mRNA CAPPING ENZYMES

Abstract

mRNA capping enzymes are provided. In one aspect, the mRNA capping enzymes comprise a hybrid of: (i) a first enzyme having TPase and GTase activity, and (ii) a methylation domain of a second enzyme, the methylation domain having MTase activity. Methods for making and using the same are also provided.

Description

mRNA CAPPING ENZYMESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Patent Application No. 63 / 657,280, filed on June 7, 2024, U.S. Provisional Patent Application No. 63 / 549,879, filed on February 5, 2024, and U.S. Provisional Patent Application No. 63 / 607,434, filed on December 7, 2023, each of which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] A Sequence Listing has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on December 8, 2024, is named ATUM-mRNA-PCT.xml and is 185,230 bytes in size.BACKGROUND

[0003] All eukaryotic mRNA contains a cap structure: an N7-methylated guanosine linked to the first nucleotide of the RNA via a reverse 5’ to 5’ triphosphate linkage (see Figure 1). The mRNA cap plays an essential role in cap-dependent initiation of protein synthesis. It also acts as the anchor for the recruitment of initiation factors that initiate protein synthesis and the 5’ to 3’ looping of mRNA during translation. 2’-0 methylation is central to the non-self-discrimination of innate immune response against foreign RNA.

[0004] As such, it is highly desirable to add a cap to synthetic RNA in many applications. One method to cap synthetic RNAs (e.g., RNAs transcribed in vitro) includes using an RNA capping enzyme. Enzymatic steps involved in RNA capping include: (1) RNA triphosphatase activity (TPase) to remove the y-phosphate from 5’ triphosphate, generating a diphosphate 5’ end and inorganic phosphate; (2) guanylyltransferase (GTase) activity to consume a guanosine triphosphate (GTP) molecule and form a covalent intermediate with a lysyl-N^-5’-phosphoguanosine; (3) in the presence of a 5’ diphosphate RNA, the GTase activity transfers the5 ’-phosphoguanosine (GMP) to the 5’ diphosphate, forming a 5 ’-5’ triphosphate linkage between the first base of the RNA and the capping base; (4) in the presence of S-adenosylmethionine (SAM), the guanine-N7 methyltransferase (MTase) activity adds a methyl group to the N7 amine of the guanosine cap to form a “Cap 0” structure; and (5) the m7G cap-specific 2’0 MTase modifies the 2’-0 of +1 ribose and generates a “Cap 1” structure. See Ramanathan A, Robb GB, Chan SH. mRNA capping: biological functions and applications. Nucleic Acids Res. 2016 Sep 19;44(16):7511-26. doi: 10.1093 / nar / gkw551. Epub 2016 Jun 17. PMID: 27317694; PMCID: PMC5027499, which is incorporated by reference herein in its entirety

[0005] Given the importance of RNA capping, a need exists for novel RNA capping enzymes to promote efficient enzymatic RNA capping reactions.SUMMARY

[0006] In one aspect, an RNA capping enzyme is provided, the RNA capping enzyme comprising a hybrid of: (i) a first enzyme having TPase and GTase activity, and (ii) a methylation domain of a second enzyme, the methylation domain having MTase activity.

[0007] In another aspect, a composition is provided, the composition comprising: an uncapped target RNA; an RNA capping enzyme comprising a hybrid of: (i) a first enzyme having TPase and GTase activity, and (ii) a methylation domain of a second enzyme, the methylation domain having MTase activity; a buffering agent; and a methyl group donor.

[0008] In another aspect, a method is provided for capping RNA in vitro, the method comprising: contacting: an RNA sample comprising an uncapped target RNA; an RNA capping enzyme comprising a hybrid of: (i) a first enzyme having TPase and GTase activity, and (ii) amethylation domain of a second enzyme, the methylation domain having MTase activity; GTP; a buffering agent; and a methyl group donor.

