Enzymatic RNA capping method

Abstract

To provide a method for efficiently capping RNA in vitro.SOLUTION: In some embodiments, the capping reaction can be done at high temperature using Vaccinia capping enzyme or variants thereof. In other embodiments, the capping reaction may comprise capping enzymes from amoebic large viruses, such as Faust, Mimivirus, or Moumouvirus, or variants thereof. Compositions and kits for practicing the methods are also provided.SELECTED DRAWING: None

Description

[Background technology]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 890,821, filed August 23, 2019, which is incorporated herein by reference in its entirety.

[0002] Addition of a cap to uncapped synthetic RNA is important for efficient protein expression in many eukaryotic cells. Furthermore, uncapped RNA (RNA with at least a 5'-triphosphate) has been reported to activate the innate immune response (Pichlmair et al., Science, 2006, 314:997-1001; Diamond et al., Cytokine & Growth Factor Reviews, 2014, 25:543-550). Thus, adding a cap to synthetic RNA is highly desirable for many therapeutic applications (e.g., protein replacement therapy, as well as prophylactic or therapeutic vaccination).

[0003] Currently, two methods are used to cap RNA. In the first method, synthetic RNA (e.g., in vitro transcribed RNA) is converted into capped RNA using an RNA capping enzyme. In the other method (commonly referred to as "co-transcriptional capping"), a cap analog, such as anti-reverse cap analog (ARCA), and a capped dinucleotide are added to the in vitro transcription reaction. In the co-transcriptional method, the cap is incorporated into the RNA molecule by co-transcription during in vitro transcription.

[0004] Compared with the co-transcriptional capping method, the enzymatic RNA capping method can achieve a high yield of capped RNA. However, the enzymatic RNA capping reaction is inefficient, so a large amount of enzyme is used, or the capped RNA must be purified from the uncapped RNA. Furthermore, the efficiency of the enzymatic RNA capping reaction (expressed as the percentage of capped RNA after the completion of the capping reaction) can vary depending on the RNA sequence, and the difference is generally attributed to the RNA structure (see, for example, Fuchs, RNA., 2016, 22:1454-66). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Pichlmair et al., Science, 2006, 314:997-1001 [Non-patent document 2] Diamond et al., Cytokine & Growth Factor Reviews, 2014, 25:543~550 [Non-patent document 3] Fuchs, RNA., 2016, 22:1454~66 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there remains a need for more efficient methods for adding caps to synthetic RNAs. [Means for solving the problem]

[0007] (Summary of the Invention) The present disclosure provides, among other things, methods for efficiently capping RNA in vitro. In some embodiments, the method may include contacting (i) an RNA sample containing uncapped target RNA, (ii) an RNA capping enzyme containing an amino acid sequence at least 90% identical (e.g., at least 95% identical) to SEQ ID NO: 1, 7, or 20, (iii) guanosine triphosphate (GTP) or modified GTP, (iv) a buffer, and optionally (v) a methyl group donor, at a temperature ranging from 40°C to 60°C to form (e.g., efficiently form) a capped target. The efficiency, determined by yield of capped RNA (50%) / enzyme concentration (nM), may be improved by at least two-fold or at least three-fold compared to the capping efficiency of the enzyme at 37°C. The uncapped target RNA may optionally contain no modified nucleotides or may contain one or more modified nucleotides (e.g., pseudouridine).

[0008] In some embodiments, a method may include contacting (i) an RNA sample containing uncapped target RNA, (ii) a single-stranded RNA capping enzyme having RNA triphosphatase (TPase) activity, guanylyltransferase (GTase) activity, and guanine-N7 methyltransferase (N7 MTase) activity, (iii) GTP or modified GTP, (iv) a buffer, and optionally (v) a methyl group donor, at a temperature ranging from 37°C to 60°C to form (e.g., effectively form) capped target RNA. A single-stranded RNA capping enzyme (e.g., an RNA capping enzyme from a giant virus such as Faustovirus, Mimivirus, or Moumouvirus) may comprise: (a) an amino acid sequence at least 90% identical (e.g., at least 95% identical) to (x) SEQ ID NO:2, (y) SEQ ID NO:3, and / or (z) SEQ ID NO:4; or (b) an amino acid sequence at least 90% identical (e.g., at least 95% identical) to (x) SEQ ID NO:5 and / or (y) SEQ ID NO:6. For example, an RNA capping enzyme from a giant virus such as Faustovirus, Mimivirus, or Moumouvirus may comprise an amino acid sequence at least 90% identical (e.g., at least 95% identical) to (a) SEQ ID NO:2, (b) SEQ ID NO:3, (c) SEQ ID NO:4, (d) SEQ ID NO:5, and / or (e) SEQ ID NO:6. An RNA capping enzyme from the genus Faustovirus (which is an example of a single-stranded RNA capping enzyme) may comprise an amino acid sequence that is at least 90% identical to (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, (d) SEQ ID NO:10, (e) SEQ ID NO:11, and / or (f) SEQ ID NO:12.

[0009] In some embodiments, the uncapped target RNA can be at least 200 nt in length (e.g., at least 300 nt, at least 500 nt, or at least 1,000 nt) and may encode a polypeptide such as a therapeutic protein or a therapeutic vaccine. Target RNAs with secondary structure, including therapeutic RNAs, can be more efficiently capped using the methods of the present disclosure. Efficiency can be defined as the yield of capped RNA (50%) / enzyme concentration (nM). In any embodiment, the method can include contacting at a first temperature (e.g., 37°C to 60°C) and increasing or decreasing the temperature (e.g., to 37°C to 60°C), where the second temperature is different from the first temperature. For example, the method can include contacting at a first temperature of 37°C for 1 to 120 minutes and increasing the temperature to 45°C or 50°C for 1 to 120 minutes. In some embodiments, the method can include increasing or decreasing the temperature to a third temperature of 37°C to 60°C for 1 to 120 minutes. The capping method, in some embodiments, can generate greater than 70% Cap0 RNA in vitro within 1 hour (eg, by co-transcription).

[0010] In some embodiments, the components and / or combinations thereof can be RNase-free, and the contacting step can optionally further include (v) one or more RNase inhibitors.

[0011] Uncapped RNA can be synthesized using solid-phase oligonucleotide synthesis chemistry or by transcribing a DNA template using a polymerase (e.g., T7 RNA polymerase or Hi-T7 RNA polymerase) in an in vitro transcription reaction, e.g., by contacting a DNA template encoding the uncapped target RNA with the polymerase.

[0012] In any embodiment, the contacting step can further comprise contacting a SAM and / or a cap 2'O methyltransferase enzyme (2'OMTase).

[0013] In any embodiment, the contacting step may include contacting (i), (ii), (iii), (iv), and (v) in a single location, e.g., within a single microfluidic surface, a single reaction tube, or other reaction vessel.

[0014] Also provided herein are compositions comprising an uncapped target RNA, a single-stranded RNA capping enzyme having TPase activity, GTase activity, and N7 MTase activity, GTP, and a buffer. In some embodiments, the composition may have a temperature ranging from 37°C to 60°C. In some embodiments, the composition may be RNase-free and may optionally include one or more RNase inhibitors. In some embodiments, the single-stranded RNA capping enzyme may comprise (a) an amino acid sequence at least 90% identical to (x) SEQ ID NO:2, (y) SEQ ID NO:3, and / or (z) SEQ ID NO:4; or (b) an amino acid sequence at least 90% identical to (x) SEQ ID NO:5 and / or (y) SEQ ID NO:6. The single-stranded RNA capping enzyme may comprise an amino acid sequence at least 90% identical to an RNA capping enzyme of Faustovirus D5b (SEQ ID NO: 7), Faustovirus E12 (SEQ ID NO: 8), Faustovirus ST1 (SEQ ID NO: 9), Faustovirus LC9 (SEQ ID NO: 10), Mimivirus (SEQ ID NO: 11), or Moumouvirus (SEQ ID NO: 12). In some embodiments, the composition may further comprise a DNA template, a polymerase (e.g., a bacteriophage polymerase), and ribonucleotides for transcribing the RNA. In some embodiments, the composition may further comprise a SAM and / or a cap 2' OMTase. In some embodiments, the uncapped single target RNA can be at least 200 nt in length (at least 300 nt, at least 500 nt, or at least 1,000 nt) and can encode a polypeptide such as a therapeutic protein or a therapeutic vaccine.

[0015] Kits are also provided. In some embodiments, the kit may include a single-stranded RNA capping enzyme having TPase activity, GTase activity, and N7 MTase activity present in a storage buffer; and a concentrated reaction buffer. In some embodiments, the kit may further include a DNA template, a polymerase, and ribonucleotides for transcribing RNA. In some embodiments, the kit may further include SAM and / or Cap 2' OMTase. Exemplary methods, compositions, and kits utilizing methyltransferase may also include SAM in the reaction mix. The single-stranded RNA capping enzyme may include (a) an amino acid sequence at least 90% identical to (x) SEQ ID NO:2, (y) SEQ ID NO:3, and / or (z) SEQ ID NO:4; or (b) an amino acid sequence at least 90% identical to (x) SEQ ID NO:5 and / or (y) SEQ ID NO:6. The single-stranded RNA capping enzyme may comprise an amino acid sequence that is at least 90% identical to an RNA capping enzyme of the genus Faustovirus D5b (SEQ ID NO: 7), Faustovirus E12 (SEQ ID NO: 8), Faustovirus ST1 (SEQ ID NO: 9), Faustovirus LC9 (SEQ ID NO: 10), Mimivirus (SEQ ID NO: 11) or Moumouvirus (SEQ ID NO: 12).

[0016] In some embodiments, the RNA capping enzyme may have an amino acid sequence that is at least 90% identical to SEQ ID NO: 20. In some embodiments, the RNA capping enzyme fusion may comprise (a) an amino acid sequence that is at least 90% identical to SEQ ID NO: 7 and (b) an amino acid sequence that is at least 90% identical to positions 1419 to 1587 of SEQ ID NO: 20. [Brief explanation of the drawings]

[0017] [Figure 1]Figure 1 shows the predicted secondary structures of the model RNA molecules used. RNA1 was designed to have minimal secondary structure at the 5' end. RNA2, RNA3, and RNA4 were designed to contain blunt ends, one-base, or two-base overhangs at their 5' ends, while exhibiting the same predicted minimum free energy of unfolding (MFE). Secondary structure and minimum free energy of unfolding were calculated at 37°C and 45°C using the RNA Fold Webserver (Vienna University, Vienna, Austria). The modeled RNA molecules have the following sequences: RNA1: SEQ ID NO: 13, RNA2: SEQ ID NO: 14, RNA3: SEQ ID NO: 15, and RNA4: SEQ ID NO: 16. [Figure 2] Figure 1 shows the RNA capping activity of 1H3C2 and VCE on RNA at different reaction temperatures. The amount of m7Gppp-capping RNA is a measure of the RNA capping activity of the enzyme. Each bar represents the average result of four independent experiments, and the error bars indicate the standard deviation. As shown in the figure, both H3C2 and VCE exhibited maximum RNA capping activity at 45°C. While the capping activity of VCE declined sharply above 50°C, H3C2 retained significant capping activity at 50°C, 52°C, and 55°C. [Figure 3A] Figure 3 shows RNA capping activity as a function of capping enzyme concentration at 37°C, 45°C, 50°C and 55°C. Figure 3A shows the activity of VCE. [Figure 3B] Figure 3B shows RNA capping activity as a function of capping enzyme concentration at 37°C, 45°C, 50°C, and 55°C. Figure 3B shows the activity of H3C2. See Table 1 for a quantitative comparison of capping activity under each condition. [Figure 4A]Figure 1 shows RNA capping activity by putative RNA capping enzymes on 150-nt RNA at various temperatures. Lanes 1-6 show capping reactions using in vitro translation products of AMN83561 (H3C2) (lane 1), AIB52055 (lane 2), SME65026 (lane 3), SMH63629 (lane 4), amino acids 1-668 of AAV50651 (the N-terminal region of the Mimivirus capping enzyme) (lane 5), or VP_007354410 (the Moumouvirus capping enzyme) (lane 6). In lane 7, purified H3C2 (20 nM) was used. The ribonucleotide sequence of the 150-nt RNA used in these experiments is: [ka] (SEQ ID NO: 17). Figure 4A shows the capping activity at 37°C. [Figure 4B] Figure 1 shows RNA capping activity by putative RNA capping enzymes on 150-nt RNA at various temperatures. Lanes 1-6 show capping reactions using in vitro translation products of AMN83561 (H3C2) (lane 1), AIB52055 (lane 2), SME65026 (lane 3), SMH63629 (lane 4), amino acids 1-668 of AAV50651 (the N-terminal region of the Mimivirus capping enzyme) (lane 5), or VP_007354410 (the Moumouvirus capping enzyme) (lane 6). In lane 7, purified H3C2 (20 nM) was used. The ribonucleotide sequence of the 150-nt RNA used in these experiments is: [ka] (SEQ ID NO: 17). Figure 4B shows the capping activity at 45°C. [Figure 4C]Figure 1 shows RNA capping activity by putative RNA capping enzymes on 150-nt RNA at various temperatures. Lanes 1-6 show capping reactions using in vitro translation products of AMN83561 (H3C2) (lane 1), AIB52055 (lane 2), SME65026 (lane 3), SMH63629 (lane 4), amino acids 1-668 of AAV50651 (the N-terminal region of the Mimivirus capping enzyme) (lane 5), or VP_007354410 (the Moumouvirus capping enzyme) (lane 6). In lane 7, purified H3C2 (20 nM) was used. The ribonucleotide sequence of the 150-nt RNA used in these experiments is: [ka] (SEQ ID NO: 17). Figure 4C shows the capping activity at 50°C. [Figure 4D] Figure 1 shows RNA capping activity by putative RNA capping enzymes on 150-nt RNA at various temperatures. Lanes 1-6 show capping reactions using in vitro translation products of AMN83561 (H3C2) (lane 1), AIB52055 (lane 2), SME65026 (lane 3), SMH63629 (lane 4), amino acids 1-668 of AAV50651 (the N-terminal region of the Mimivirus capping enzyme) (lane 5), or VP_007354410 (the Moumouvirus capping enzyme) (lane 6). In lane 7, purified H3C2 (20 nM) was used. The ribonucleotide sequence of the 150-nt RNA used in these experiments is: [ka] (SEQ ID NO: 17). Figure 4D shows the capping activity at 55°C. [Figure 4E]Figure 1 shows RNA capping activity by putative RNA capping enzymes on 150-nt RNA at various temperatures. Lanes 1-6 show capping reactions using in vitro translation products of AMN83561 (H3C2) (lane 1), AIB52055 (lane 2), SME65026 (lane 3), SMH63629 (lane 4), amino acids 1-668 of AAV50651 (the N-terminal region of the Mimivirus capping enzyme) (lane 5), or VP_007354410 (the Moumouvirus capping enzyme) (lane 6). In lane 7, purified H3C2 (20 nM) was used. The ribonucleotide sequence of the 150-nt RNA used in these experiments is: [ka] (SEQ ID NO: 17). Figure 4E shows the capping activity at 60°C. [Figure 5-1]

