Enzymatic RNA capping method

Abstract

A method for efficiently capping RNA in vitro is provided. In some embodiments, the capping reaction can be performed at high temperatures using a Vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reaction can include a capping enzyme from a large amoebic virus, such as Faustovirus, Mimivirus, or Moumouvirus, or variants thereof. Compositions and kits for carrying out the method are also provided.

Description

Background Art

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

[0002] The addition of a cap to non-capped synthetic RNA is important for efficient protein expression in many eukaryotic cells. Furthermore, uncapped RNA (at least RNA having 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, in many therapeutic applications, the addition of a cap to synthetic RNA is highly desired (e.g., in addition to protein replacement therapy, prophylactic vaccination or therapeutic vaccination).

[0003] Currently, two methods are used to cap RNA. In the first method, synthetic RNA (e.g., RNA transcribed in vitro) is converted to capped RNA using an RNA capping enzyme. In another method (generally referred to as "co-transcriptional capping"), a cap analog such as an 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 co-transcriptionally incorporated into the RNA molecule during in vitro transcription.

[0004] Compared to co-transcription capping, enzymatic RNA capping can achieve a higher yield of capped RNA. However, enzymatic RNA capping is inefficient, requiring the use of large amounts of enzymes, or necessitating the purification of capped RNA from uncapped RNA. Furthermore, the efficiency of enzymatic RNA capping (expressed as the percentage of capped RNA after completion of the capping reaction) can vary depending on the RNA sequence, and the differences are generally attributed to the RNA structure (see, e.g., 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 [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, a more efficient method for adding caps to synthetic RNA is still needed. [Means for solving the problem]

[0007] (Summary of the invention) This disclosure provides, in particular, a method for efficiently capping RNA in vitro. In some embodiments, the method may include the step of contacting (i) an RNA sample containing an uncapped target RNA, (ii) an RNA capping enzyme containing an amino acid sequence that is at least 90% identical (e.g., at least 95% identical) to SEQ ID NOs: 1, 7, or 20, (iii) guanosine triphosphate (GTP) or modified GTP, (iv) a buffer, and optionally (v) a methyl group donor at a temperature in the range of 40°C to 60°C to form a capped target (e.g., efficiently form it). The efficiency, determined by the yield of capped RNA (50%) / enzyme concentration (nM), can be at least two-fold or at least three-fold improved compared to the capping efficiency of the enzyme at 37°C. The uncapped target RNA may optionally be free of modified nucleotides or may contain one or more modified nucleotides (e.g., pseudouridine).

[0008] In some embodiments, the method may include the step of contacting (i) an RNA sample containing uncapped target RNA, (ii) a single-strand 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 in the range of 37°C to 60°C to form (e.g., efficiently form) capped target RNA. Single-chain RNA capping enzymes (e.g., RNA capping enzymes derived from giant viruses such as Faustovirus, Mimivirus, or Moumouvirus) may contain an amino acid sequence that is at least 90% identical (e.g., at least 95% identical) to (a)(x)SEQ ID NO: 2, (y)SEQ ID NO: 3, and / or (z)SEQ ID NO: 4; or (b)(x)SEQ ID NO: 5, and / or (y)SEQ ID NO: 6. For example, RNA capping enzymes from giant viruses such as Faustovirus, Mimivirus, or Moumouvirus may contain an amino acid sequence that is 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. RNA capping enzymes derived from the genus Faustovirus (an example of a single-chain RNA capping enzyme) may contain amino acid sequences that are 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 therapeutic vaccine. Target RNA having a secondary structure, including therapeutic RNA, can be more efficiently capped using the method of this disclosure. Efficiency may be defined as the yield of capped RNA (50%) / enzyme concentration (nM). In any embodiment, the method may include a step of contacting at a first temperature (e.g., 37°C to 60°C) and then increasing or decreasing the temperature (e.g., from 37°C to 60°C), where the second temperature is different from the first temperature. For example, the method may include a step of contacting at a first temperature of 37°C for 1 to 120 minutes and then increasing the temperature to 45°C or 50°C for 1 to 120 minutes. In some embodiments, the method may include a step of increasing or decreasing the temperature to a third temperature of 37°C to 60°C for 1 to 120 minutes. In some embodiments, the capping method can generate more than 70% of Cap0 RNA in vitro within one hour (for example, by co-transcription).

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

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

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

[0013] In any embodiment, the contact step may include bringing (i), (ii), (iii), (iv), and (v) into contact in a single location, for example, within a single microfluidic surface, within a single reaction test tube, or within another reaction vessel.

[0014] This specification also provides compositions comprising uncapped target RNA, a single-strand 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 range of 37°C to 60°C. In some embodiments, the composition may be RNase-free and may optionally contain one or more RNase inhibitors. In some embodiments, the single-strand RNA capping enzyme may comprise (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 single-stranded RNA capping enzyme may contain an amino acid sequence that is at least 90% identical to the RNA capping enzymes 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). In some embodiments, the composition may further include a DNA template, a polymerase (e.g., bacteriophage polymerase), and ribonucleotides for transcribing RNA. In some embodiments, the composition may further include SAM and / or cap 2'OMTase. In some embodiments, an 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 may encode a polypeptide such as a therapeutic protein or therapeutic vaccine.