[0009] In another aspect, a kit is provided, the kit comprising: an RNA capping enzyme comprising a hybrid of: (i) a first enzyme having TPase and GTase activity, and (ii) a methylation domain of a second enzyme, the methylation domain having MTase activity; wherein the RNA capping enzyme is in a storage buffering agent; and a reaction buffering agent.BRIEF DESCRIPTION OF THE FIGURES

[0010] The accompanying figures, which are incorporated in and constitute a part of the specification, are used merely to illustrate various example aspects.

[0011] Figure 1 shows Cap 0 and Cap 1 mRNA structures in eukaryotes.

[0012] Figure 2 shows example capping activity of enzymes comprising SEQ ID NOs: 1-20 and 32 (with SEQ ID NO: 20 being a positive control).

[0013] Figure 3 shows example capping activity of enzymes comprising SEQ ID NOs: 32-44.

[0014] Figure 4 shows example capping activity of enzymes comprising SEQ ID NOs: 76, 80,81, 82, 83, and 85.

[0015] Figures 5A-5F show HPLC traces of the residual cap dinucleotide of RNAs treated with capping enzymes as described herein after digestion with nuclease Pl and calf alkaline phosphatase.

[0016] Figure 6 shows example capping activity of enzymes comprising SEQ ID NOs: 1, 2, 4, 5, 7, 8, 15, 18, 20, 33-44, and QKE50463 (with SEQ ID NO: 20 and QKE50463 serving as positive controls) in the presence of Vaccinia 2’-O-methyltransferase.DETAILED DESCRIPTIONDefinitions

[0017] Use of the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0018] “Buffering agent” means an agent that allows a solution to resist changes in pH when acid or alkali is added to the solution. Suitable examples include tris(hydroxymethyl)aminomethane (Tris or THAM), (2-[4-(2-hydroxyethyl)piperazin-l- yl]ethanesulfonic acid) (HEPES), [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 3-(N-morpholino)propanesulfonic acid (MOPS), tricine, and 2-(N- morpholino)ethanesulfonic acid (MES).

[0019] “Fusion” or “hybrid” means two or more polypeptides, subunits, or proteins covalently joined to one another (e.g., by a peptide bond). For example, a fusion may comprise a non- naturally occurring combined polypeptide chain comprising two proteins or two protein domains joined directly to each other by a peptide bond or joined through a peptide linker. In the context of an mRNA capping enzyme as disclosed herein, a fusion protein may be a hybrid of a portion of one enzyme and a portion of a second enzyme.

[0020] “Target RNA” means a polyribonucleotide of interest. A target RNA may be uncapped. If desired or required, a target RNA may be contacted with a de-capping enzyme, for example, as a co-treatment with or pre-treatment before capping.

[0021] “Uncapped” means an RNA that does not have a cap and that can be used as a substrate for a capping enzyme. Uncapped RNA typically has a tri- or di-phosphorylated 5’ end. RNAs transcribed in vitro have a triphosphate group at the 5’ end.

[0022] The disclosure refers to several proteins for which it provides an example “SEQ IDNO:.” Unless otherwise apparent from the context, reference to a protein should be understood as including the specific SEQ ID NO, as well as allelic and species variants thereof having at least 90, 95, or 99% identity thereto. Examples of allelic and species variants can be found in the SwissProt and other databases.

[0023] Unless defined otherwise herein, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the relevant art. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2nd Ed., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY, 1991, provide one of skill with a general dictionary of many of the terms used herein. Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. The terms defined immediately below are more fully defined by reference to the specification as a whole.Study Design

[0024] Putative capping enzymes were identified from analyses of natural sequence databases. Genes for each were synthesized, cloned into an expression vector, and expressed in E. coli strain BL21. Each protein was purified by immobilized metal affinity chromatography and assayed for the ability to catalyze RNA cap formation.