[0023] Figure 1 shows conserved regions showing 90% or more sequence identity among the amino acid sequences of Faustovirus D5b, E12, ST1, and LC9 (SEQ ID NOS: 7-10) capping enzymes that map to AMH83561 (H3C2). The conserved regions are designated Fausto_CR_01, Fausto_CR_03, and Fausto_CR_05. The functional domains of the H3C2 capping enzyme, i.e., TPase, GTase, and N7 MTase, are indicated. [Figure 5-2]

[0023] Figure 1 shows conserved regions showing 90% or more sequence identity among the amino acid sequences of Faustovirus D5b, E12, ST1, and LC9 (SEQ ID NOS: 7-10) capping enzymes that map to AMH83561 (H3C2). The conserved regions are designated Fausto_CR_01, Fausto_CR_03, and Fausto_CR_05. The functional domains of the H3C2 capping enzyme, i.e., TPase, GTase, and N7 MTase, are indicated. [Figure 5-3]

[0023] Figure 1 shows conserved regions showing 90% or more sequence identity among the amino acid sequences of Faustovirus D5b, E12, ST1, and LC9 (SEQ ID NOS: 7-10) capping enzymes that map to AMH83561 (H3C2). The conserved regions are designated Fausto_CR_01, Fausto_CR_03, and Fausto_CR_05. The functional domains of the H3C2 capping enzyme, i.e., TPase, GTase, and N7 MTase, are indicated. [Figure 5-4]

[0023] Figure 1 shows conserved regions showing 90% or more sequence identity among the amino acid sequences of Faustovirus D5b, E12, ST1, and LC9 (SEQ ID NOS: 7-10) capping enzymes that map to AMH83561 (H3C2). The conserved regions are designated Fausto_CR_01, Fausto_CR_03, and Fausto_CR_05. The functional domains of the H3C2 capping enzyme, i.e., TPase, GTase, and N7 MTase, are indicated. [Figure 6-1] Figure 1 shows conserved regions showing 90% or greater sequence identity between the amino acid sequences of Acanthanomeba polyphaga mimivirus (AAV50651, SEQ ID NO: 11) and Acanthanomeba polyphaga moumouvirus capping enzyme, which map to YP_007354410 (Acanthanomeba polyphaga moumouvirus capping enzyme; moumou CE) (SEQ ID NO: 12). The conserved regions are designated moumou_CR_03 (SEQ ID NO: 6) and moumou_CR_04 (SEQ ID NO: 7). The functional domains of moumou CE, i.e., the TPase, GTase, and N7 MTase, are indicated in this alignment by dashed lines above the aligned sequences. The conserved regions are highlighted. [Figure 6-2]Figure 1 shows conserved regions showing 90% or greater sequence identity between the amino acid sequences of Acanthanomeba polyphaga mimivirus (AAV50651, SEQ ID NO: 11) and Acanthanomeba polyphaga moumouvirus capping enzyme, which map to YP_007354410 (Acanthanomeba polyphaga moumouvirus capping enzyme; moumou CE) (SEQ ID NO: 12). The conserved regions are designated moumou_CR_03 (SEQ ID NO: 6) and moumou_CR_04 (SEQ ID NO: 7). The functional domains of moumou CE, i.e., the TPase, GTase, and N7 MTase, are indicated in this alignment by dashed lines above the aligned sequences. The conserved regions are highlighted. [Figure 6-3] Figure 1 shows conserved regions showing 90% or greater sequence identity between the amino acid sequences of Acanthanomeba polyphaga mimivirus (AAV50651, SEQ ID NO: 11) and Acanthanomeba polyphaga moumouvirus capping enzyme, which map to YP_007354410 (Acanthanomeba polyphaga moumouvirus capping enzyme; moumou CE) (SEQ ID NO: 12). The conserved regions are designated moumou_CR_03 (SEQ ID NO: 6) and moumou_CR_04 (SEQ ID NO: 7). The functional domains of moumou CE, i.e., the TPase, GTase, and N7 MTase, are indicated in this alignment by dashed lines above the aligned sequences. The conserved regions are highlighted. [Figure 7]This figure shows that RNA capping reactions performed at 45°C facilitate efficient capping of hairpin RNAs. At 37°C (white bars), both H3C2 and VCE efficiently capped RNA1, which has minimal 5' secondary structure (see Figure 1). At 37°C, RNA2, RNA3, and RNA4 (see Figure 1), which have stable 5' secondary structure, were not capped or only poorly capped (white bars). However, H3C2 efficiently capped all four RNAs at 45°C (gray bars). At 45°C, VCE efficiently capped RNA1 and RNA2 and partially capped RNA3 and RNA4 (gray bars). [Figure 8] This figure shows that RNA capping reactions performed at 45°C facilitate efficient capping of long RNAs. Increasing concentrations of H3C2 or VCE were incubated with 400 nM fluc RNA (1.7 kb) in 10 μL of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl, 1 mM DTT, pH 8) in the presence of 0.5 mM GTP and 0.1 mM SAM at 37°C or 45°C for 30 minutes. The first 24 nt of fluc RNA was then cleaved by oligo-induced RNase H cleavage. The extent of capping within the cleaved 24 nt fragment was analyzed by electrophoresis on a 15% urea polyacrylamide gel. Band intensities were quantified and used to calculate the percentage of capping under each condition. The calculated values ​​were graphed against enzyme concentration and fitted to the modified Hill equation to derive the enzyme concentration at which 50% capping was achieved (Cap50). For H3C2, the Cap50 values ​​were 33.6 nM and 0.96 nM at 37°C and 45°C, respectively. For VCE, the Cap50 values ​​were 103 nM and 2.97 nM at 37°C and 45°C, respectively. For both H3C2 and VCE, reactions at 45°C substantially reduced the amount of enzyme required to cap 50% of the substrate RNA compared to reactions at 37°C. [Figure 9]This figure shows that RNA capping reactions performed at 45°C facilitate more efficient capping of long RNAs other than fluc RNA than at 37°C. 100 nM H3C2 or VCE was incubated with 500 nM cluc RNA (1.8 kb) or CFTR RNA (4.0 kb) in 10 μL of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl, 1 mM DTT, pH 8) in the presence of 0.5 mM GTP and 0.1 mM SAM at 37°C or 45°C for 30 minutes. Defined 5' fragments were cleaved using thermostable RNase H (New England Biolabs, Ipswich, MA) and purified using a combination of AMPure® XP Beads (Beckman Coulter, Indianapolis, IN) and Streptavidin Dynabeads® (Thermo Fisher Scientific, Waltham, MA). Intact mass analysis of the purified 5' fragments was performed by Novatia LLC (Newtown, PA) using a high-resolution LC / MS workflow. The mass intensities of the capped and uncapped species were used to calculate the percentage of capped RNA. [Figure 10]This figure shows that high reaction temperatures enhance the enzymatic activity of Vaccinia virus (Vaccinia) RNA Cap 2'O methyltransferase (MTase). 5 μM of chemically synthesized Cap-025-nt RNA (m7Gppp25mer) was reacted with 200 μM SAM in 20 μL of 1× RNA capping buffer in the presence of 100 U of Vaccinia virus (Vaccinia) Cap 2'O MTase (New England Biolabs, Ipswich, MA) at the indicated temperatures for 30 minutes. The reaction was stopped by heating at 70°C for 10 minutes. RNA was purified from reaction components, digested into nucleosides and cap structures, and analyzed by UPLC. By performing reactions at 45°C and 50°C, more Cap-1 RNA can be generated than at 37°C. [Figure 11] This figure shows that "one-pot" generation of Cap-1 structures is more efficient at 45°C than at 37°C. In this experiment, 5 μM of chemically synthesized 5' triphosphate ribonucleotide oligo (ppp25mer) was incubated with 200 U of Vaccinia Cap 2' OMTase in 40 μL of 1× RNA capping buffer in the presence of 50 nM H3C2 or VCE, 1 mM GTP, and 0.2 mM SAM at 37°C or 45°C for 30 minutes. The relative quantities of reactants and products were derived from direct LC / MS analysis. As shown in this figure, performing the one-pot enzymatic reaction at 45°C yields more Cap-1 structured RNA than at 37°C. [Figure 12]This figure shows that one-step capped RNA synthesis for a 1.7 kb fluc transcript using commercially available enzymes and the recommended reaction temperature does not result in significant amounts of Cap-0 or Cap-1 RNA. The reactions were carried out at 37°C for 1 hour using the indicated components at the recommended enzyme concentrations (see Example 7 for details). After DNase I treatment, transcription output was measured using the Qubit RNA BR kit. Products of the capped RNA synthesis reaction were analyzed by LC / MS. The mass intensities of corresponding masses were used to estimate the proportion of transcripts containing different 5' groups. Data points are the average of triplicate experiments unless otherwise indicated. [Figure 13] Figure 1 shows a one-step method for synthesizing capped fluc transcripts using a high concentration of H3C2 capping enzyme and high temperature. One-step capped RNA synthesis was carried out at 37 or 45°C for 1 hour using T7 RNA polymerase with 0.5 mM H3C2 RNA capping enzyme. The proportions of ppp-capped RNA, pp-capped RNA, Gppp-capped RNA, and m7Gppp-capped RNA were estimated using capillary electrophoresis. Data points are the average of duplicate experiments. [Figure 14] Figure 1 shows one-step Cap-0 or Cap-1 RNA synthesis using high capping enzyme concentrations at 45 or 50°C. Products of the capping RNA synthesis reaction were analyzed by LC / MS. Mass intensities of corresponding masses were used to estimate the proportion of transcripts containing different 5' groups. Data points are the average of triplicate experiments unless otherwise indicated. [Figure 15]Figure 1 shows one-step Cap-0 RNA synthesis using H3C2:T7 fusion protein at 37°C or 45°C. The fusion protein yielded 98% Cap-0 transcripts at 45°C for 1 hour. Products of the capping RNA synthesis reaction were analyzed by LC / MS. Mass intensities of corresponding masses were used to estimate the percentage of transcripts containing different 5' groups. Data points are the average of duplicate experiments unless otherwise indicated. [Figure 16] Figure 1 shows that the capping enzymes VCE and H3C2 efficiently capped the 1.8 kb cluc / A120 transcript. 500 nM of unmodified or pseudouridine-containing cluc / A120 transcript was incubated with 100 nM H3C2 or VCE in 10 mL of 1x RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl, 1 mM DTT, pH 8) in the presence of 0.5 mM GTP and 0.1 mM SAM at 37°C for 30 minutes. The capping reaction was analyzed by RNase H / intact LC / MS workflow as described in Example 9. VCE and H3C2 resulted in cluc / A120 transcripts containing 68.4% or 100% m7Gppp-capping pseudouridine, respectively. Notably, both capping enzymes cap a greater proportion of pseudouridine-containing cluc / A120 transcripts than their uridine counterparts. [Figure 17]Figure 1 shows the efficiency of Cap-1 formation with large amounts of RNA. A total of 250 pmoles (145 mg) of cluc / A120 in vitro transcripts of approximately 1800 nt RNA were capped for 1 hour at 45°C in a 500 μL reaction containing 1× RNA capping buffer (50 mM Tris-HCl, pH 8.0, 5 mM KCl, 1 mM MgCl, 1 mM DTT), 0.5 mM GTP, 0.1 mM SAM, 10 U / μL Vaccinia Cap 2'O methyltransferase, and 500 nM H3C2. The efficiency of Cap-1 formation was assessed by RNase H / intact LC / MS as described in Example 10. Under these reaction conditions, 54.8% of the pseudouridine-containing cluc / A120 transcripts were Cap-1, whereas 47.7% of the uridine counterparts were Cap-1. Optimization of reaction conditions (described in Example 10) may improve the yield of Cap-1 formation. Results are the average of duplicate experiments. [Figure 18] Figure 1 shows that Cap-1 cluc / A120 transcripts are functionally active in vivo. Cap-1 cluc / A120 transcripts containing uridine or pseudouridine were transfected into HEK293 cells. The translation efficiency of capped cluc / A120 transcripts was measured 4 hours posttransfection as the relative luciferase activity of HEK293 cells transfected with capped cluc / A120 transcripts compared with HEK293 cells transfected with uncapped cluc / A120 transcripts. Luciferase assays showed that capped transcripts containing rU and capped transcripts containing pseudouridine exhibited 10- and 20-fold higher luciferase activity than uncapped controls, respectively, indicating that RNAs capped with H3C2 and vaccinia 2' methyltransferase were functionally active at 45°C. [Figure 19]This figure shows that multiple temperature steps can improve the transcription output and capping efficiency of single-pot capped RNA synthesis. Single-pot capped RNA synthesis reactions using T7 RNA polymerase paired with VCE or H3C2 capping enzymes were performed for a 1.7 kb fluc transcript as described in Example 11. In this case, the reaction was incubated at 37°C for 30 minutes, followed by a second incubation at temperatures between 28°C and 50°C for 30 minutes. The transcription output was assessed using the DNase I / Qubit method. The percentage of capping was estimated by RNase H / Klenow fill-in and urea-PAGE. Samples showing the highest capping rates were subjected to intact LC / MS analysis. For VCE, the efficiency of m7Gppp transcript formation peaked when the second reaction temperature was 45°C, whereas for H3C2, the efficiency of m7Gppp formation peaked when the second reaction temperature was 50°C. On the other hand, transcription output by T7 RNAP, when paired with VCE or H3C2, reflects low transcription activity at the lower and upper temperature limits tested (28°C and 50°C, respectively), peaking at 45°C. Thus, tandem temperature steps of 30 min at 37°C followed by 30 min at 45°C provided the optimal balance between transcription output and capping efficiency in this example. Modifications to reaction conditions, such as reaction time, reaction temperature, and number of temperature steps, may further improve transcription yield and the proportion of m7G-capped transcripts. DETAILED DESCRIPTION OF THE INVENTION