[0015] Kits are also provided. In some embodiments, the kit may include a single-strand RNA capping enzyme having TP-ase activity, GT-ase activity and N7 MT-ase activity, present in a storage buffer; and a concentrated reaction buffer. In some embodiments, the kit may further include a DNA template, polymerase and ribonucleotide for transcribing RNA. In some embodiments, the kit may further include SAM and / or cap 2'OMT-ase. Examples of methods, compositions and kits utilizing methyltransferase may also include SAM in the reaction mix. The single-strand RNA capping enzyme may include an amino acid sequence that is at least 90% identical to (a)(x)SEQ ID NO: 2, (y)SEQ ID NO: 3 and / or (z)SEQ ID NO: 4; or (b)(x)SEQ ID NO: 5 and / or (y)SEQ ID NO: 6. Single-chain RNA capping enzymes may contain amino acid sequences that are at least 90% identical to those of RNA capping enzymes 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 include (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 1419-1587 of SEQ ID NO: 20. [Brief explanation of the drawing]

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

Chemical formula

Chemical formula

[0018] The aspects of this disclosure may be further understood in light of the embodiments, section headings, drawings, descriptions, and examples, but none of these should be understood to limit the entire scope of this disclosure in any way. Accordingly, the claims expressed below should be understood with the scope and spirit of this disclosure in mind.

[0019] Each of the individual embodiments described and illustrated herein has its own distinct components and features, which may be readily separated from, or readily combined with, any of the features of some other embodiments, without departing from the scope or spirit of this teaching. Any of the listed methods may be performed in the order of the listed events, or in any other logically possible order. The disclosed reaction conditions, including but not limited to reaction temperature, reaction duration, and the concentrations of reactants (e.g., enzymes, substrates) and / or reactants, may vary.

[0020] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains. Furthermore, certain terms used herein are defined in light of embodiments of this disclosure and for the purpose of clarity and ease of reference.

[0021] The sources for commonly understood terms and symbols may include standard treatises and textbooks such as Kornberg and Baker, "DNA Replication," 2nd edition (WHFreeman, New York, 1992); Lehninger, "Biochemistry," 2nd edition (Worth Publishers, New York, 1975); Strachan and Read, "Human Molecular Genetics," 2nd edition (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 edition, John Wiley and Sons, New York (1994); and Hale and Markham, "Harper Collins Dictionary of Biology," Harper Perennial, NY (1991).

[0022] Unless otherwise explicitly indicated by context, the singular forms “one (a)” and “one (an)” as used herein and in the accompanying claims should be noted to include multiple referents. Therefore, for example, the term “one protein” refers to one or more proteins, i.e., a single protein and multiple proteins. Furthermore, it should be noted that the claims may be drafted to exclude optional elements. Thus, this statement is intended to be used as an antecedent for the use of exclusionary terminology, such as “simply” or “only,” or for “negative” limitation, relating to the enumeration of claim elements.

[0023] A numerical range includes the number that defines the range. Every number encompasses the midpoint of an integer greater than or equal to the integer and the midpoint of an integer less than or equal to the integer; that is, the number 2 is understood to encompass the range from 1.5 to 2.5, and so on. The number 2.5 encompasses the range from 2.45 to 2.55. Unless otherwise specified, when numerical values ​​for a sample are presented, each number alone may represent the midpoint of a range of values, and together may represent the endpoint of a range.

[0024] In the context of this disclosure, “buffer” means an agent that, when an acid or alkali is added to the solution, enables the solution to resist changes in pH. Examples of suitable non-naturally occurring buffers that may be used in the compositions, kits, and methods of the present invention include, for example, Tris, HEPES, TAPS, MOPS, Tricin, or MES.

[0025] In the context of this disclosure, “capping” refers to the enzymatic addition of the Nppp- portion to the 5' end of the RNA, where N is a nucleotide such as G or modified G. Modified G may have a methyl group at the N7 position of the guanine ring, or a label may be added at the 2 or 3 position of the ribose, in which case the label may be an oligonucleotide, a detection label such as a fluorophore, or a capture portion such as biotin or desthiobiotin, in which case the label may be optionally linked to the ribose of the nucleotide, for example, by a linker. See, for example, WO2015 / 085142. Depending on which enzyme is present in the capping reaction and / or whether SAM is present, 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).

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

[0027] In the context of this disclosure, “fusion” means two or more polypeptides, subunits, or proteins that are covalently linked to each other (for example, by peptide bonds). For example, a protein fusion may refer to a non-spontaneous polypeptide containing the protein of interest, covalently linked to a reporter protein. Alternatively, a fusion may include a combination of non-spontaneous polypeptide chains containing two proteins or two protein domains that are directly linked to each other by peptide bonds or linked 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 a DNA-directed RNA polymerase (typically bacteriophage polymerase) to produce a product containing RNA molecules copied from the template.

[0029] In the context of this disclosure, “Faustovirus RNA capping enzyme” refers to a single-strand RNA capping enzyme capable of capping RNA (e.g., having detectable TP-ase activity, GT-ase activity, and N7 MT-ase activity), which includes an enzyme having at least 90% identity to, for example, 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 an RNA capping enzyme derived from Faustovirus D5b (SEQ ID NO: 7). Unless otherwise expressly stated, Faustovirus RNA capping enzymes for the purposes of the examples and illustrations disclosed herein, having similar properties, effects, and / or benefits, may be interchangeable with each other.

[0030] In the context of this disclosure, “H3C2 fusion” refers to a fusion of a bifunctional enzyme capable of synthesizing RNA from a template polynucleotide and capping RNA, comprising an RNA polymerase (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase) and a Faustovirus RNA capping enzyme positioned at the N-terminus or C-terminus of the polymerase. The H3C2 fusion may further include a leader and / or linker (e.g., between the polymerase and H3C2). The H3C2 fusion may have 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 with, for example, SEQ ID NO: 20. The H3C2:T7 RNA polymerase fusion is an example of an H3C2 fusion.

[0031] In the context of this disclosure, “H3C2 variant” means, in each case, an enzyme capable of capping H3C2, H3C2 fusions, and RNA, which comprises 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 NOs. 7, 8, 9, 10, 11, and / or 12.

[0032] In the context of this disclosure, “modified nucleotides” (including references to modified NTPs, modified ATPs, modified GTPs, modified CTPs, and modified UTPs) means any non-canonical nucleoside, nucleotide, or their corresponding phosphorylated forms. Modified nucleotides may include one or more skeletal modifications or base modifications. Examples of modified nucleotides include dl, dU, 8-oxo-dG, dX, and THF. Further examples of modified nucleotides include those disclosed in U.S. Patent Publications US20170056528A1, US20160038612A1, US2015 / 0167017A1, and US20200040026A1. Modified nucleotides may include spontaneously occurring nucleotides or non-spontaneously occurring nucleotides.