[0025] mRNA capping includes of a series of reactions. First, the gamma phosphate is removed from the 5 ’-triphosphate of the mRNA. Then a GMP residue is transferred from GTP to the diphosphate terminus of the RNA to create a 5 ’-5’ triphosphate linkage. Additionally, the cap guanosine is methylated at N7 to create a structure referred to as Cap 0. The net result of these triphosphatase and guanylyltransferase activities is an extension of the RNA in length, which canbe assayed by polyacrylamide gel electrophoresis, capillary electrophoresis, or other common methods. Methylation of the cap may be analyzed by chromatography and / or mass spectrometry of the RNA, digestion products thereof, or derivatives thereof.

[0026] Putative capping enzymes were first assayed for the ability to transfer a GMP residue to the RNA by incubating the enzyme with purified triphosphate mRNA and GTP and assessing the extension of the RNA by gel electrophoresis (see Table 1 and Figure 2).

[0027] The N7 methylation activity generally resides in a domain distinct from that for the triphosphatase and guanylyltransferase activities. Using sequence and structural analyses, N7- methylation domains were predicted for each enzyme, and several fusion (hybrid) enzymes were created where methylation domains from enzymes showing strong methylation were used to replace those of enzymes showing poor methylation. Multiple such hybrids showed improved overall activity compared to the natural sequences from which they were derived (see Table 1 and Figures 3 and 4). Methylation domains that showed strong methylation include those represented by SEQ ID NOs: 57-63 and 86-89. Hybridization of multiple guanylyltransferase and N7- methyltransferase domains was critical to find suitable combinations. In some hybrids, methylation domains from capping enzymes showing active methyltransferase activity showed no activity when fused to guanylyltransferase domains from another enzyme (e.g., SEQ ID NOs: 36, 37, 41, and 42).

[0028] RNAs showing cap addition were further analyzed for cap N7-methylation by digesting the RNA with Nuclease Pl and calf intestinal phosphatase and analyzing the products by HPLC. The results are shown in Figures 5A-5F for SEQ ID NOs: 1, 5, 7, 8, 38, 40, 76, 81-83, and 85. Peaks corresponding to the N7-methylated and unmethylated cap dinucleotides based on standards are indicated

[0029] Several enzymes showed significant guanylyl transfer under the conditions tested.Enzymes that showed guanylyl transfer varied in their catalysis of N7-methylation (see Table 1). Some enzymes with high guanylyl transfer showed low cap methylation, and some with low guanylyl transfer showed high cap methylation. Three enzymes with insignificant guanylyltransferase activity were shown to catalyze the N7-methylation of non-methylated RNA caps generated by non-methylating capping enzymes, indicating that these proteins have an active methylation activity.

[0030] Inclusion of a cap 2’-0 methyltransferase such as Vaccinia 2 ’-methyltransferase (which methylates the mRNA 5 ’-terminal residue at the 2’-0 position) in the reactions can generate Cap 1 transcript at high efficiency. No significant differences in relative activities were observed when reactions were performed in the presence or absence of Vaccinia 2’- methyltransferase (see Table 1 and Figure 6).

[0031] Depending on which enzyme is used and the other components in the reaction mix, disclosed methods may be used to make RNAs that have a Gppp cap, a 7-methylguanylate cap (i.e., a m7Gppp cap, or Cap 0), or an RNA that has an m7Gppp cap that has addition modifications in the first and / or second nucleotides in the RNA (i.e., Cap 1 and “Cap 2”; see Fechter J. Gen. Vir. 2005 86: 1239-49, which is incorporated by reference herein in its entirety). For example, if there is no SAM in the reaction mix, then RNAs that have a Gppp cap may be produced. If the reaction mix comprises SAM in addition to the capping enzyme, then Cap 0 RNA may be produced. If the reaction mix comprises other enzymes, e.g., cap 2’-OMTase in addition to SAM, then Cap 1 and / or Cap 2 RNA may be produced. A reaction mix may comprise other components in addition to those explicitly described above. With reference to Figures 5A-5F, then, the area under the curve corresponding to the designation of “m7GpppG” is a measure of the amount of 7-methylguanylatecapped or Cap 0 RNA produced relative to the “GpppG” or unmethylated, guanylate-capped RNA produced.EXAMPLES