[0018] Aspects of the present disclosure may be further understood in light of the embodiments, section subheadings, drawings, descriptions, and examples, none of which should be understood to limit the overall scope of the present disclosure in any way. Accordingly, the claims set forth below should be read with the scope and spirit of the present disclosure in mind.

[0019] Each of the individual embodiments described and exemplified herein has distinct components and features that may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present teachings. Any recited method may be carried out in the order of events recited, or in any other order that is logically possible. Disclosed reaction conditions, including, but not limited to, reaction temperature, reaction duration, reaction component (e.g., enzyme, substrate) and / or reactant concentration, may be varied.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Moreover, certain terms are defined herein in the context of embodiments of the present disclosure and for clarity and ease of reference.

[0021] Authorities for commonly understood terms and symbols may include standard treatises and textbooks such as Kornberg and Baker, "DNA Replication," 2nd ed. (W.H. Freeman, New York, 1992); Lehninger, "Biochemistry," 2nd ed. (Worth Publishers, New York, 1975); Strachan and Read, "Human Molecular Genetics," 2nd ed. (Wiley-Liss, New York, 1999); Eckstein, ed., "Oligonucleotides and Analogs: A Practical Approach" (Oxford University Press, New York, 1991); Gait, ed., "Oligonucleotide Synthesis: A Practical Approach" (IRL Press, Oxford, 1984); Singleton et al., "Dictionary of Microbiology and Molecular biology," 2nd ed., John Wiley and Sons, New York (1994), and Hale and Markham, "Harper Collins Dictionary of Biology," Harper Perennial, NY (1991).

[0022] Please note that, as used in this specification and the appended claims, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise. Thus, for example, the term "a protein" refers to one or more proteins, i.e., a single protein as well as multiple proteins. Please further note that the claims may be drafted to exclude optional elements. As such, this statement is intended to serve as a predicate for the use of exclusionary terminology, such as "solely," "only," or a "negative" limitation, in connection with the recitation of claim elements.

[0023] Numerical ranges are inclusive of the numbers defining the range. All numbers include the midpoints of integers above and below the integer, i.e., the number 2 is understood to include 1.5 to 2.5. The number 2.5 includes 2.45 to 2.55, etc. Unless otherwise specified, when sample numerical values ​​are presented, each number alone represents the midpoint within the range of values ​​and, together, may represent the endpoints of the range.

[0024] In the context of the present disclosure, a "buffer" refers to an agent that allows a solution to resist a change in pH when an acid or alkali is added to the solution. Examples of suitable non-naturally occurring buffers that can be used in the compositions, kits, and methods of the present invention include, for example, Tris, HEPES, TAPS, MOPS, Tricine, or MES.

[0025] In the context of this disclosure, "capping" refers to the enzymatic addition of an Nppp moiety to the 5' end of an RNA, where N is a nucleotide such as G or a modified G. The modified G may have a methyl group at the N7 position of the guanine ring or a label attached at the 2- or 3-position of the ribose; in some embodiments, the label may be an oligonucleotide, a detection label such as a fluorophore, or a capture moiety such as biotin or desthiobiotin, optionally linked to the ribose of the nucleotide, e.g., by a linker. See, e.g., WO 2015 / 085142. The cap may have a Cap-0 structure, a Cap-1 structure, or a Cap-2 structure (reviewed in Ramanathan, Nucleic Acids Res., 2016, 44:7511-7526), ​​depending on which enzyme is present in the capping reaction and / or whether SAM is present.

[0026] In the context of this disclosure, a "DNA template" refers to a double-stranded DNA molecule to be transcribed in an in vitro transcription reaction. The DNA template has a promoter (e.g., a T7 promoter, a T3 promoter, or an SP6 promoter) recognized by an RNA polymerase upstream of the transcribed region.

[0027] In the context of the present disclosure, a "fusion" refers to two or more polypeptides, subunits, or proteins covalently linked to each other (e.g., by peptide bonds). For example, a protein fusion may refer to a non-naturally occurring polypeptide comprising a protein of interest covalently linked to a reporter protein. Alternatively, a fusion may include a combination of a non-naturally occurring polypeptide chain comprising two proteins or two protein domains directly linked to each other by peptide bonds or via a peptide linker.

[0028] In the context of this disclosure, "in vitro transcription" (IVT) refers to a cell-free reaction in which a double-stranded DNA (dsDNA) template is copied by an RNA polymerase (typically a bacteriophage polymerase) directed by the DNA to produce a product containing an RNA molecule copied from the template.

[0029] In the context of the present disclosure, "Faustovirus RNA capping enzyme" refers to a single-stranded RNA capping enzyme (e.g., having detectable TPase, GTase, and N7 MTase activity) capable of capping RNA, including, for example, an enzyme having at least 90% identity to Faustovirus D5b (SEQ ID NO: 7), Faustovirus E12 (SEQ ID NO: 8), Faustovirus ST1 (SEQ ID NO: 9), or Faustovirus LC9 (SEQ ID NO: 10). "H3C2 RNA capping enzyme," "H3C2 capping enzyme," or "H3C2" refers to the RNA capping enzyme from Faustovirus D5b (SEQ ID NO: 7). Unless expressly stated otherwise, for purposes of illustration and examples disclosed herein, Faustovirus RNA capping enzymes with similar properties, effects and / or benefits may be interchangeable with each other.

[0030] In the context of the present disclosure, "H3C2 fusion" refers to a fusion comprising an RNA polymerase (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase), a bifunctional enzyme capable of synthesizing RNA from a template polynucleotide and capping RNA, and a Faustovirus RNA capping enzyme located N-terminal or C-terminal to the polymerase. The H3C2 fusion may further comprise a leader and / or linker (e.g., between the polymerase and H3C2). The H3C2 fusion may have, for example, an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 20. An H3C2:T7 RNA polymerase fusion is an example of an H3C2 fusion.

[0031] In the context of the present disclosure, "H3C2 variant" refers to H3C2, H3C2 fusions and enzymes capable of capping RNA that comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 7, 8, 9, 10, 11 and / or 12, in each case.

[0032] In the context of this disclosure, "modified nucleotide" (including reference to modified NTP, modified ATP, modified GTP, modified CTP, and modified UTP) refers to any non-canonical nucleoside, nucleotide, or their corresponding phosphorylated forms. Modified nucleotides can include one or more backbone or base modifications. Examples of modified nucleotides include dl, dU, 8-oxo-dG, dX, and THF. Further examples of modified nucleotides include the modified nucleotides disclosed in U.S. Patent Publication Nos. US20170056528A1, US20160038612A1, US2015 / 0167017A1, and US20200040026A1. Modified nucleotides can include naturally occurring nucleotides or non-naturally occurring nucleotides.

[0033] In the context of this disclosure, "non-naturally occurring" refers to a polynucleotide, polypeptide, carbohydrate, lipid, or composition that does not exist in nature. Such a polynucleotide, polypeptide, carbohydrate, lipid, or composition can differ in one or more respects from a naturally occurring polynucleotide, polypeptide, carbohydrate, lipid, or composition. For example, a polymer (e.g., a polynucleotide, polypeptide, or carbohydrate) can differ in the type and sequence of its components (e.g., nucleotide sequence, amino acid sequence, or sugar molecule). A polymer can differ from a naturally occurring polymer in terms of the molecule(s) to which it is linked. For example, a "non-naturally occurring" protein can differ from a naturally occurring protein in its secondary, tertiary, or quaternary structure by having chemical bonds (e.g., covalent bonds, including peptide bonds, phosphate bonds, disulfide bonds, ester and ether bonds, and other bonds) to a polypeptide (e.g., a fusion protein), lipid, carbohydrate, or any other molecule. Similarly, a "non-naturally occurring" polynucleotide or nucleic acid may contain one or more other modifications (e.g., the addition of a label or other moiety) at the 5'-end, 3'-end, and / or between the 5'-end and 3'-end of the nucleic acid (e.g., methylation). A "non-naturally occurring" composition may differ from a naturally occurring composition in one or more of the following ways: (a) having components that are not combined in nature; (b) having components in concentrations not found in nature; (c) excluding one or more components that would otherwise be found in the naturally occurring composition; (d) having a form not found in nature, e.g., a dried form, a lyophilized form, a crystalline form, an aqueous form; and (e) having one or more additional components (e.g., a buffer, detergent, dye, solvent, or preservative) beyond those found in nature. All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0034] In the context of the present disclosure, an "RNA sample" or "sample" refers to a composition that may or may not contain target RNA. For example, an RNA sample may be known to contain or suspected to contain such target RNA, and / or may be a composition that is being assessed for the presence of target RNA. An RNA sample may include naturally occurring target RNA (e.g., target RNA extracted from a cell, tissue, or organism), target RNA produced by in vitro transcription, and / or chemically synthesized RNA.

[0035] In the context of the present disclosure, "single-stranded RNA capping enzyme" refers to a capping enzyme in which a single polypeptide chain contains TPase activity, GTase activity, and N7 MTase activity. The capping enzymes of the Faustovirus, Mimivirus, and Moumouvirus genera are examples of single-stranded RNA capping enzymes. H3C2 fusions are further examples of single-stranded RNA capping enzymes. VCE is a heterodimer and thus is not a single-stranded RNA capping enzyme.