[0033] In the context of this disclosure, “non-spontaneous” means polynucleotides, polypeptides, carbohydrates, lipids, or compositions that do not exist in nature. Such polynucleotides, polypeptides, carbohydrates, lipids, or compositions may differ in one or more respects from naturally occurring polynucleotides, polypeptides, carbohydrates, lipids, or compositions. For example, polymers (e.g., polynucleotides, polypeptides, or carbohydrates) may differ in the type and sequence of their constituent elements (e.g., nucleotide sequences, amino acid sequences, or sugar molecules). Polymers may differ from naturally occurring polymers in light of the molecule(s) to which they are linked. For example, “non-spontaneous” proteins may differ from naturally occurring proteins in their secondary, tertiary, or quaternary structure by having chemical bonds (e.g., peptide bonds, phosphate bonds, disulfide bonds, ester bonds, ether bonds, and other covalent bonds) to polypeptides (e.g., fusion proteins), lipids, carbohydrates, or any other molecule. Similarly, “non-naturally occurring” polynucleotides or nucleic acids may contain one or more other modifications (e.g., labeling or addition of other parts) (e.g., methylation) at the 5' end, 3' end, and / or between the 5' and 3' ends of the nucleic acid. A “non-naturally occurring” composition may differ from a naturally occurring composition in one or more of the following ways: (a) having components not combined in nature; (b) having components at concentrations not found in nature; (c) excluding one or more components that, unless otherwise specified, would be found in a naturally occurring composition; (d) having a form not found in nature, e.g., a dried form, a freeze-dried form, a crystalline form, an aqueous form; and (e) having one or more additional components beyond those found in nature (e.g., buffers, detergents, dyes, solvents, or preservatives). All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference.

[0034] In the context of this disclosure, “RNA sample” or “sample” means 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 an RNA sample may be a composition that is assessed for the presence of target RNA. An RNA sample may include naturally occurring target RNA (e.g., target RNA extracted from cells, tissues or organisms), target RNA produced by in vitro transcription, and / or chemically synthesized RNA.

[0035] In the context of this disclosure, “single-chain RNA capping enzyme” refers to a capping enzyme in which a single polypeptide chain contains TP-ase activity, GT-ase activity, and N7-MT-ase activity. Faustovirus, Mimivirus, and Moumouvirus capping enzymes are examples of single-chain RNA capping enzymes. H3C2 fusions are further examples of single-chain RNA capping enzymes. VCE is a heterodimer and, as such, is not a single-chain RNA capping enzyme.

[0036] In the context of this disclosure, “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 certain amounts of RNA products in such mixtures may be cleaved. A single uncapped target RNA species may, in some cases, 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 contain not only 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 this disclosure, “target RNA” means the polyribonucleotide of interest. The polyribonucleotide may be, or may include, the therapeutic RNA or its precursor (e.g., the uncapped precursor of capped therapeutic RNA). The target RNA may arise from intracellular transcription or from in vitro transcription. The target RNA may be present in a mixture, e.g., in an in vitro transcription reaction mixture, in cells, or in cell lysates. The target RNA may be uncapped. Where desired or required, the target RNA may be contacted with a decapping enzyme, for example, as a co-treatment with capping, or as a pre-treatment before capping.

[0038] In the context of this disclosure, “uncapped” means (a) RNA without a cap and (b) RNA that can be used as a substrate for a capping enzyme. Uncapped RNA typically has a triphosphorylated 5' end or a diphosphorylated 5' end. RNA transcribed in vitro has a triphosphate group at its 5' end.

[0039] In the context of this disclosure, “variant” means a protein having an amino acid sequence that differs from the naturally occurring amino acid sequence (i.e., having 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] This specification provides various in vitro RNA capping methods. 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 RNA with secondary structures (see Figure 2 and Tables 1 and 2 below). For example, when used to cap protein-coding in vitro transcription RNA, the same amount of capping product can be produced at 45°C using less than 1 / 30th the amount of enzyme compared to 37°C (see Table 6). In addition, incubation at 45°C allows VCE to efficiently cap uncapped RNA oligonucleotides with secondary structures at the 5' end (e.g., the 5' end which is expected to base pair with another sequence in the molecule, resulting in a blunt end or a 3' or 5' overhang of one or two nucleotides, as illustrated in Figure 1). Such RNA is extremely inefficient to cap using VCE at 37°C (see Figure 9). Therefore, in some embodiments, the method may include a step of incubating a reaction mix containing RNA, VCE or its variants, GTP or modified GTP, SAM, and a buffer at a temperature in the range of 42°C to 47°C, for example, 44°C to 46°C, to efficiently attach the cap structure to the uncapped target RNA. These embodiments of the method are particularly useful for RNA that has or is expected to have a secondary structure, such as RNA longer than 200 nt and RNA oligonucleotides that have a secondary structure at the 5' end, which are transcribed in vitro.

