pseudouridine, N 1 -methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5-methyluridine, pseudoisocytosine, 5-methoxycytosine, 2-thiocytosine, 5-hydroxycytosine, N 4 -methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, N 1 -methylguanine, O 6- methylguanine, 1-methyl -guanosine, N 2 -methyl-guanosine (m 2 G), N 2 ,N 2 -dimethyl-guanosine (m 2,2 G), 2-methyl-2′-O-methyl-guanosine (m 2 Gm), N 2 ,N 2 -dimethyl-2′-O-methyl-guanosine (m 2,2 Gm) 1-methyl-2′-O-methyl-guanosine, N 2 ,N 7 -dimethyl-2′-O-methyl-guanosine (m 2,7 Gm), or isoguanineadenine
wherein:
(i) x can be any integer from 0-8,
wherein the sugar in the nucleotides may be selected from ribose, deoxyribose, and comprise of modifications including 2′-O-alkyl, 2′-O-methoxyethyl, 2′-O allyl, 2′-O alkylamine, 2′-fluororibose, or 2′-deoxyribose;
the phosphate groups in one or more nucleotides can be substituted for phosphorothioates, phosphorodithioate, alkylphosphonate, arylphosphonate, or N-phosphoramidate linkages;
(ii) the polynucleotide cap can be a salt or solvated form; and
(iii) the polynucleotide cap can be a single stereoisomer or plurality of stereoisomers of one or more of the compounds described by Formula 1 or a salt or salts thereof.
12. The method according to claim 11 , wherein the O- alkyl is an O-methyl.
13. The method according to claim 11 , wherein x is 1.
14. The method according to claim 13 , wherein R3 is 7 methylguanine, R1 and R2 are hydroxyl groups.
15. The method according to claim 13 , wherein R3 is 7 methylguanine, R1 and R2 are O-alkyl.
16. The method according to claim 1 , wherein the mixture further comprises a nucleic acid template.
17. The method according to claim 1 , wherein the mixture further comprises a DNA template.
18. The method according to claim 1 , wherein the co-transcriptionally capping further comprises co-transcriptionally capping the RNA at a temperature that is in the range of 30° C. to 70° C.
19. The method according to claim 1 , wherein the co-transcriptionally capping further comprises co-transcriptionally capping the RNA at a temperature that is in the range of 50° C. to 65° C.
20. The method according to claim 1 , wherein the ribonucleotide triphosphates (rNTPs) and/or modifications thereof further comprises at least one unmodified ribonucleotide triphosphate and at least one modified ribonucleotide triphosphate, wherein the at least one modified ribonucleotide optionally comprises a modification in its sugar group, its phosphate group, and/or its base.