[0036] In the context of the present disclosure, a "single uncapped target RNA species" refers to a mixture of target RNA molecules having essentially the same sequence. Transcripts produced by in vitro transcription and RNA oligonucleotides produced by solid-phase synthesis are exemplary single uncapped target RNA species. It is recognized that a certain amount of RNA products in such a mixture may be cleaved. A single uncapped target RNA species may optionally contain modified nucleotides (e.g., non-canonical nucleotides not found in nature). RNA preparations from cells contain a complex mixture of naturally occurring RNA molecules with different sequences, and such preparations not only contain uncapped target RNA species, but also a wide variety of non-target RNAs. In some embodiments, the uncapped target RNA species is a single RNA species.

[0037] In the context of the present disclosure, "target RNA" refers to a polyribonucleotide of interest. The polyribonucleotide may be or include a therapeutic RNA or a precursor thereof (e.g., an uncapped precursor of a capped therapeutic RNA). The target RNA may result from intracellular or in vitro transcription. The target RNA may be present in a mixture, e.g., an in vitro transcription reaction mixture, a cell, or a cell lysate. The target RNA may be uncapped. If desired or required, the target RNA may be contacted with a decapping enzyme, e.g., as a simultaneous treatment with capping, or as a pretreatment prior to capping.

[0038] In the context of this disclosure, "uncapped" refers to (a) RNA that does not have a cap and (b) RNA that can be used as a substrate for a capping enzyme. Uncapped RNA typically has a triphosphate or diphosphorylated 5' end. In vitro transcribed RNA has a triphosphate group at the 5' end.

[0039] In the context of the present disclosure, a "variant" refers to a protein having an amino acid sequence that differs from a naturally occurring amino acid sequence (i.e., has less than 100% sequence identity to the amino acid sequence of the naturally occurring protein), but is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the naturally occurring amino acid sequence.

[0040] Various in vitro RNA capping methods are provided herein. Some embodiments of the methods are based, in part, on the discovery that VCE of SEQ ID NO: 1 significantly increases activity at 45°C compared to 37°C, particularly for RNAs with secondary structure (see Figure 2 and Tables 1 and 2 below). For example, when used to add a cap to in vitro transcribed RNA encoding a protein, the same amount of capped product can be produced using less than 30-fold less enzyme at 45°C compared to 37°C (see Table 6). In addition, incubation of the reaction at 45°C allows VCE to efficiently add a cap to uncapped RNA oligonucleotides that have secondary structure at the 5' end (e.g., a 5' end that is predicted to base-pair with another sequence within the molecule, resulting in a blunt end or a 1- or 2-nucleotide 3' or 5' overhang, as illustrated in Figure 1). Such RNAs are highly inefficiently capped using VCE at 37°C (see Figure 9). Thus, in some embodiments, the method may include incubating a reaction mix containing RNA, VCE or a variant thereof, GTP or modified GTP, SAM, and a buffer at a temperature ranging from 42° C. to 47° C., e.g., 44° C. to 46° C., to efficiently add a cap structure to the uncapped target RNA. These embodiments of the method are particularly useful for in vitro transcribed RNAs that have or are predicted to have secondary structure, such as RNAs greater than 200 nt in length and RNA oligonucleotides with secondary structure at their 5' ends.

[0041] Another embodiment of the method is based, in part, on the discovery that the RNA capping enzyme from Faustovirus D5b (SEQ ID NO: 7; an example of an H3C2 capping enzyme, abbreviated herein as "H3C2") is capable of capping RNA significantly more efficiently than VCE at nearly all temperatures tested (see Figure 2 and Tables 1 and 6 below) and can be used to efficiently add a cap to in vitro-transcribed RNA at 45°C, similar to VCE. For example, at 45°C, less than one-thirtieth the amount of Faustovirus D5b(H3C2) can be used to generate the same amount of capping product as at 37°C (see Table 6). Additionally, incubating the reaction at 45°C allows Faustovirus D5b(H3C2) to efficiently add a cap to uncapped RNA oligonucleotides that have secondary structure at the 5' end. Such RNA is highly inefficiently capped using Faustovirus D5b (H3C2) at 37°C (see Figure 9). Capping enzymes from other giant viruses of amoeba, including Faustovirus ST1, Faustovirus LC9, Faustovirus E12, Mimivirus, and Moumouvirus, have also been examined and found to be active at elevated temperatures. Thus, in some embodiments, a method can include incubating a reaction mix containing a sample containing RNA, a single-stranded capping enzyme, GTP or modified GTP, and a buffer at a temperature ranging from 37°C to 60°C, e.g., 37°C to 42°C, 42°C to 47°C, 47°C to 52°C, or 52°C to 60°C, to efficiently add a cap structure to uncapped target RNA in vitro.These embodiments of the method are particularly useful for RNAs that have or are predicted to have secondary structure, such as in vitro transcribed RNAs greater than 200 nt in length and RNA oligonucleotides that have secondary structure at their 5' ends.

[0042] More efficient capping of RNA substrates may aid in capping with less enzyme added to the capping reaction, producing more capped RNA (as a percentage of RNA in the reaction) using the same amount of enzyme, terminating the reaction earlier, and / or more efficiently capping RNAs with secondary structure at the 5' end.

[0043] The disclosed reaction conditions can be varied, including, but not limited to, reaction temperature, reaction duration, concentrations of reaction components (e.g., SAM, inorganic pyrophosphatase, NTP, transcription template), and enzymes (e.g., capping enzymes, polymerases, and fusions thereof). For example, H3C2:T7 RNA polymerase fusion proteins can further improve the efficiency of Cap-0 RNA synthesis in relation to the proportion of Cap-0 transcripts and transcription output. Additionally, incorporation of a cap 2'O methyltransferase, such as vaccinia virus cap 2'O methyltransferase, into the reaction can generate Cap-1 transcripts at high efficiency. Depending on which enzyme is used and the other components in the reaction mix, the disclosed methods can be used to generate Gppp-capped RNA, 7-methylguanylic acid-capped RNA (i.e., m7Gppp-capped or "Cap 0"), or m7Gppp-capped RNA with additional modifications within the first and / or second nucleotide of the RNA (i.e., "Cap 1" and "Cap 2"; see Fechter, J. Gen. Vir., 2005, 86:1239-49). For example, if SAM is not present in the reaction mix, Gppp-capped RNA can be generated. If the reaction mix contains SAM in addition to the capping enzyme, Cap 0 RNA can be generated. If the reaction mix contains other enzymes in addition to SAM, such as Cap 2' OMTase, Cap 1 RNA and / or Cap 2 RNA can be generated. The reaction mix can include other components in addition to those explicitly described above.

[0044] In some embodiments, the uncapped RNA in the reaction mix may be prepared by solid-phase oligonucleotide synthesis chemistry (see, e.g., Li et al., J. Org. Chem., 2012, 77:9889-9892), in which case the RNA in the sample may have a length ranging from 10 to 500 bases, e.g., 20 to 200 bases. In other embodiments, the uncapped RNA in the reaction mix may be prepared in a cell-free in vitro transcription (IVT) reaction in which a double-stranded DNA template containing a promoter for RNA polymerase (e.g., a T7 promoter, a T3 promoter, or an SP6 promoter) upstream of the transcribed region is copied by an RNA polymerase (typically a bacteriophage polymerase) directed by the DNA to generate a product containing the RNA molecule copied from the template. In either embodiment, the RNA sample to be capped in this method contains a single RNA molecular species (synthetic oligonucleotide or transcript). In addition, the reaction mix can be RNase-free and can optionally contain one or more RNase inhibitors. The RNA in the sample may contain non-naturally occurring sequences of nucleotides, and in some embodiments, may contain non-naturally occurring nucleotides. In some embodiments, the in vitro transcription reaction may utilize thermostable mutants of T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase (see, e.g., PCT / US2017 / 013179 and U.S. Application No. 15 / 594,090). In these embodiments, the RNA may be transcribed at temperatures higher than 44°C (e.g., at least 45°C, at least 50°C, at least 55°C, or at least 60°C, but not higher than about 70°C or 75°C) to reduce the immunogenicity of the RNA (see, e.g., WO2018 / 236617). In some cases, uncapped RNA can be capped immediately after it is made, for example, by adding an RNA capping enzyme and GTP / modified GTP, if necessary, to the in vitro transcription reaction after the reaction has run its course.In some embodiments, the RNA produced in an in vitro transcription reaction may be purified prior to capping.

[0045] In some embodiments, the RNA in the sample may be therapeutic RNA. In these embodiments, the product of the method may be used without purification of the capped RNA from the uncapped RNA. In these embodiments, the product of the reaction may be combined with a pharmaceutically acceptable excipient to create a formulation, where a "pharmaceutically acceptable excipient" is any solvent compatible with administration to a living mammal via transdermal, oral, intravenous, or other administration means used in the art. Examples of pharmaceutically acceptable excipients include those described in US2017 / 0119740. The formulation may be administered in vivo, for example, to a subject, examples of which include a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Depending on the subject, RNA (modified or unmodified) may be introduced by directly injecting the RNA into cells or indirectly through the medium surrounding the cells. Administration may be performed by standardized methods. RNA may be naked RNA or may be formulated into a form suitable for administration to a subject, e.g., a human. Formulations may include liquid formulations (solutions, suspensions, dispersions), topical formulations (gels, ointments, drops, creams), and liposomal formulations (such as those described in US9,629,804B2; US2012 / 0251618A1; WO2014 / 152211; US2016 / 0038432A1). The cells into which the RNA product is introduced may be in vitro cells (i.e., cells cultured in vitro on a synthetic medium). Thus, the RNA product may be an RNA product transfected into a cell. The cells into which the RNA product is introduced may be in vivo (cells that are part of a mammal). Thus, introduction can be achieved in vivo by administering the RNA product to a subject. The cells into which the RNA product is introduced can be ex vivo (tissue removed from a mammal, e.g., cells that are part of a soft tissue or tissue isolated from the blood of a mammal).

[0046] To produce mRNA for therapeutic applications, it may be desirable or required to synthesize large quantities of uniformly capped mRNA transcripts in a cost-effective and streamlined manner that is scalable to support multigram production. Current approaches for mRNA production are expensive because they use mRNA cap analogs in in vitro transcription reactions. Alternatively, separate reactions are required to generate 5'-triphosphate RNA by in vitro transcription followed by enzymatic mRNA capping (a more complex process that is more difficult to scale up). One-step in vitro synthesis of capped RNA using T7 RNA polymerase and a capping enzyme could be a streamlined manufacturing process. A single-pot reaction with both enzymes could reduce or eliminate the otherwise prohibitive cost of synthetic mRNA cap analogs and reduce the complexity of scaling up this workflow to support multigram production (and beyond).

[0047] method Provided herein are methods for efficiently producing capped RNA. In some embodiments, the methods include thermoactive Hi-T7 RNA polymerase and H3C2 RNA capping enzyme, and unexpectedly, thermoactive Vaccinia mRNA cap 2'O methyltransferase. Surprisingly, reaction temperatures substantially higher than 37°C allow for the synthesis of capped mRNA in a single reaction. In some embodiments, RNA transcription and capping can be performed in a single reaction at elevated temperatures (e.g., 40°C to 60°C). For example, RNA polymerase and a separate capping enzyme, with or without Vaccinia cap 2'O methyltransferase, can be used simultaneously in a capped RNA synthesis reaction. Combination of RNA transcription and capping can be achieved, for example, by a fusion protein containing H3C2, a single-subunit RNA capping enzyme, and T7 RNA polymerase or Hi-T7 RNA polymerase. The methods described herein can achieve high levels of synthesis of Cap-0 RNA or Cap-1 RNA.

[0048] In some embodiments, capping methods may include contacting an RNA polymerase (e.g., T7 RNA polymerase, thermoactive Hi-T7 RNA polymerase, and / or H3C2 fusion), a polynucleotide (e.g., DNA or RNA) encoding a target RNA, a capping enzyme (e.g., VCE, H3C2, H3C2 fusion), NTPs, and a buffer, optionally in the presence or absence of SAM. Nucleoside triphosphates (NTPs) may include unmodified ATP, modified ATP (m6ATP, m1ATP), unmodified CTP, modified CTP (e.g., m5CTP), unmodified GTP, modified GTP, unmodified UTP, modified UTP (e.g., pseudouridine triphosphate, m1 pseudouridine triphosphate), NTPs containing 2'O-methylation, and / or combinations thereof. In some embodiments, capping methods can include contacting a capping enzyme (e.g., VCE, H3C2, H3C2 fusion), target RNA, and guanosine triphosphate (GTP) and / or modified GTP in the presence or absence of a SAM. In some embodiments, capping methods can include contacting a capping enzyme (e.g., VCE, H3C2, H3C2 fusion), target RNA, and guanosine triphosphate (GTP) and / or modified GTP with an O-methyltransferase (e.g., heat-activated Vaccinia mRNA cap 2' O-methyltransferase).