[0032] Disclosed reaction conditions may be varied, including, without limitation, reaction temperature, reaction duration, reaction component concentrations (e.g., SAKE inorganic pyrophosphatase, NTPs, transcript template), and enzymes (e.g., capping enzymes, polymerases, and fusions thereof)Example 1 : Identification and production of putative capping enzymes

[0033] Putative capping enzymes were identified by analysis of genomic sequence databases. Protein sequences showing homology to at least one domain known to catalyze one of the activities required for mRNA capping (i.e., TPase, GTase, and MTase activities) were chosen for biochemical characterization.

[0034] Genes for each putative capping enzyme were designed for high expression in E. coli and cloned into the expression vector pD861-SR. Several of the genes were cloned to produce N- terminal and C-terminal 6xHis-tagged proteins to assess tag position tolerance.

[0035] E. coli strain BL21 was transformed with each resulting expression vector, and strains were induced for expression in mid-log growth phase with 4 mM rhamnose for 20 h at 24 °C. Cells were lysed, and soluble protein was harvested using standard methods (Epicentre T1075).

[0036] Soluble proteins were purified from lysate by immobilized nickel affinity chromatography and buffer exchanged into 100 mM NaCl, 50 mM arginine, 0.1 mM TCEP, and40 mM Tris-HCl, pH 8.0.Example 2: Assay of capping activities

[0037] 5’-triphosphate-RNA was generated by in vitro transcription of a linearized DNA template with T7 polymerase using a commercially available kit (NEB E2040S). The resulting RNA was incubated with each putative capping enzyme to assess GTase and MTase activities.

[0038] Capping reactions included 25 nM purified putative capping enzyme, 10 ug triphosphate-RNA, 0.5 mM GTP, 0.2 mM SAM, 10U RNasin, 5 mM KC1, 1 mM MgCh, 1 mM DTT, 0.02% Pol oxamer 188, and 50 mM Tris-HCl pH 8.0 in a total volume of 10 pl. For reactions including 2’-O-methyltransferase, reactions included an additional 40U of Vaccinia 2’- methyltransferase (NEB M0366S). Reactions were incubated for 60 min at 37 °C, and RNA was purified using a Monarch RNA purification kit (NEB T2050L).

[0039] GTase activity was assayed by size analysis of RNA fragments generated by sitespecific cleavage with a DNA enzyme (DNAzyme2) that yields a 28-base 5 ’-terminal RNA fragment with uncapped RNA (Santaro, S.W. and Joyce, G.F., Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4262-6.doi: 10.1073 / pnas.94.9.4262). DNAzyme2 was annealed to the RNA, and cleavage reactions were incubated for 3 h at 37 °C. Reactions included 0.4 pM mRNA, 2 pM DNAzyme2, 10 mM MgCh, 10 mM MnCh, and 50 mM Tris-HCl, pH 7.5. Resulting fragments were analyzed on a denaturing 15% polyacrylamide TBE-Urea gel stained with SybrGold. GTase activity is indicated by slowed migration in electrophoresis. The appearance of a slower migrating band indicates the degree to which the RNA has been modified with a capping 5 ’-guanosine residue.

[0040] N7-methylation and 2’-0 methylation of the introduced cap were assayed by chromatographic analysis after digestion of the RNA by nuclease Pl and calf intestinal phosphatase (Galloway, et al, Open Biol. 2020 Feb; 10(2): 190306. doi: 10.1098 / rsob.190306. Epub2020 Feb 26). This enzyme treatment completely digests the RNA sequence to nucleosides plus phosphate but leaves cap structures intact. The digestion was analyzed by HPLC to quantify the relative amounts of N7-methylated and non-methylated cap dinucleotides.