[0041] Other embodiments of the method are based in part on the discovery that an RNA capping enzyme derived from Faustovirus D5b (SEQ ID NO: 7; an example of an H3C2 capping enzyme, hereafter referred to as "H3C2") can cap RNA significantly more efficiently than VCE at almost all test temperatures (see Figure 2 and Tables 1 and 6 below), and, like VCE, can be used to efficiently attach caps to in vitro transcribed RNA at 45°C. For example, at 45°C, the same amount of capping product can be produced using less than 1 / 30th the amount of Faustovirus D5b (H3C2) compared to 37°C (see Table 6). In addition, incubating the reaction at 45°C allows Faustovirus D5b (H3C2) to efficiently attach caps to uncapped RNA oligonucleotides that have a secondary structure at the 5' end. Such RNAs are extremely inefficient to cap using Faustovirus D5b(H3C2) at 37°C (see Figure 9). Capping enzymes from other giant amoeba viruses, including Faustovirus ST1, Faustovirus LC9, Faustovirus E12, Mimivirus, and Moumouvirus, were also investigated and found to be active at high temperatures. Thus, in some embodiments, the method may include the step of incubating a reaction mix containing an RNA-containing sample, a single-chain capping enzyme, GTP or modified GTP, and a buffer at a temperature in the range of 37°C to 60°C, for example, 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 an uncapped target RNA in vitro.These embodiments of the method are particularly useful for RNAs that have or are expected to have a secondary structure, such as RNAs longer than 200 nt and RNA oligonucleotides that have a secondary structure at their 5' end, which are transcribed in vitro.

[0042] More efficient capping of RNA substrates can be achieved by reducing the amount of enzyme added to the capping reaction product, generating more capping RNA (as a percentage of RNA in the reaction product) using the same amount of enzyme, terminating the reaction earlier, and / or more efficiently capping RNA that has a secondary structure at its 5' end.

[0043] Without limitation, the disclosed reaction conditions, including reaction temperature, reaction duration, concentration of reactants (e.g., SAM, inorganic pyrophosphatase, NTP, transcript template), and enzymes (e.g., capping enzymes, polymerases, and their fusions), may vary. 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. In addition, the incorporation of cap 2'O methyltransferases, such as vaccinia virus cap 2'O methyltransferase, into the reactants can produce Cap-1 transcripts with high efficiency. Depending on which enzyme is used and other components in the reaction mix, the disclosed method may be used to produce RNA having a Gppp cap, RNA having a 7-methylguanylate cap (i.e., m7Gppp cap or "cap 0"), or RNA having an m7Gppp cap with additional modifications within the first and / or second nucleotides 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, RNA having a Gppp cap may be produced. If the reaction mix contains SAM in addition to the capping enzyme, cap 0 RNA may be produced. If the reaction mix contains other enzymes in addition to SAM, such as cap 2'OMTase, cap 1 RNA and / or cap 2 RNA may be produced. The reaction mix may contain other components in addition to those explicitly described above.

[0044] In some embodiments, the uncapped RNA in the reaction mix may be prepared by a solid-phase oligonucleotide synthesis chemical reaction (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, for example, from 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., T7 promoter, T3 promoter, or SP6 promoter) upstream of the region to be transcribed is copied by a DNA-directed RNA polymerase (typically bacteriophage polymerase) to produce a product containing RNA molecules copied from the template. In any embodiment, the RNA sample to be capped in this method contains a single RNA molecule (synthetic oligonucleotide or transcript). In addition, the reaction mix may be RNase-free and may optionally contain one or more RNase inhibitors. The RNA in the sample may contain non-natural nucleotide sequences, and in some embodiments, may contain non-spontaneously occurring nucleotides. In some embodiments, the in vitro transcription reaction may utilize thermostable variants of T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase (see, e.g., PCT / US2017 / 013179 and U.S. Patent Application No. 15 / 594,090). In these embodiments, the RNA may be transcribed at temperatures above 44°C (e.g., at least 45°C, at least 50°C, at least 55°C, or at least 60°C, and about 70°C or 75°C or lower) to reduce the immunogenicity of the RNA (see, e.g., WO2018 / 236617). In some cases, uncapped RNA can be capped immediately after its creation by, for example, adding RNA capping enzymes and GTP / modified GTP to the in vitro transcript after the reaction has progressed.In some embodiments, the RNA produced in the in vitro transcription reaction may be purified before 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 pharmaceutically acceptable excipients to prepare a formulation, where “pharmaceutically acceptable excipient” means any solvent that is suitable for administration to a living mammal via transdermal, oral, intravenous, or other means of administration used in the art. Examples of pharmaceutically acceptable excipients include, for example, those described in US2017 / 0119740. The formulation may be administered in vivo to subjects, for example, humans or any non-human animals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horses, or primates). Depending on the target, RNA (modified or unmodified RNA) may be introduced by direct injection into cells, or indirectly through a surrounding medium. Administration may be carried out by standardized methods. RNA may be naked RNA, or it may be formulated into a form suitable for administration to the target, e.g., humans. Formulations may include liquid formulations (solutions, suspensions, dispersions), topical formulations (gels, ointments, droplets, creams), and liposomal formulations (such as those described in US9,629,804B2; US2012 / 0251618Al; 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). Therefore, the RNA product may be an RNA product that is transfected into cells. The cells into which the RNA product is introduced may be in vivo (cells that are part of a mammal). Therefore, introduction can be achieved in vivo by administering the RNA product to the target. The cells into which the RNA product is introduced can exist ex vivo (tissues extracted from mammals, e.g., cells that are part of soft tissue or tissues isolated from mammalian blood).

[0046] For therapeutic applications, the synthesis of large quantities of uniformly capped mRNA transcripts may be desired or required in a cost-effective and rational manner that is scalable to support the synthesis of several grams. Current methods for mRNA production are expensive because they use mRNA capping analogs in the in vitro transcription reaction. Alternatively, separate reactants are required to produce 5'-triphosphate RNA by enzymatic mRNA capping (a more complex process that is more difficult to scale up) following in vitro transcription. A one-step in vitro synthesis of capped RNA using T7 RNA polymerase and a capping enzyme could be a rational manufacturing process. A single-container reaction using both enzymes can reduce or eliminate the otherwise exorbitant cost of synthetic mRNA capping analogs and alleviate the complexity of scaling up this workflow to support the synthesis of several grams (and more).