[0049] In some embodiments, the contacting step can be performed in a single location (e.g., in a single step), for example, on any surface (e.g., a plate or bead) or in any container (e.g., a test tube, flask, vial, column, vessel, bioreactor, or other space). In some embodiments, the contacting step can include contacting some or all of the target elements in any desired order or simultaneously. In some embodiments, the contacting step can include contacting some or all of the target elements in the presence of a buffer. For example, the contacting step can include contacting a capping enzyme (e.g., VCE, H3C2, H3C2 fusion), a target RNA, GTP, and a buffer in the presence or absence of a SAM, in any order or simultaneously. In some embodiments, the contacting step may further include contacting at a temperature of 40°C to 60°C (e.g., 40°C, 42°C, 44°C, 45°C, 46°C, 48°C, 50°C, 52°C, 54°C, 55°C, 56°C, 58°C, or 60°C) for any desired time, for example, 1 minute to 120 minutes (e.g., 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, or 120 minutes). In any of the embodiments disclosed herein, the contacting step may further include contacting at a first temperature of 25°C to 60°C and cooling or heating to a second temperature of 25°C to 60°C that is different from the first temperature, where optionally at least one of the first temperature or the second temperature is 40°C or higher. The first temperature may be selected to generate, stabilize, and / or maintain a desired amount of uncapped target RNA, and the second temperature may be selected to generate, stabilize, and / or maintain a desired amount of capped target RNA. Each temperature may be maintained for any desired period of time (e.g., 1 minute to 120 minutes).For example, the first temperature can be 37°C for a first period of 1 to 30 minutes, and the second temperature can be 28°C, 32°C, 37°C, 45°C, or 50°C for a second period of 1 to 30 minutes. In any of the disclosed embodiments, the contacting step can include directly contacting one object with another object, bringing two objects together on a surface or in a container in sufficient proximity so that they can physically and / or chemically interact, with or without further shaking or mixing. The contacting step can include an initial act of bringing one object into direct contact or proximity with another object and / or maintaining conditions that permit or favor contact of the two objects with each other.

[0050] In some embodiments, a method can include synthesizing RNA (e.g., template-directed or undirected RNA) in vitro to produce an uncapped target RNA and capping the target RNA (e.g., in a single-step reaction). Synthesizing RNA from a template can include, for example, contacting an RNA polymerase (e.g., T7 RNA polymerase, thermoactive Hi-T7 RNA polymerase, and / or an H3C2 fusion) with a template (e.g., an RNA template or a DNA template), one or more NTPs, and a buffer, optionally in the presence or absence of SAM, to produce the uncapped target RNA. Nucleoside triphosphates (NTPs) can include unmodified ATP, modified ATP (m6ATP, m1ATP), unmodified CTP, modified CTP (e.g., m5CTP), unmodified GTP, modified GTP, unmodified UTP, modified UTP (e.g., pseudouridine triphosphate, m1 pseudouridine triphosphate), NTPs containing 2'O-methylation, and / or combinations thereof. Capping the target RNA can include, for example, contacting H3C2 (e.g., H3C2 or an H3C2 fusion) with the target RNA and guanosine triphosphate (GTP) and / or modified GTP to produce a capped target RNA. In some embodiments, the capping method can include contacting the target RNA with the H3C2 fusion at a temperature between 25°C and 60°C. In some embodiments, the capping method can include contacting the target RNA with a decapping enzyme to remove an existing cap and / or confirm that it is uncapped.

[0051] Capping can include, for example, contacting a capping enzyme (e.g., VCE, H3C2, H3C2 fusion) with a target RNA, an O-methyltransferase (e.g., a heat-active Vaccinia mRNA cap 2' O-methyltransferase), a SAM, guanosine triphosphate (GTP), a modified GTP, and / or a buffer. For example, capping can include contacting a capping enzyme, a target RNA, GTP (optionally with or without modified GTP), and optionally a buffer. Capping can include contacting a capping enzyme, a target RNA, GTP (optionally with or without modified GTP), an O-methyltransferase, a SAM, and optionally a buffer in a single container (e.g., in a single step). Capping can include capping a pre-existing target RNA and / or synthesizing or capping a target RNA by contacting, in a single container (e.g., in a single step), for example, a target RNA template (DNA or RNA) encoding the target RNA, a polymerase (e.g., T7 RNA polymerase, thermoactive Hi-T7 RNA polymerase and / or H3C2 fusion), NTPs (optionally incorporating or excluding one or more modified NTPs), a capping enzyme (e.g., VCE, H3C2, H3C2 fusion), and / or a buffer.

[0052] composition Various compositions related to the disclosed methods are also provided. In some embodiments, the composition (e.g., a reaction mix) can include a target RNA (e.g., in an RNA sample) and / or a DNA template encoding the target RNA. In some embodiments, the composition can include a single-stranded RNA capping enzyme having TPase activity, GTase activity, and N7 MTase activity, GTP, SAM, and a buffer. The composition can have a temperature ranging from 37°C to 60°C, e.g., 37°C to 42°C, 42°C to 47°C, 47°C to 52°C, or 52°C to 60°C. In some embodiments, the composition can be free of RNase activity as a result of RNase being inhibited, inactivated, or absent. For example, an RNase-free composition can include one or more RNase inhibitors. In some embodiments, the composition can further include a DNA template operably linked to a promoter for transcribing RNA, a bacteriophage polymerase to induce transcription at the promoter, and NTPs. In some embodiments, the composition can include a capped 2' OMTase. The single-stranded RNA capping enzyme may comprise (a) an amino acid sequence at least 90% identical to SEQ ID NO: 2, 3, or 4; or (b) an amino acid sequence at least 90% identical to SEQ ID NO: 5 or 6. Compositions according to some embodiments comprise, in order from the N-terminus to the C-terminus of the H3C2 fusion, (a) a Faustovirus RNA capping enzyme (e.g., H3C2 RNA capping enzyme) and an RNA polymerase, or (b) an RNA polymerase and an H3C2 RNA capping enzyme, wherein the H3C2 fusion may optionally further comprise a leader (e.g., operably disposed within the fusion) and / or a linker disposed between the H3C2 and the RNA polymerase. In some embodiments, any or all of the components mentioned in this paragraph may be combined in a single container or otherwise incorporated. Components, e.g., components in a single container, may be present in a dried form (e.g., anhydrous and / or lyophilized). The container with one or more of the mentioned ingredients may also contain an aqueous medium.

[0053] kit The present disclosure also provides a kit for carrying out the method described above. In some embodiments, the kit may contain any one or more of the components listed above. For example, the kit may contain a single-stranded RNA capping enzyme having TPase activity, GTase activity, and N7 MTase activity, and the enzyme may be present in a storage buffer and a reaction buffer (which may be, for example, a 5x or 10x concentrated reaction buffer). In some embodiments, the kit may include a bacteriophage polymerase and NTPs for transcribing RNA from a DNA template. In addition, the kit may include a SAM and cap 2' OMTase. As is clear from the above discussion, the enzyme may include an amino acid sequence at least 90% identical to (a) SEQ ID NO: 2, 3, or 4; or (b) an amino acid sequence at least 90% identical to SEQ ID NO: 5 or 6.

[0054] If desired, various components of the kit may be present in separate containers, or certain compatible components may be pre-combined into a single container. In addition to the above-mentioned components, the subject kits may further include instructions for using the kit components to practice the subject methods, i.e., instructions for capping RNA. [Example]

[0055] All reagents are available from New England Biolabs (Ipswich, Mass.) and / or the indicated suppliers.

[0056] [Example 1] RNA capping activity of H3C2 and VCE at high temperature 10 nM of purified VCE RNA capping enzyme or purified H3C2 RNA capping enzyme was incubated with 500 nM of RNA1 (Figure 1) containing a fluorescent group (FAM) at its 3' end in 10 μL of 1x RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl, 1 mM DTT, pH 8.0) in the presence of 0.5 mM GTP and 0.1 mM SAM at temperatures ranging from 23°C to 65°C for 30 minutes. The reaction was quenched by the addition of 1 μL of 50 mM EDTA followed by heat inactivation at 70°C for 10 minutes. The quenched reaction was analyzed by capillary electrophoresis using an Applied Biosystems 3730xl Genetic Analyzer. After 30 minutes of capping, the peak areas corresponding to m7Gppp-RNA1, unmethylated Gppp-RNA1, uncapped pp-RNA1, and uncapped ppp-RNA1 were quantified and used to calculate the percentage of m7Gppp-RNA1. In Figure 2, each bar represents the average of four independent experiments. Error bars represent the standard deviation for four replicates. At low enzyme concentrations (10 nM), both H3C2 and VCE exhibited the highest capping activity at 45°C, whereas H3C2 retained higher capping activity than VCE even at 50°C and 55°C.

[0057] To better quantify the RNA capping activity of VCE and H3C2 at different reaction temperatures, enzymes across multiple dilutions were incubated under the same reaction conditions at 37°C, 45°C, 50°C, or 55°C, respectively, for 30 minutes. Calculated values ​​for the percentage of m7Gppp-RNA1 were plotted against enzyme concentration and fitted to a Hill equation to determine the enzyme concentration at which 50% capping was achieved (Cap 50) was derived. As shown in Figures 3A-3B and Table 1, conversion of 50% of 0.5 μM ppp-RNA1 to m7Gppp-RNA1 at 37°C, 45°C, 50°C, or 55°C in 30 minutes required 3.06 nM, 0.94 nM, 4.08 nM, and 30.00 nM VCE, respectively. Furthermore, at 50°C or less, 400 nM or more VCE capped >90% of 0.5 μM RNA in 30 minutes. For H3C2, 0.69 nM, 0.67 nM, 2.15 nM, or 21.3 nM of enzyme could convert 50% of ppp-RNA1 to m7Gppp-RNA1 at 37°C, 45°C, 50°C, or 55°C in 30 minutes, respectively. In addition, 50 nM H3C2 effectively caps >90% of ppp-RNA1 at 55°C or below for 30 minutes.

[0058] [Table 1]

[0059] Example 2: Identification and testing of H3C2 orthologues H3C2, an RNA capping enzyme purified from Faustovirus D5b (Genbank® accession number: AMN83561), was active in vitro at temperatures below 60°C. Sequence identity identified several putative orthologs in other Faustovirus strains. The gene products of putative RNA capping enzymes from other Faustovirus strains and related giant viruses, Acanthanomeba polyphaga mimivirus and Acanthanomeba polyphaga moumouvirus, were found to be active in RNA capping at temperatures between 55°C and 60°C. The ORFs of the identified orthologous genes were synthesized and inserted into a T7 expression vector, placing target gene expression under the control of the T7 promoter. The T7 expression cassette was amplified by PCR using Q5® High Fidelity 2x Master Mix (New England Biolabs, Ipswich, MA) according to the manufacturer's recommendations. The amplified T7 expression cassette was purified using the Monarch® PCR & DNA cleanup kit (New England Biolabs, Ipswich, MA) according to the manufacturer's instructions. The purified T7 expression cassette was subjected to in vitro transcription / translation using the PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs, Ipswich, MA) according to the manufacturer's instructions. The PURExpress reaction products (Figures 4A-4E), containing gene products with the indicated GenBank accession numbers, were used in RNA capping assays. Briefly, 2 μL of PURExpress product, 0.4 mM of 150 nt in vitro transcribed RNA, 4 μM GTP, 0.1 mM SAM, and traces of 1 μL of PURExpress product were added to 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl, 1 mM DTT, pH 8.0). 32A 50 μL RNA capping reaction consisting of P-α-GTP was assembled on ice. The reaction was divided into five equal portions, each of which was incubated for 30 minutes at the indicated temperature. The reaction was stopped by adding 10 μL of 2× RNA Loading Dye (New England Biolabs, Ipswich, MA) to the reaction and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. Capping activity was indicated by an X-ray signal migrating at the position of the indicated substrate RNA. As shown in Figures 4A to 4E, the putative RNA capping enzymes from Faustovirus ST1 (Genbank accession number: SME65026; lane 3) and Faustovirus LC9 (SMH63629; lane 4), the N-terminal fraction of Mimivirus capping enzyme (amino acids 1 to 668 of Genbank accession number: AAV50651; Benarroch et al., 2008; lane 5), and the N-terminal fraction of Moumouvirus capping enzyme (Genbank accession number: AAV50651; lane 6) were isolated. The RNA capping enzyme (Genbank accession number: YP_007354410; lane 6) caps 150-mer RNAs at temperatures below 55°C, whereas the RNA capping enzymes from Faustovirus D5b (Genbank accession number: AMN83561; F13C2; lane 1), Faustovirus E12 (Genbank accession number: AIB52055; lane 2), and purified F13C2 (lane 7) cap 150-mer RNAs at temperatures below 60°C.