[0041] Columns A-D of Table 1 show, respectively, the Genbank Reference, the expressed protein molecular weight, the SEQ ID NOs for the proteins expressed (N-terminal or C-terminal 6xHis-tagged proteins), and the SEQ ID NOs for the expressed proteins without the 6xHis-tag. Column E of Table 1 indicates the capping activity. Column F of Table 1 indicates the N7- methyltransferase activity. Column G of Table 1 indicates the capping activity in the presence of 2’-O- methyltransferase.Table 1No <5% reaction observed5-49% reaction observed>50% reaction observedNT Not Tested

[0042] Other than with respect to SEQ ID NOs: 15 (N-terminal +) and 18 (C-terminal ++), the position of the 6xHis-tag did not significantly impact the capping activity.

[0043] Figure 2 shows the capping activity of enzymes comprising SEQ ID NOs: 1-20 and 32(with SEQ ID NO: 20 being a positive control) in the absence of 2’-O-methyltransferase. Figure 3 shows the capping activity of enzymes comprising SEQ ID NOs: 32-44 in the absence of 2’-O- methyltransferase. SEQ ID NOs: 15, 18, and 33-43 represent hybrids containing a methylation domain selected from SEQ ID NOs: 57-63. Figure 4 shows example capping activity of enzymes comprising SEQ ID NOs: 76, 80, 81, 82, 83, and 85 in the absence of 2’-O-methyltransferase. SEQ ID NOs: 80-83 and 85 represent hybrids containing a methylation domain selected from SEQ ID NOs: 86-89.

[0044] Selected RNAs showing cap addition were further analyzed for cap N7-methylation by digesting the RNA with Nuclease Pl and calf intestinal phosphatase and analyzing the products by HPLC. The results are shown in Figures 5A-5F for SEQ ID NOs: 1, 5, 7, 8, 38, 40, 76, 81-83, and 85. Peaks corresponding to the N7-methylated (m7GpppG) and unmethylated (GpppG) cap dinucleotides based on standards are indicated.

[0045] Figure 6 shows the capping activity of enzymes comprising SEQ ID NOs: 1, 2, 4, 5, 7, 8, 15, 18, 20, 33-44, and QKE50463 (with SEQ ID NO: 20 and QKE50463 serving as positive controls) in the presence of 2’-O-methyltransferase. In each case, guanylyl transfer is indicated by the shift in the size of the cleavage product to a slower migrating band. The degree of capping is estimated from the percentage of RNA fragments that are slower shifted as determined by gel image densitometry.

Claims

CLAIMSWhat is claimed is:

1. A composition, comprising: an uncapped target RNA; an RNA capping enzyme comprising a hybrid of:(i) a first enzyme having TPase and GTase activity, and(ii) a methylation domain of a second enzyme, the methylation domain having MTase activity; a buffering agent; and a methyl group donor.

2. The composition of claim 1, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 57, 58, 87, 86, 61, 62, 63, 88, or 89.

3. The composition of claim 1, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 57, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 45 or 50.

4. The composition of claim 1, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 58, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 47 or 52.

5. The composition of claim 1, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 87, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 71 or 74.

6. The composition of claim 1, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 86, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 69 or 72.

7. The composition of claim 1, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 61, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29.

8. The composition of claim 1, wherein the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29, 45, 47, 50, 52, 69, 71, 72, or 74.

9. The composition of any one of claims 1-8, wherein the composition further comprises an RNase inhibitor.

10. The composition of any one of claims 1-8, wherein the composition further comprises a DNA template, a bacteriophage polymerase, and ribonucleotide triphosphates, for transcribing the DNA template to form the uncapped target RNA.

11. The composition of any one of claims 1-8, wherein the methyl group donor comprises S- adenosyl methionine (SAM).

12. The composition of any one of claims 1-8, wherein the composition further comprises a cap 2’-O-methyltransferase enzyme.

13. A composition, comprising: an uncapped target RNA; an RNA capping enzyme, wherein the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 24, 25, 21, or 22; a buffering agent; and a methyl group donor.