[0047] method This specification provides a method for efficiently generating capped RNA. In some embodiments, the method comprises a thermally active Hi-T7 RNA polymerase and an H3C2 RNA capping enzyme, and unexpectedly, a thermally active vaccinia virus (Vaccinia) mRNA cap 2'O methyltransferase. Surprisingly, reaction temperatures substantially higher than 37°C enable the synthesis of capped mRNA with a single reactant. In some embodiments, the RNA transcription and capping operations can be carried out in a single reactant at high temperatures (e.g., 40°C to 60°C). For example, RNA polymerases and individual capping enzymes, with or without vaccinia virus (Vaccinia) cap 2'O methyltransferase, can be used simultaneously in the capped RNA synthesis reaction. The combination of RNA transcription and capping operations can be achieved, for example, by a fusion protein containing H3C2, a single-subunit RNA capping enzyme, and a T7 RNA polymerase or Hi-T7 RNA polymerase. The methods described herein can achieve high levels of Cap-0 RNA or Cap-1 RNA synthesis.

[0048] In some embodiments, the capping method may optionally include the step of contacting an RNA polymerase (e.g., T7 RNA polymerase, thermally active Hi-T7 RNA polymerase and / or H3C2 fusion), a polynucleotide encoding a target RNA (e.g., DNA or RNA), a capping enzyme (e.g., VCE, H3C2, H3C2 fusion), an NTP, and a buffer in the presence or absence of SAM. The nucleoside triphosphate (NTP) 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, ml pseudouridine triphosphate), NTPs containing 2'O methylation, and / or combinations thereof. In some embodiments, the capping method may include the step of contacting a capping enzyme (e.g., VCE, H3C2, H3C2 fusion), target RNA, and guanosine triphosphate (GTP) and / or modified GTP in or out of the presence of SAM.

[0049] In some embodiments, the contacting step may be carried out in a single location (e.g., in a single step), for example, on any surface (e.g., a plate or beads), or in any container (e.g., a test tube, flask, vial, column, container, bioreactor, or other space). In some embodiments, the contacting step may include bringing some or all of the target elements into contact in any desired order or synchronously. In some embodiments, the contacting step may include bringing some or all of the target elements into contact in the presence of a buffer. For example, the contacting step may include bringing a capping enzyme (e.g., VCE, H3C2, H3C2 fusion), target RNA, GTP, and a buffer in any order or synchronously, with or without the presence of SAM. In some embodiments, the contact 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 contact step may further include contacting at a first temperature of 25°C to 60°C and then cooling or heating to a second temperature of 25°C to 60°C that is different from the first temperature, in which case at least one of the first or second temperature is 40°C or higher. A first temperature may be selected to generate, stabilize, and / or retain a desired amount of uncapped target RNA, and a second temperature may be selected to generate, stabilize, and / or retain a desired amount of capped target RNA. Each temperature may be maintained for any desired time (e.g., 1 minute to 120 minutes).For example, the first temperature can be 37°C over 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 over a second period of 1 to 30 minutes. In any of the disclosed embodiments, the contact step may include bringing one object into direct contact with another object, and bringing the two objects together in close proximity so that they may interact physically and / or chemically, with or without further shaking or mixing, on a surface or in a container. The contact step may include an initial action of bringing one object into direct contact or close proximity with another object and / or maintaining conditions that allow or preferentially allow contact between the two objects.

[0050] In some embodiments, the method may include the steps of synthesizing RNA (e.g., template-directed RNA or undirected RNA) in vitro to produce uncapped target RNA and capping the target RNA (e.g., a single-step reaction). The step of synthesizing RNA from a template may include, for example, optionally in the presence or absence of SAM, contacting RNA polymerase (e.g., T7 RNA polymerase, thermally active Hi-T7 RNA polymerase and / or H3C2 fusion) with a template (e.g., RNA template or DNA template), one or more NTPs and a buffer to produce uncapped target RNA. 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, ml pseudouridine triphosphate), NTPs containing 2'O methylation and / or combinations thereof. The step of capping the target RNA may 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 capped target RNA. In some embodiments, the capping method may include contacting the target RNA with an H3C2 fusion at a temperature of 25°C to 60°C. In some embodiments, the capping method may include contacting the target RNA with a decapping enzyme to remove any existing caps and / or to confirm that it is not capped.

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

[0052] composition Various compositions related to the disclosed method are also provided. In some embodiments, the composition (e.g., a reaction mix) may include target RNA (e.g., in an RNA sample) and / or a DNA template encoding the target RNA. In some embodiments, the composition may include a single-strand RNA capping enzyme having TP-ase activity, GT-ase activity and N7-MT-ase activity, GTP, SAM, and a buffer. The composition may have a temperature range of 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 may be RNase-free as a result of the RN-ase being inhibited, inactivated, or absent. For example, an RNase-free composition may include one or more RNase inhibitors. In some embodiments, the composition may further include a promoter-operably linked DNA template for transcribing RNA, a bacteriophage polymerase and NTP for inducing transcription at the promoter site. In some embodiments, the composition may include a cap 2'-OMT-ase. The single-chain RNA capping enzyme may include (a) an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, 3, or 4; or (b) an amino acid sequence that is at least 90% identical to SEQ ID NO: 5 or 6. Compositions according to certain embodiments include, 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 include a leader (e.g., operably positioned within the fusion) and / or a linker positioned between the H3C2 and the RNA polymerase. In certain embodiments, any or all of the components mentioned in this paragraph may be combined in a single container or incorporated in other forms. Components, e.g., components in a single container, may also exist in a dry form (e.g., anhydrous form and / or lyophilized form). A container containing one or more of the mentioned ingredients may also contain an aqueous medium.

[0053] kit This disclosure also provides a kit for carrying out the method described above. In some embodiments, the kit may contain one or more of the components listed above. For example, the kit may contain a single-strand RNA capping enzyme having TP-ase activity, GT-ase activity and N7 MT-ase activity, and the enzyme may be present in a storage buffer and a reaction buffer (e.g., a 5-fold or 10-fold concentrated reaction buffer). In some embodiments, the kit may include a bacteriophage polymerase and an NTP for transcribing RNA from a DNA template. In addition, the kit may include a SAM and a cap 2'OMT-ase. As will become clear from the above discussion, the enzyme may include an amino acid sequence that is at least 90% identical to (a) SEQ ID NO: 2, 3, or 4; or (b) SEQ ID NO: 5 or 6.