[0060] [Example 3] Sequence analysis Sequence alignment analysis revealed that three regions, 60 or 111 amino acids in length, exhibited greater than 90% amino acid sequence identity among the four Faustovirus RNA capping enzymes active at or below 55°C (Figure 5). The conserved region, Fausto_CR_01, spans 60 amino acids within the TPase domain of F13C2. Fausto_CR_03 spans 111 amino acids at the N-terminus of the guanosine N7 methyltransferase domain. Fausto_CR_05 spans 60 amino acids at the C-terminus of the guanosine N7 methyltransferase domain of F13C2. The amino acid sequence of Fausto_CR_01 (corresponding to amino acids 43-102 of the F13C2 sequence) is FDKLKPDGEITTTMRVSNADGMAREITFGGGVKTGEMFVKKQNICVFDVVDIFSYKVAVS (SEQ ID NO: 2). The amino acid sequence of Fausto_CR_03 (corresponding to amino acids 537-647 of the F13C2 sequence) is GFYGNNYKIASDIYLNYIDVFNFDDLWKYNPGYFEKNKSDIYIAPNKYRRYLIKSLFNKYIKNAKWVIDAAAGRGADLHLYKAECVENLLAIDIDPTAISELIRRRNEITGDVFNFDDLWKYNPGYFEKNKSDIYIAPNKYRRYLIKSLFNKYIKNAKWVIDAAAGRGADLHLYKAECVENLLAIDIDPTAISELIRRRNEITG (SEQ ID NO: 3). The amino acid sequence of Fausto_CR_05 (corresponding to amino acids 778-873 of the F13C2 sequence) is KYSIKRLYDSDKLTKTGQKIAVLLPMSGEMKEEPLCNIKNIISMARKMGLDLVESANFSV (SEQ ID NO: 4).

[0061] The following table summarizes the sequence identities between the amino acid sequences of Faustovirus capping enzymes.

[0062] [Table 2]

[0063] [Table 3]

[0064] FIG. 6 shows an alignment of various Faustovirus RNA capping enzyme sequences showing the sequences of the TPase, GTase and MTase regions as well as the conserved domains.

[0065] Sequence alignment analysis also revealed that two regions, 67 and 111 amino acids long, share greater than 90% sequence identity between the RNA capping enzymes of Acanthomeba polyphaga mimivirus and Acanthomeba polyphaga moumouvirus, both of which are active at temperatures below 55°C (Fig. 7). The conserved region, moumou_CR_03 (corresponding to amino acids 576–642 of the Moumouvirus sequence), spans 67 amino acids between the GTase domain and the guanosine N7 methyltransferase domain. The conserved region, moumou_CR_04 (corresponding to amino acids 672–782 of the Moumouvirus sequence), spans 111 amino acids N-terminal to the guanosine N7 methyltransferase domain. The amino acid sequence of moumou_CR_03 is INDNTVVEFIFDNFKIDMDDPYKWIPIRTRYDKTESVQKYHKKYGNNLHIANRIWKTITNPITEDII (SEQ ID NO: 5). The amino acid sequence of moumou_CR_04 is YYQKNTSNAAGMRAFNNFIKSNMITTYCKDGDKVLDIGCGRGGDLIKFIHAGIEEYVGIDIDNNGLYVINDSAFNRYKNLKKTIKNIPPMTFINADARGLFNLEAQEKILP (SEQ ID NO: 6).

[0066] The following table summarizes the sequence identity between the amino acid sequence of the Mimivirus RNA capping enzyme and the amino acid sequence of the Moumouvirus RNA capping enzyme.

[0067] [Table 4]

[0068] [Table 5]

[0069] FIG. 8 shows an alignment of Mimivirus and Moumouvirus sequences showing the sequences of the TPase, GTase and MTase regions as well as the conserved domains.

[0070] Example 4: RNA capping reaction at 45°C enhances capping efficiency in short model hairpin RNAs To assess capping efficiency, short RNAs, as illustrated in Figure 1, were designed as described herein. A control RNA (RNA1) was designed to have a short hairpin structure and an unstructured 5' end with an MFE of -1.5 kcal / mol at 37°C or -0.73 kcal / mol at 45°C (Figure 1). RNA2, RNA3, and RNA4 were designed to form stable hairpin structures with a theoretical minimum free energy (MFE) of unfolding of -7.2 kcal / mol at 37°C or -5.4 kcal / mol at 45°C (RNAFold server: rna.tbi.univie.ac.at). RNA2, RNA3, and RNA4, each with a minimum free energy structure, were further designed to have a blunt end, a one-base overhang, or a two-base overhang at their 5' ends (Figure 1). RNAs were generated by in vitro transcription using T7 RNA polymerase. For the capping reaction, 50 nM of H3C2 or VCE was incubated with 500 nM of RNA substrate in 10 μL of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl, 1 mM DTT, pH 8.0) in the presence of 0.5 mM GTP and 0.1 mM SAM at 37°C or 45°C for 30 min. At 37°C, H3C2 capped 100% of RNA1, which was predicted to have an unstructured 5′ end, and 22% of RNA4, which was predicted to have a 2-nt overhang at the 5′ end (Figure 9). H3C2 was unable to cap RNA2 and RNA3 under these conditions. At 45°C, H3C2 capped 100% of all four RNA substrates. At 37° C., VCE was able to cap only 80% of RNA1. At 45° C., VCE capped 100% of RNA1 and RNA2 and approximately 60% of RNA3 and RNA4.

[0071] [Example 5] RNA capping reaction at 45°C enhances capping efficiency for long RNAs A 1766-nt RNA containing the firefly luciferase gene (flucfluc) was generated by in vitro transcription using T7 RNA polymerase. 400 nM fluc RNA was incubated with the indicated concentrations of H3C2 or VCE in 10 μL of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl, 1 mM DTT, pH 8) in the presence of 0.5 mM GTP and 0.1 mM SAM at 37°C or 45°C for 30 minutes. A targeting oligo (TO) designed to direct RNase activity to excise a 25-nt 5' fragment was then added to each capping reaction to a final concentration of 2.5 μM. The reaction mixture was then heated to 80°C for 30 seconds to inactivate the capping enzyme, followed by slow cooling to room temperature. Thermostable RNase H (New England Biolabs, Ipswich, MA) was added to each reaction at a final concentration of 0.5 U / μL, followed by incubation at 37°C for 1 hour. The RNase H reactions were then analyzed by electrophoresis through a 15% urea polyacrylamide gel. Gels were stained using SYBR® Gold (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions and then scanned using an Amersham® Typhoon® RGB (GE Healthcare, Marlborough, MA) scanner using the Cy2 channel. The intensities of capped and uncapped bands were normalized to the intensity of TO in the same lane. The normalized values ​​were used to calculate the percentage of capped RNA under each condition. Calculated values ​​were graphed against enzyme concentration and fitted to a derivative of the Hill equation to determine the enzyme concentration at which 50% capping was achieved (Cap 50 ) was derived. For H3C2, Cap 50 The values ​​were 33.6 nM and 0.96 nM at 37°C and 45°C, respectively. 50The values ​​were 103 nM and 2.97 nM at 37° C. or 45° C., respectively (FIG. 10 and Table 6 below). For both enzymes, substantially less enzyme was required to achieve 50% capping when the capping reaction was performed at 45° C.

[0072] [Table 6]

[0073] The activity of these enzymes can be expressed in milliunits per μL of reaction as shown below:

[0074] [Table 7]

[0075] To demonstrate that temperature-dependent capping efficiency enhancement can also be achieved with other long RNA molecules, we investigated the capping efficiency of H3C2 and VCE using in vitro transcripts of Cypridina luciferase (clue; 1823 nt) and cystic fibrosis transmembrane receptor (CFTR; 4712 nt) with mass spectrometry readout. Briefly, 0.5 μM of clue in vitro transcript or CFTR in vitro transcript was incubated with 100 nM of H3C2 or VCE in a 100 μL reaction mixture in the presence of 0.1 mM SAM and 0.5 mM GTP at 37°C or 45°C for 30 min. The reaction was terminated by heating to 80°C for 30 s in the presence of 2.5 μM targeting oligo, consisting of 5' deoxynucleotides and 3' ribonucleotides and a TEG-desthiobiotin group. The reaction was cooled to 25°C at a rate of 0.1°C / sec. The reaction was then subjected to RNase H cleavage by incubation with thermostable RNase H (New England Biolabs, Ipswich, MA) at a final concentration of 0.5 U / μL for 1 hour at 37°C. The 5' fragment of the RNase H cleavage reaction was then purified using size selection using AMPure® XP Beads and target selection using streptavidin magnetic beads. Briefly, 100 μL of the RNase H cleavage reaction was added to 200 μL of AMPure® XP Beads and incubated at room temperature for 5 minutes. The beads were then placed under a magnetic field, and the clarified supernatant was collected and added to the pre-cleaned AMPure® XP beads from 200 μL of bead suspension. After incubation at room temperature for 5 minutes, the beads were placed under a magnetic field. The clarified supernatant was added to 200 μL of pre-cleaned beads from Dynabeads® MyOne Streptavidin C1.After washing four times with 200 μL of wash buffer (5 mM Tris, pH 7.5, 0.5 mM EDTA, 1 M NaCl), the bound RNA was eluted by incubating the purified beads with 50 μL of biotin elution buffer (1 mM biotin, 5 mM Tris, pH 7.5, 0.1 M NaCl, 0.1 M NaCl) for 1 hour at 37° C. The eluted RNA was then analyzed by LC / MS performed by an external contractor, Novatia LLC, to determine the relative mass of the target. The degree of RNA capping was assessed by the mass intensity ratio of capped to uncapped RNA species.

[0076] As shown in Figure 11, both H3C2 and VCE exhibit greater capping activity on clue RNA and CFTR RNA at 45°C than at 37°C.

[0077] Example 6: Efficient one-pot enzymatic synthesis of Cap-1 structures on high-temperature triphosphate RNA To verify whether Vaccinia Cap 2' OMTase is active at elevated temperatures, reactions containing 5 μM of chemically synthesized Cap-0 RNA1 (25 nt) and 200 μM SAM in 20 μL of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl, 1 mM DTT, pH 8.0) were preheated to 37°C, 45°C, or 50°C for 1 min before adding 100 U of Vaccinia Cap 2' OMTase (New England Biolabs, Ipswich, MA). The reaction proceeded for 30 min at the preheat temperature and was then terminated by heating to 70°C for 10 min. RNA was purified from reaction components using an Oligo Clean-up and Concentration Kit (Norgen Biotek, Thorold, Canada). The purified RNA was then digested into nucleotides and cap structures by incubating with 2 μL of Nucleotide Digestion Mix (New England Biolabs, Ipswich, MA) in a 20 μL reaction containing Nucleotide Digestion Mix reaction buffer (50 mM sodium acetate, pH 5.4, 1 mM ZnCl) for 1 hour at 37°C. The nucleotide digestion reactions were then analyzed by Agilent 1290 Infinity II UHPLC (Agilent Technologies, Santa Clara, CA) on a Waters XSelect™ HSS T3 XP column (Waters Corporation, Milford, MA) (2.1 × 100 mm, 2.5 mm) with a gradient mobile phase consisting of methanol and 10 mM potassium phosphate buffer (pH 7.0). As shown in Figure 12, at 45°C and 50°C, more Cap-0 RNA was methylated than at 37°C.

[0078] To demonstrate one-pot enzymatic synthesis of Cap-1 structures on high-temperature 5'-triphosphate RNA, 5 μM of chemically synthesized 5'-triphosphate RNA1 was incubated with 200 U of vaccinia Cap 2' OMTase (final concentration: 5 U / μL) in a 40 μL reaction containing 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl, 1 mM DTT, pH 8.0) in the presence of 50 nM H3C2 or VCE, 1 mM GTP, and 0.2 mM SAM at 37°C or 45°C for 30 min. Reactions were analyzed directly by LC / MS to determine mass and relative mass. When paired with 50 nM VCE, 5 U / μL of 2′ OMTase produced only approximately 50% Cap-01 RNA at 37°C, whereas 100% Cap-1 RNA was produced at 45°C (FIG. 13). When paired with 50 nM H3C2, 5 U / mL of vaccinia 2′ OMTase produced approximately 90% Cap-01 RNA at 37°C and 100% Cap-1 RNA at 45°C (FIG. 13). Thus, performing the reaction at 45°C nearly doubled the yield of Cap-1 RNA in the presence of VCE and increased the yield of Cap-1 RNA in the presence of H3C2.