14. The composition of claim 13, wherein the composition further comprises an RNase inhibitor.

15. The composition of claim 13, wherein the composition further comprises a DNA template, a bacteriophage polymerase, and ribonucleotide triphosphates, for transcribing the DNA template to form the uncapped target RNA.

16. The composition of claim 13, wherein the methyl group donor comprises S-adenosyl methionine (SAM).

17. The composition of any one of claims 13-16, wherein the composition further comprises a cap 2’-O-methyltransferase enzyme.

18. An RNA capping enzyme comprising a hybrid of: (i) a first enzyme having TPase and GTase activity, and (ii) a methylation domain of a second enzyme, the methylation domain having MTase activity.

19. The RNA capping enzyme of claim 18, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 57, 58, 87, 86, 61, 62, 63, 88, or 89.

20. The RNA capping enzyme of claim 18, wherein the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 45 or 50.

21. The RNA capping enzyme of claim 18, wherein the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 47 or 52.

22. The RNA capping enzyme of claim 18, wherein the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 71 or 74.

23. The RNA capping enzyme of claim 18, wherein the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 69 or 72.

24. The RNA capping enzyme of claim 18, wherein the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 29.

25. A polynucleotide that encodes for the RNA capping enzyme of any one of claim 18-24.

26. A method for capping RNA in vitro, the method comprising: contacting:(i) an RNA sample comprising an uncapped target RNA;(ii) an RNA capping enzyme comprising a hybrid of:(a) a first enzyme having TPase and GTase activity, and(b) a methylation domain of a second enzyme, the methylation domain having MTase activity;(iii) guanosine triphosphate (GTP);(iv) a buffering agent; and(iv) a methyl group donor.

27. The method of claim 26, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 57, 58, 87, 86, 61, 62, 63, 88, or 89.

28. The method of claim 26, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 57, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 45 or 50.

29. The method of claim 26, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 58, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 47 or 52.

30. The method of claim 26, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 87, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 71 or 74.

31. The method of claim 26, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 86, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 69 or 72.

32. The method of claim 26, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 61, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29.

33. The method of claim 26, wherein the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29, 45, 47, 50, 52, 69, 71, 72, or 74.

34. The method of claim 26, wherein the contacting further comprises contacting at a temperature of about 37° C.

35. The method of claim 26, wherein the contacting further comprises contacting an RNase inhibitor.

36. The method of claim 26, further comprising synthesizing the uncapped RNA by contacting a DNA template encoding the uncapped RNA and a polymerase to produce the uncapped RNA.

37. The method of claim 26, wherein the methyl group donor is S-adenosyl methionine.

38. The method of any one of claims 26-37, wherein the contacting further comprises contacting a cap 2’-O-methyltransferase enzyme.

39. A kit, comprising:(i) an RNA capping enzyme comprising a hybrid of:(a) a first enzyme having TPase and GTase activity, and(b) a methylation domain of a second enzyme, the methylation domain having MTase activity; wherein the enzyme is in a storage buffering agent; and(ii) a reaction buffering agent.

40. The kit of claim 39, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 57, 58, 87, 86, 61, 62, 63, 88, or 89.

41. The kit of claim 39, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 57, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 45 or 50.

42. The kit of claim 39, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 58, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 47 or 52.

43. The kit of claim 39, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 87, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 71 or 74.

44. The kit of claim 39, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 86, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 69 or 72.

45. The kit of claim 39, wherein the methylation domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 61, and the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29.

46. The kit of claim 39, wherein the RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29, 45, 47, 50, 52, 69, 71, 72, or 74.47.

48. The kit of claim 39, wherein the kit further comprises an RNA polymerase and ribonucleotides for transcribing a template polynucleotide encoding a target RNA.

49. The kit of claim 39, wherein the kit further comprises S-adenosyl methionine.

50. The kit of claim 39, wherein the kit further comprises guanosine triphosphate (GTP).

51. The kit of claim 39, wherein the kit further comprises cap 2 ’-O-m ethyltransferase enzyme.

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