[0054] Depending on the preference, the various components of the kit may be present in individual containers, or components of a particular suitability may be pre-combined into a single container. In addition to the components described above, the kit may further include instructions for performing the method using the kit's components, i.e., instructions for capping RNA. [Examples]

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

[0056] [Example 1] RNA capping activity of H3C2 and VCE at high temperatures 10 nM purified VCE RNA capping enzyme or purified H3C2 RNA capping enzyme, along with 500 nM RNA1 (Figure 1) containing a fluorescent group (FAM) at its 3' end, was incubated for 30 minutes at a temperature ranging from 23°C to 65°C in 10 μL of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8.0) in the presence of 0.5 mM GTP and 0.1 mM SAM. The reaction was quenched by thermal inactivation at 70°C for 10 minutes, following the addition of 1 μL of 50 mM EDTA. The quenched reaction product was analyzed by capillary electrophoresis using an Applied Biosystems 3730xl Genetic Analyzer. In Figure 2, used to quantify the peak areas corresponding to m7Gppp-RNA1, unmethylated Gppp-RNA1, uncapped pp-RNA1, and uncapped ppp-RNA1 after a 30-minute capping reaction and to calculate the proportion of m7Gppp-RNA1, each bar represents the mean of four independent experiments. The error bar represents the standard deviation for the four replicates. At low enzyme concentrations (10 nM), both H3C2 and VCE exhibited the highest level of capping activity at 45°C, while H3C2 maintained 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 at multiple dilutions were incubated under the same reaction conditions at 37°C, 45°C, 50°C, or 55°C for 30 minutes, respectively. The calculated percentage of m7Gppp-RNA1 was graphed against the enzyme concentration and applied to the derivation formula from Hill's equation to determine the enzyme concentration at which 50% capping is achieved (Cap 50The following was derived. As shown in Figures 3A-3B and Table 1, the conversion of 50% of 0.5 μM ppp-RNA1 to m7Gppp-RNA1 at 37°C, 45°C, 50°C, or 55°C for 30 minutes requires VCEs of 3.06 nM, 0.94 nM, 4.08 nM, and 30.00 nM, respectively. Furthermore, at temperatures below 50°C, VCEs of 400 nM or more cap >90% of 0.5 μM RNA in 30 minutes. For H3C2, enzymes of 0.69 nM, 0.67 nM, 2.15 nM, or 21.3 nM can convert 50% of ppp-RNA1 to m7Gppp-RNA1 at 37°C, 45°C, 50°C, or 55°C for 30 minutes, respectively. In addition, 50 nM H3C2 effectively caps >90% of ppp-RNA1 at temperatures below 55°C 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 under in vitro conditions at temperatures below 60°C. Several putative orthologs from other Faustovirus strains were identified by sequence identity. 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. ORFs of the identified ortholog genes were synthesized and inserted into T7 expression vectors to place the expression of the target gene under the control of the T7 promoter. Following the manufacturer's recommendations, the T7 expression cassette was amplified by PCR using Q5® High Fidelity 2× Master Mix (New England Biolabs, Ipswich, MA). Following the manufacturer's instructions, the amplified T7 expression cassette was purified using the Monarch® PCR & DNA cleanup kit (New England Biolabs, Ipswich, MA). Following 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). The PURExpress reaction products (Figures 4A-4E) containing the gene product with the indicated Genbank accession number were used in the RNA capping assay. In short, the mixture consists of 2 μL of PURExpress product, 0.4 mM 150 nt in vitro transcript RNA, 4 μM GTP, 0.1 mM SAM, and trace amounts of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8.0), 1× RNA capping buffer, 2 μL of PURExpress product, 0.4 mM 150 nt in vitro transcript RNA, 4 μM GTP, 0.1 mM SAM, and trace amounts of 32A 50 μL RNA capping reaction mixture, composed of P-α-GTP, was assembled on ice. The reaction mixture was divided into five equal parts, and each was incubated at the indicated temperature for 30 minutes. The reaction was stopped by adding 10 μL of 2× RNA Loading Dye (New England Biolabs, Ipswich, MA) to the reaction mixture, and the results were analyzed by denatured polyacrylamide electrophoresis and autoradiography. Capping activity is indicated by a moving X-ray signal at the indicated substrate RNA position. As shown in Figures 4A-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 1-668 of Genbank accession number: AAV50651; Benarroch et al., 2008; lane 5), and Moumouvirus capping enzymes are also present. While the enzyme (Genbank accession number: YP_007354410; lane 6) caps 150-mer RNA at temperatures below 55°C, RNA capping enzymes derived 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 RNA at temperatures below 60°C.

[0060] [Example 3] Sequence analysis Sequence alignment analysis revealed that three regions, each 60 or 111 amino acids long, exhibited over 90% amino acid sequence identity among four Faustovirus RNA capping enzymes that were active 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 (Sequence ID 3). The amino acid sequence of Fausto_CR_05 (corresponding to amino acids 778-873 of the F13C2 sequence) is KYSIKRLYDSDKLTKTGQKIAVLLPMSGEMKEEPLCNIKNIISMARKMGLDLVESANFSV (Sequence ID 4).

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

[0062] [Table 2]

[0063] [Table 3]

[0064] Figure 6 shows the alignment of various Faustovirus RNA capping enzyme sequences, including the sequences of the TP-ase, GT-ase, and MT-ase regions, in addition to the conserved domain.