[0079] Example 7: One-step synthesis of Cap-0 RNA and Cap-1 RNA using separate RNA polymerases, RNA capping enzymes, and cap 2'O methyltransferases A. One-step capped RNA synthesis by the Vaccinia capping enzyme is inefficient 5 U / μl T7 RNA polymerase (New England Biolabs; Cat. No. M0251), Hi-T7 RNA polymerase (New England Biolabs; Cat. No. M0658), 1 U / μl Vaccinia capping enzyme (New England Biolabs; Cat. No. M2080), and 5 U / μl Vaccinia mRNA cap 2'O methyltransferase (New England Biolabs; Cat. No. M0366), adapted for transcription and capping of the 1.7 kb fluc transcript (SEQ ID NO: 18), were used as recommended by the manufacturer for each reaction. Reactions contained 1x T7 RNA polymerase buffer (40 mM Tris-HCl, pH 7.9, 10 mM NaCl, 1 mM DTT, 2 mM spermidine) when using T7 RNA polymerase, or 1x Hi-T7 RNA polymerase buffer (40 mM Tris-HCl, pH 7.9, 60 mM NaCl, 1 mM DTT, 2 mM spermidine) when using Hi-T7 RNA polymerase. Reactions were supplemented with 19 mM MgCl2, 5 mM each of ATP, UTP, GTP, CTP, and 0.5 mM SAM. Reactions were carried out at 37°C for 1 hour, the recommended reaction temperature and duration for T7 RNA polymerase and Vaccinia capping enzyme.

[0080] To assess the yield of in vitro transcription after 1 hour of incubation for each reaction, 1 μL of the reaction mix was added to 4 μL of a solution containing 1× DNase I reaction buffer (10 mM Tris-HCl, pH 7.6, 2.5 mM MgCl 0.5 mM CaCl ) and 0.5 U / μL DNase I (New England Biolabs) and incubated at 37°C for 30 minutes. The DNase I reactions were then analyzed for total RNA concentration using a Qubit RNA Broad Range kit (Thermo Fisher) according to the manufacturer's instructions. To verify the size and integrity of the transcripts, the DNase I reactions were analyzed using 2% E-Gel (Thermo Fisher), with images captured using a Typhoon RGB scanner (GE Healthcare). The results are shown as transcription output in Figure 14.

[0081] To analyze the extent of RNA capping, the one-step capped RNA synthesis was analyzed by RNase H cleavage followed by intact LC / MS analysis. Briefly, the one-step capped RNA synthesis was terminated by heating at 80°C for 30 seconds in the presence of 2.5 μM targeting oligo (TO-1), consisting of 5' deoxynucleotides and 3' ribonucleotides and a TEG-desthiobiotin group (SEQ ID NO: 19). The reaction was cooled to 25°C at a rate of 0.1°C / second. The reaction was then subjected to RNase H cleavage by incubation at 37°C for 1 hour with thermostable RNase H (New England Biolabs; product number: M0523) at a final concentration of 0.5 U / μL. To facilitate analysis of the 5' group using capillary electrophoresis, FAM-labeled nucleotides were added to the 3' end of the RNase H-cleaved fragments. Briefly, 5 μL of the RNase H reaction was collected and added to 5 μL of a solution containing 2×NEBuffer2, 0.5 mM dATP, 0.05 mM FAM-12-dCTP (Perkin Elmer), and 0.25 U / μL DNA polymerase I large (Klenow) fragment (New England Biolabs). The reaction was incubated at 37°C for 1 hour. In some cases, a small fraction of the Klenow reaction was analyzed on a urea-PAGE to verify successful capping. Briefly, 2 μL of the Klenow reaction was collected and added to 8 μL of 2×RNA loading dye (New England Biolabs). The mixture was then analyzed by electrophoresis through a 15% urea polyacrylamide gel. Gel images were collected using a Typhoon RGB scanner (GE Healthcare) using the Cy2 channel.

[0082] To analyze the 5'-group state of the coupled transcription / capping reaction product using capillary electrophoresis or mass spectrometry, the Klenow reaction product (the RNase H cleavage product still annealed to the desthiobiotinylated targeting oligo) was then purified by size selection using AMPure® XP Beads (Thermo Fisher Scientific) and then selected using streptavidin magnetic beads. Briefly, 45 μL of nuclease-free water was added to 5 μL of the RNase H cleavage reaction, which was then added to 100 μL of NEBNext® Sample Purification Beads (New England Biolabs) and incubated at room temperature for 5 minutes. The beads were then placed adjacent to a magnet at room temperature for 2 minutes. The clarified supernatant was collected and added to pre-cleared NEBNext® Sample Purification Beads derived from 100 μL of the bead suspension. After incubation at room temperature for 5 minutes, the beads were placed next to a magnet for 2 minutes at room temperature. The clarified supernatant was added to 50 μL of pre-cleaned beads from Dynabeads® MyOne Streptavidin C1 (Thermo Fisher). After washing four times with 50 μL of low-fume wash buffer (5 mM Tris, pH 7.5, 0.5 mM EDTA, 60 mM NaCl), the bound RNA was eluted by incubating the cleaned beads in 10 μL of nuclease-free water. The eluted RNA was then analyzed by capillary electrophoresis on an Applied Biosystems 3130xl Genetic Analyzer (16 capillary array) or an Applied Biosystems 3730xl Genetic Analyzer (96 capillary array) using the GeneScan® 120 LIZ dye Size Standard (Applied Biosystems).Reaction products were analyzed using PeakScanner software (Thermo Fisher Scientific) and a suite of in-house software. Peak areas corresponding to m7Gppp-RNase H-cleaved transcripts, unmethylated Gppp-RNase H-cleaved transcripts, uncapped pp-RNase H-cleaved transcripts, and ppp-RNase H-cleaved transcripts were quantified and used to calculate the proportion of capped RNA synthesis in ppp, pp, Gppp, and m7Gppp transcripts.

[0083] Mass spectrometry was used to determine the relative abundance of m7GpppGm-capped RNA (Cap-1), m7G-capped RNA (Cap-0), unmethylated G-capped RNA, and uncapped RNA. The intact masses of the relevant molecular species were determined using LC / MS performed by an external contractor, Novatia LLC (Newtown, PA), or an in-house facility. The degree of RNA capping was assessed by the mass intensity ratio of the relevant RNase H cleavage products.

[0084] Figure 14 shows the results of one-step transcription using currently available commercially available reaction conditions. Comparing bar 1 and bar 2, the presence of VCE did not significantly affect the transcription output of T7 RNA polymerase, resulting in no detectable m7GpppG (Cap-0) transcripts. A small percentage (5.4%) of unmethylated G-capped RNA, an undesired capping reaction intermediate, was detected. Approximately 50% of the transcripts also contained a 5' diphosphate group, another capping reaction intermediate. When vaccinia virus cap 2'O methyltransferase was incorporated into the reaction, as shown in bar 3, the transcription output was not significantly affected, and m7GpppGm (Cap-1) transcripts were detected. Approximately 50% of the transcripts had a 5' diphosphate group, and 15% had an unmethylated G-capped intermediate. When Hi-T7 RNA polymerase was used instead of T7 RNA polymerase, similar results to those observed in the T7 RNA polymerase reaction were observed (the presence of VCE did not significantly affect Hi-T7 RNA polymerase transcription, resulting in no detectable levels of Cap-0 transcripts). Addition of vaccinia virus cap 2'O methyltransferase to the reaction did not result in Cap-1 transcripts. These results indicate that currently standard enzyme reagents and reaction conditions for in vitro mRNA synthesis (i.e., RNA synthesis followed by mRNA capping) do not support the production of capped mRNA in a single reaction in vitro (i.e., one-step capped RNA synthesis).

[0085] The B. Faustovirus RNA capping enzyme, H3C2, efficiently caps transcripts in a one-step reaction in vitro Because the reagents and conditions in Example 7A were ineffective in producing capped transcripts in a one-step in vitro reaction, new enzymes and reaction conditions were examined for their ability to perform one-step capped RNA synthesis in vitro. Figure 13 shows the results of one-step capped RNA synthesis using high concentrations of H3C2 capping enzyme at 37°C or 45°C.

[0086] At 37°C, 0.5 mM H3C2 capping enzyme resulted in 12% m7Gppp-capped transcripts, with 24.1% unmethylated G-capped transcripts, as measured by capillary electrophoresis. On the other hand, at 45°C, 61% of transcripts had m7Gppp caps, with only 1.4% having unmethylated G-caps. At both temperatures, transcription output was 50-60% of that without H3C2 capping enzyme. Thus, increasing the H3C2 concentration and reaction temperature improves the yield of Cap-0 transcription in one-step capped RNA synthesis using T7 RNA polymerase and H3C2 RNA capping enzyme.

[0087] The effects of further expanding the range of enzyme reagents and reaction temperatures were investigated by performing Cap-0, one-step capped RNA synthesis at 45°C and 50°C using different combinations of T7 RNA polymerase, Hi-T7 RNA polymerase, VCE, H3C2, and Vaccinia virus (Vaccinia) cap 2'O methyltransferase. Specifically, one-step capped RNA synthesis was performed as described in Example 7A using 0.5 mM VCE capping enzyme or H3C2 capping enzyme in their respective reaction buffers, as indicated, with 5 U / μL T7 RNA polymerase or Hi-T7 RNA polymerase in the presence or absence of 5 U / μL Vaccinia virus (Vaccinia) mRNA cap 2'O methyltransferase. Reactions were carried out at 45 or 50°C for 1 hour.

[0088] Figure 14 summarizes the results of this extended investigation. In bars 1–5, capped RNA synthesis reactions were performed at 45°C using T7 RNA polymerase. In the presence of H3C2 capping enzyme, 84% of transcripts were m7Gppp-capped (Cap-0), and 8% were unmethylated G-capped (bar 2). In the presence of both H3C2 and Cap2'O ​​methyltransferase, 87% of transcripts were m7GpppGm-capped (Cap-87), and 6% were unmethylated G-capped; Cap-0 was not detected (bar 3). Using VCE, 76% of transcripts had the Cap-0 structure, and 24% had the unmethylated G-cap structure (bar 4). In the presence of both VCE and cap 2'O methyltransferase, 55% of the transcripts had the Cap-1 structure, 36% had the Cap-0 structure, and 8% had an unmethylated G-cap structure. At 45°C, RNA yield was not significantly affected when H3C2 or VCE was present in the reaction. However, when either cap 2'O methyltransferase or a capping enzyme was present, RNA yield decreased from 0.14 mg / μL to 0.09 mg / μL. At 45°C, the percentage of capped transcripts in capped RNA synthesis reactions using Hi-T7 RNA polymerase (bars 6–10) was smaller than that in capped RNA synthesis reactions using T7 RNA polymerase (bars 1–5). H3C2 capping enzyme and VCE capping enzyme yielded 45% (bar 7) and 50% (bar 9) of Cap-0 transcripts, respectively. H3C2 with cap 2'O methyltransferase resulted in 44% Cap-1 transcripts, 39% unmethylated G-capping transcripts, and no detectable Cap-0 transcripts (bar 8). VCE with cap 2'O methyltransferase resulted in 19% Cap-1 transcripts, 22% Cap-0 transcripts, and 42% unmethylated G-capping transcripts (bar 10).Interestingly, the transcription output of reactions performed at 45°C using Hi-T7 RNA polymerase in the presence of capping enzyme or Cap 2'O methyltransferase was not significantly affected (bars 6–10). When capped RNA synthesis reactions were performed at 50°C using Hi-T7 RNA polymerase, the H3C2 capping enzyme resulted in 80% Cap-0 transcripts and 20% unmethylated G-capped transcripts (bar 12). In the presence of both H3C2 and Cap 2'O methyltransferase, 71% of transcripts contained the Cap-1 structure, 2% contained the Cap-0 structure, and 26% contained the unmethylated G-cap structure (bar 13). Using VCE, 55% of transcripts had the unmethylated G-cap structure, and Cap-0 was not detected. When both VCE and cap 2'O methyltransferase were present, 23% of the transcripts had the Cap-0 structure and 31% had the unmethylated G-cap structure.

[0089] In summary, this example provides one-step reaction conditions (Table 7) for using H3C2 RNA capping enzyme together with T7 RNA polymerase or Hi-T7 RNA polymerase to generate Cap-0 RNA in vitro at an efficiency of 80% or less. This example also describes one-step reaction conditions (Table 8) for using H3C2 RNA capping enzyme, vaccinia virus mRNA cap 2'O methyltransferase together with T7 RNA polymerase or Hi-T7 RNA polymerase to generate Cap-1 RNA at an efficiency of 70-80%.

[0090] [Table 8]

[0091] [Table 9]

[0092] Example 8: One-step synthesis of Cap-0 RNA using H3C2:T7 RNA polymerase fusion protein A fusion construct was prepared to generate a protein consisting of H3C2 RNA capping enzyme and T7 RNA polymerase linked by a presumably flexible linker sequence (SEQ ID NO: 20). The fusion protein was encoded by a DNA sequence (SEQ ID NO: 21) under the control of the T7 promoter or tac promoter, expressed in E. coli, and purified using standard chromatographic methods.