[0065] Sequence alignment analysis also revealed that two regions, 67 amino acid length and 111 amino acid length, exhibited more than 90% sequence identity between the Acanthanomeba polyphaga mimivirus and the Acanthanomeba polyphaga moumouvirus RNA capping enzyme, which are active below 55°C (Figure 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. moumou_CR_04 (corresponding to amino acids 672-782 of the Moumouvirus sequence) spans 111 amino acids at the N-terminus of the guanosine N7 methyltransferase domain. The amino acid sequence of moumou_CR_03 is INDNTVVEFIFDNFKIDMDDPYKWIPIRTRYDKTESVQKYHKKYGNNLHIANRIWKTITNPITEDII (Sequence ID 5). The amino acid sequence of moumou_CR_04 is YYQKNTSNAAGMRAFNNFIKSNMITTYCKDGDKVLDIGCGRGGDLIKFIHAGIEEYVGIDIDNNGLYVINDSAFNRYKNLKKTIKNIPPMTFINADARGLFNLEAQEKILP (Sequence ID 6).

[0066] The table below summarizes the sequence identity between the amino acid sequences of Mimivirus RNA capping enzymes and Moumouvirus RNA capping enzymes.

[0067] [Table 4]

[0068] [Table 5]

[0069] Figure 8 shows the alignment of Mimivirus and Moumouvirus sequences, including the sequences of the TP-ase, GT-ase, and MT-ase regions, in addition to the conserved domain.

[0070] [Example 4] RNA capping reaction at 45°C enhances capping efficiency in short-chain model hairpin RNA. To assess the capping efficiency, short RNAs exemplified 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 a stable hairpin structure with a theoretical minimum free energy (MFE) 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 were further designed to have their respective minimum free energy structures with blunt ends, one-nucleotide or two-nucleotide overhangs at their 5' ends (Figure 1). RNA was generated by in vitro transcription using T7 RNA polymerase. For the capping reaction, 50 nM H3C2 or VCE was incubated with 500 nM RNA substrates in 10 μL of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 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 minutes. At 37°C, H3C2 capped 100% of RNA1, which was expected to have an unstructured 5' end, and 22% of RNA4, which was expected to have a two-base overhang at the 5' end (Figure 9). Under these conditions, H3C2 was unable to cap RNA2 and RNA3. 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 RNA. A 1766nt RNA containing the firefly luciferase gene (flucfluc) was generated by in vitro transcription using T7 RNA polymerase. 400nM of 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 MgCl2, 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. Next, a targeting oligonucleotide (TO) designed to align the RNase and excise a 25nt 5' fragment, with a final concentration of 2.5 μM, was added to each capping reaction product. The reaction was then heated at 80°C for 30 seconds to inactivate the capping enzyme, and then slowly cooled to room temperature. The thermostable RNase H (New England Biolabs, Ipswich, MA) was added to each reactant at a final concentration of 0.5 U / μL, followed by incubation at 37°C for 1 hour. The RNase H reactants were then analyzed by electrophoresis through a 15% urea-polyacrylamide gel. Following the manufacturer's instructions, the gel was stained with SYBR® Gold (Thermo Fisher Scientific, Waltham, MA) and then scanned using an Amersham® Typhoon® RGB (GE Healthcare, Marlborough, MA) scanner with a Cy2 channel. The intensities of the capping and non-capping bands were normalized against the TO intensity in the same lane. Using the normalized values, the percentage of capped RNA under each condition was calculated. The calculated values ​​were graphed against enzyme concentration and applied to the derivation from Hill's equation to determine the enzyme concentration (Cap) at which 50% capping is achieved. 50 ) was derived. For H3C2, Cap 50 The values ​​were 33.6 nM and 0.96 nM at 37°C and 45°C, respectively. For VCE, Cap 50The values ​​were 103 nM and 2.97 nM at 37°C and 45°C, respectively (Figure 10 and Table 6 below). For both enzymes, a substantially small amount of enzyme was required to achieve 50% capping when the capping reaction was carried out at 45°C.

[0072] [Table 6]

[0073] As shown below, the activity of these enzymes can be expressed in milliliters per 1 μL of reactant:

[0074] [Table 7]

[0075] To demonstrate that temperature-dependent enhancement of capping efficiency can be achieved in other long-chain RNA molecules, the capping efficiency of H3C2 and VCE was studied by mass spectrometry readout using in vitro transcripts of Cypridina luciferase (clue; 1823nt) and cystic fibrosis transmembrane receptor (CFTR; 4712nt). Briefly, 0.5 μM of the clue 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 minutes. The reaction was stopped by heating at 80°C for 30 seconds in the presence of 2.5 μM of a targeting oligo consisting of a 5' deoxynucleotide, a 3' ribonucleotide, and a TEG-desthiobiotin group. The reaction mixture was cooled to 25°C at a rate of 0.1°C / second. The reaction mixture was then cleaved by thermally stable RNase H (New England Biolabs, Ipswich, MA) in an incubation at 37°C for 1 hour at a final concentration of 0.5 U / μL. The 5' fragment of the RNase H-cleaved reaction mixture was then purified using size selection with AMPure® XP Beads and target selection with streptavidin magnetic beads. Briefly, 100 μL of the RNase H-cleaved reaction mixture 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 pre-cleaned AMPure® XP beads derived from a 200 μL 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-purified beads derived from Dynabeads® MyOne Streptavidin C1.After four washes with 200 μL of washing buffer (5 mM Tris, pH 7.5, 0.5 mM EDTA, 1 M NaCl), the purified beads were incubated with 50 μL of biotin elution buffer (1 mM biotin, 5 mM Tris, pH 7.5, 0.1 M NaCl, 0.1 M NaCl) at 37°C for 1 hour to elute the bound RNA. The eluted RNA was then analyzed by LC / MS performed by Novatia LLC, an external contractor, to determine the relative mass of the target. The degree of RNA capping was evaluated by the mass intensity ratio of capped RNA molecules to uncapped RNA molecules.