[0093] A one-step capped RNA synthesis reaction for the 1.7 kb fluc transcript was carried out in the presence of 0.5 mM H3C2:T7 RNA polymerase fusion in 1x T7 RNA polymerase buffer supplemented with 19 mM MgCl2, 5 mM each of ATP, UTP, GTP, and CTP, and 0.1 mM SAM, as indicated in Example 7. For comparison, reactions containing 5 U / μL T7 RNA polymerase with or without 0.5 mM H3C2 capping enzyme were performed. Reactions were carried out at 37 or 45°C for 1 hour. RNA yield and the proportion of capped and uncapped transcripts were measured and analyzed as indicated in Example 7.

[0094] The results are shown in Figure 15. When reactions were performed at 37°C, the H3C2:T7 fusion protein yielded 75% m7Gppp-capped (Cap-0) transcripts and 20% unmethylated G-capped transcripts (bar 1). Under the same conditions, the individual enzymes, H3C2 alone and T7 RNA polymerase alone, yielded 12% Cap-0 transcripts and 27% unmethylated G-capped transcripts (bar 2). Transcription output decreased from 0.45 mg / μL in the presence of T7 RNA polymerase alone (bar 3) to 0.39 mg / μL when H3C2 was added, and further decreased to 0.19 mg / μL when the H3C2:T7 fusion was used for transcription. At 45°C, the H3C2:T7 RNA polymerase fusion yielded 95% Cap-0 transcripts and 5% unmethylated G-capped transcripts (bar 4), compared with 60% Cap-0 transcripts and 6% unmethylated G-capped transcripts (bar 5) when the individual enzymes were used. Similar to reactions performed at 37°C, transcription output decreased from 0.31 mg / μL (bar 6) when T7 RNA polymerase alone was present to 0.18 mg / μL when H3C2 was added or 0.12 mg / μL when the H3C2:T7 fusion was used for transcription.

[0095] Thus, under the reaction conditions described, this example demonstrates efficient (e.g., as high as 98%), in vitro, one-step Cap-0 RNA synthesis using an H3C2:T7 RNA polymerase fusion protein.

[0096] Example 9: RNA capping enzyme efficiently caps in vitro transcripts containing pseudouridine Approximately 1800-nt RNA encoding Cypridina luciferase protein (cluc / A120) with a 120-nt poly(A) tail was synthesized by in vitro transcription using T7 RNA polymerase (New England Biolabs Inc.) in the presence of pseudouridine triphosphate (Trilink Biotechnologies) or unmodified uridine triphosphate (New England Biolabs Inc.). 500 nM of unmodified or pseudouridine cluc / A120 transcript was incubated with 100 nM H3C2 or VCE in 10 mL of 1x RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8) in the presence of 0.5 mM GTP and 0.1 mM SAM at 37°C for 30 min. A targeting oligo (TO) designed to direct RNase to excise the 5' fragment was then added to each capping reaction to achieve a final TO concentration of 2.5 mM. Each reaction was then heated to 80°C for 30 seconds and slowly cooled to room temperature to allow the TO to anneal with the transcript and inactivate the capping enzyme. Thermostable RNase H (New England Biolabs) was added to each reaction at a final concentration of 0.5 U / μL and incubated at 37°C for 1 hour. The reaction products were then purified and analyzed by intact LC / MS as described in Example 7. As shown in Figure 16, VCE and H3C2 capped 68.4% and 100% of pseudouridine-containing cluc / A120 transcripts with m7Gppp, respectively. Notably, both capping enzymes cap a greater proportion of pseudouridine-containing cluc / A120 transcripts than their uridine counterparts.

[0097] Example 10: Scale-up and functional assay of Cap-1 capping A total of 250 picomoles (145 mg) of approximately 1800 nt cluc / A120 in vitro transcripts were capped for 1 h at 45°C in a 500 μL reaction containing 1× RNA capping buffer (50 mM Tris-HCl, pH 8.0, 5 mM KCl, 1 mM MgCl, 1 mM DTT), 0.5 mM GTP, 0.1 mM SAM, 10 U / μL Vaccinia Cap 2'O-methyltransferase (NEB M0366), and 500 nM H3C2. The efficiency of Cap-1 formation was assessed by RNase H / intact LC / MS as described in Example 7. RNA was purified using acidic phenol and chloroform, followed by ethanol precipitation and rehydration in nuclease-free water. RNA concentration was estimated using the Qubit RNA (Broad Range) kit (ThermoFisher). Translation efficiency of capped cluc / A120 transcripts was measured as relative luciferase activity by HEK293 cells transfected with capped cluc / A120 transcripts compared to HEK293 cells transfected with uncapped cluc / A120 transcripts. HEK293 cells were transfected with 250 ng of purified cluc / A120 transcripts using Lipofectamine MessengerMax transfection reagent (ThermoFisher). Luciferase activity was assayed 4 hours posttransfection in 10 μL of culture medium supernatant using the BioLux® Cypridina Luciferase Assay Kit (New England Biolabs Inc., Ipswich). Luminescence was measured using a Centro LB960 microplate luminometer from Berthold Technologies. As shown in Figure 17, the scaled-up capping reaction achieved 47.7% and 54.8% Cap-1 formation on cluc / A120 containing rU and pseudouridine, respectively, consistent with the results in Example 5 and Figure 11.Triplicate luciferase assay results showed that translation efficiency from purified capped transcripts containing rU and pseudouridine exhibited high luciferase activity (Figure 18). Note that modifications to reaction conditions, such as reaction volume, enzyme and component concentrations, reaction time, and reaction temperature, can improve the efficiency of Cap-1 formation and the translation efficiency of the resulting Cap-1 RNA when large amounts of RNA are used.

[0098] Example 11: Single-vessel capped RNA synthesis with multiple temperature steps As indicated in Example 7, single-pot capped RNA synthesis reactions for the 1.7 kb fluc transcript were performed using 5 U / ul of T7 RNA polymerase (New England Biolabs; Cat. No. M0251) with or without 500 nM of Vaccinia capping enzyme (New England Biolabs; Cat. No. M2080) or H3C2 capping enzyme, incubated at 37°C for 30 minutes, followed by an additional 30-minute incubation at 28°C, 32°C, 37°C, 45°C, or 50°C. Reactions contained 1x T7 RNA polymerase buffer (40 mM Tris-HCl, pH 7.9, 10 mM NaCl, 1 mM DTT, 2 mM spermidine) supplemented with 19 mM MgCl2, 5 mM each of ATP, UTP, GTP, CTP, and 0.5 mM SAM. Transcription output was analyzed by DNase I / Qubit quantification as described in Example 7. The capping efficiency of the reactions was assessed by RNase H cleavage followed by Klenow FAM-dCTP fill-in and urea-PAGE as indicated in Example 7. Selected reactions were further analyzed by intact mass spectrometry. As shown in Figure 19, for VCE, the m7Gppp formation efficiency peaked when the second reaction temperature was 45°C, whereas for H3C2, the m7Gppp formation efficiency peaked when the second reaction temperature was 50°C. Transcription output by T7 RNAP, on the other hand, peaked at 45°C when paired with VCE or H3C2, reflecting low transcription activity at the lower and upper temperature limits tested (28°C and 50°C, respectively). Thus, tandem temperature steps of 30 min at 37°C followed by 30 min at 45°C provided the optimal balance between transcription output and capping efficiency in this example. Modifications to reaction conditions, such as reaction time, reaction temperature, and number of temperature steps, may further improve transcription yield and the proportion of m7G-capped transcripts.

Claims

1. 1. A method for capping RNA in vitro, comprising: (i) an RNA sample containing uncapped target RNA; (ii) an RNA capping enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 7, or 20; (iii) guanosine triphosphate (GTP) or modified GTP (iv) a buffering agent; and (v) Methyl group donor at a temperature of 40°C to 60°C to form capped target RNA. A method comprising:

2. 1. A method for efficiently capping RNA in vitro, comprising: (i) an RNA sample containing uncapped target RNA; (ii) a single-stranded RNA capping enzyme having RNA triphosphatase (TPase) activity, guanylyltransferase (GTase) activity, and guanine-N7 methyltransferase (N7 MTase) activity; (iii) guanosine triphosphate (GTP) or modified GTP (iv) a buffering agent; and (v) Methyl group donor at a temperature of 37°C to 60°C to form capped target RNA. A method comprising:

3. 3. The method of claim 1 or 2, wherein the uncapped target RNA is at least 200 nt in length.

4. 4. The method of claim 1, wherein the contacting step further comprises raising or lowering the temperature to a second temperature between 37°C and 60°C, the second temperature being different from the first temperature.

5. A single-stranded RNA capping enzyme (a) an amino acid sequence that is at least 90% identical to (x) SEQ ID NO:2, (y) SEQ ID NO:3, and / or (z) SEQ ID NO:4; or (b) an amino acid sequence that is at least 90% identical to (x) SEQ ID NO: 5 and / or (y) SEQ ID NO: 6 The method of claim 2 , comprising:

6. 3. The method of claim 2, wherein the single-stranded RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, (d) SEQ ID NO:10, (e) SEQ ID NO:11, or (f) SEQ ID NO:

12.

7. 7. The method of any of claims 1-6, wherein (i), (ii), (iii), and (iv) are RNase-free, and the contacting step optionally further comprises (v) contacting with one or more RNase inhibitors.

8. The method of any one of claims 1 to 7, further comprising the step of synthesizing uncapped RNA using solid phase oligonucleotide synthesis chemistry.

9. 9. The method of any one of claims 1 to 8, further comprising synthesizing uncapped RNA by contacting a DNA template encoding the uncapped RNA with a polymerase to produce the uncapped RNA.

10. The method of any one of claims 1 to 9, wherein the methyl group donor is S-adenosylmethionine, and the contacting step further comprises (vi) contacting with a cap 2'O methyltransferase enzyme.

11. The method of any one of claims 1 to 10, wherein the uncapped target RNA comprises one or more pseudouridines.

12. 12. The method of any of claims 1 to 11, wherein the contacting step further comprises contacting (i), (ii), (iii), (iv) and optionally (v) at a single location.

13. (i) uncapped target RNA; (ii) a single-stranded RNA capping enzyme having RNA triphosphatase (TPase) activity, guanylyltransferase (GTase) activity, and guanine-N7 methyltransferase (N7 MTase) activity; (iii) guanosine triphosphate (GTP); (iv) a buffering agent; and (v) Methyl group donor A composition comprising:

14. The composition of claim 13 having a temperature in the range of 37°C to 60°C.

15. 13. The composition of claim 12, which is RNase-free and optionally comprises (v) one or more RNase inhibitors.

16. A single-stranded RNA capping enzyme (a) an amino acid sequence that is at least 90% identical to (x) SEQ ID NO:2, (y) SEQ ID NO:3, and / or (z) SEQ ID NO:4; or (b) an amino acid sequence that is at least 90% identical to (x) SEQ ID NO: 5 and / or (y) SEQ ID NO: 6 The composition according to any one of claims 13 to 15, comprising:

17. 17. The composition of any of claims 13 to 16, wherein the single-stranded RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, (d) SEQ ID NO:10, (e) SEQ ID NO:11, or (f) SEQ ID NO:

12.

18. The composition of any one of claims 13 to 17, further comprising a DNA template, a bacteriophage polymerase, and ribonucleotide triphosphates for transcribing RNA.

19. 19. The composition of any of claims 13 to 18, optionally comprising (vi) S-adenosylmethionine (SAM) and (vii) a cap 2'O methyltransferase enzyme.

20. 20. The composition of any one of claims 13 to 19, wherein the uncapped target RNA comprises one or more pseudouridines.

21. a single-stranded RNA capping enzyme having RNA triphosphatase (TPase) activity, guanylyltransferase (GTase) activity, and guanine-N7 methyltransferase (N7 MTase) activity, present in a storage buffer; and Reaction Buffer Kit including:

22. 21. The kit of claim 20, further comprising a bacteriophage polymerase and ribonucleotides for transcribing a template polynucleotide encoding a target RNA.

23. 23. The kit of claim 21 or 22, further comprising S-adenosylmethionine (SAM), a cap 2'O methyltransferase enzyme (2'OMTase), or both SAM and 2'OMTase.

24. A single-stranded RNA capping enzyme (a) an amino acid sequence that is at least 90% identical to (x) SEQ ID NO:2, (y) SEQ ID NO:3, and / or (z) SEQ ID NO:4; or (b) an amino acid sequence that is at least 90% identical to (x) SEQ ID NO: 5 and / or (y) SEQ ID NO: 6 The kit according to any one of claims 20 to 23, comprising:

25. The kit of any of claims 20 to 23, wherein the single-stranded RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, (d) SEQ ID NO:10, (e) SEQ ID NO:11, or (f) SEQ ID NO:

12.

26. An RNA capping enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:

20.

27. An RNA capping enzyme fusion comprising (a) an amino acid sequence that is at least 90% identical to SEQ ID NO:7 and (b) an amino acid sequence that is at least 90% identical to positions 1419 to 1587 of SEQ ID NO:20.

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