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

[0077] [Example 6] Efficient one-pot enzymatic synthesis of Cap-1 structure on high-temperature triphosphate RNA. To verify whether vaccinia virus cap 2'OMTase is active at high temperatures, a reaction mixture containing 5 μM of chemically synthesized Cap-0 RNA1 (25 nts) and 200 μM of SAM in 20 μL of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8.0) was preheated at 37°C, 45°C, or 50°C for 1 minute before adding 100 U of vaccinia virus cap 2'OMTase (New England Biolabs, Ipswich, MA). The reaction was allowed to proceed at the preheating temperature for 30 minutes, and then stopped by heating at 70°C for 10 minutes. RNA was purified from the reaction components using the Oligo Clean-up and Concentration Kit (Norgen Biotek, Thorold, Canada). Next, the purified RNA was digested into nucleotides and cap structures by incubation at 37°C for 1 hour in 20 μL of reaction mixture containing nucleotide digestion mix buffer (50 mM sodium acetate, pH 5.4, 1 mM ZnCl2) along with 2 μL of nucleotide digestion mix (New England Biolabs, Ipswich, MA). The nucleotide digestion reaction products 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, more Cap-0 RNA was methylated at 45°C and 50°C than at 37°C.

[0078] To support the one-pot enzymatic synthesis of the Cap-1 structure on high-temperature 5' triphosphate RNA, chemically synthesized 5 μM 5' triphosphate RNA1 was incubated with 200 U of vaccinia virus (Vaccinia) cap 2'OMTase (final concentration 5 U / μL) in a 40 μL reaction mixture containing 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8.0) at 37°C or 45°C for 30 minutes in the presence of 50 nM H3C2 or VCE, 1 mM GTP, and 0.2 mM SAM. The reaction mixture was directly analyzed by LC / MS to determine its mass and relative mass. When paired with 50 nM VCE, 5 U / μL 2'OMTase produced only about 50% Cap-01 RNA at 37°C, while producing 100% Cap-01 RNA at 45°C (Figure 13). When paired with 50 nM H3C2, 5 U / mL vaccinia virus (Vaccinia) 2'OMTase produced about 90% Cap-01 RNA at 37°C and 100% Cap-01 RNA at 45°C (Figure 13). Therefore, performing the reaction at 45°C nearly doubled the Cap-01 RNA yield in the presence of VCE and increased the Cap-01 RNA yield 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 capping RNA synthesis using the vaccinia virus capping enzyme is inefficient. As recommended by the manufacturers for each reactant, we used 5 U / μl of T7 RNA polymerase (New England Biolabs; model number: M0251), Hi-T7 RNA polymerase (New England Biolabs; model number: M0658), 1 U / μl of vaccinia virus capping enzyme (New England Biolabs; model number: M2080), and 5 U / μl of vaccinia virus mRNA cap 2'O methyltransferase (New England Biolabs; model number: M0366), which are suitable for the transcription and capping of a 1.7 kb fluc transcript (SEQ ID NO: 18). The reaction mixture contained either 1×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 1×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. The reaction was supplemented with 19 mM MgCl2 and 5 mM each of ATP, UTP, GTP, and CTP, and 0.5 mM SAM. The reaction was carried out at 37°C for 1 hour, which is the recommended reaction temperature and duration for T7 RNA polymerase and vaccinia virus capping enzymes.

[0080] For each reactant, to evaluate the yield of in vitro transcription after a 1-hour incubation, 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 MgCl2, 0.5 mM CaCl2) and 0.5 U / μL DNase I (New England Biolabs), and incubated at 37°C for 30 minutes. Subsequently, the DNase I reactants were analyzed for total RNA concentration using the 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 reactants were analyzed using 2% E-Gel (Thermo Fisher) along with images acquired using a Typhoon RGB scanner (GE Healthcare). The results are shown as transcription outputs in Figure 14.

[0081] To analyze the degree of RNA capping, one-step capping RNA synthesis was examined by intact LC / MS analysis following cleavage with RNase H. Briefly, step-by-step capping RNA synthesis was stopped by heating at 80°C for 30 seconds in the presence of 2.5 μM of targeting oligo (TO-1), which consists of a 5' deoxyribonucleotide, a 3' ribonucleotide, and a TEG-desthiobiotin group (SEQ ID NO: 19). The reaction mixture was cooled to 25°C at a rate of 0.1°C / second. The reaction mixture was then subjected to RNase H cleavage by incubation at 37°C for 1 hour with thermally stable RNase H (New England Biolabs; model 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 cleavage fragments. Briefly, 5 μL of the RNase H reaction product 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 of a large DNA polymerase I (Klenow) fragment (New England Biolabs). The reaction product was incubated at 37°C for 1 hour. In some cases, a small fraction of the Klenow reaction product was analyzed on a urea PAGE to verify the success of capping. Briefly, 2 μL of the Klenow reaction product 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) with a Cy2 channel.

[0082] To analyze the 5' group state of the coupled transcription / capping reaction products using capillary electrophoresis or mass spectrometry, the Klenow reaction products (RNase H cleavage products as annealed to desthiobiotinylated targeting oligos) were purified by size selection using AMPure® XP Beads (Thermo Fisher), and then selected using streptavidin magnetic beads. Briefly, 45 μL of nuclease-free water was added to 5 μL of RNase H cleavage reaction product, 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 left to stand adjacent to a magnet at room temperature for 2 minutes. The clarified supernatant was collected and added to pre-purified NEBNext® Sample Purification Beads derived from 100 μL of bead suspension. Next, the beads were incubated at room temperature for 5 minutes, and then left to stand next to a magnet at room temperature for 2 minutes. The clarified supernatant was added to 50 μL of pre-cleaned beads derived from Dynabeads® MyOne Streptavidin C1 (Thermo Fisher). After four washes with 50 μL of low-fume concentration washing buffer (5 mM Tris, pH 7.5, 0.5 mM EDTA, 60 mM NaCl), the cleaned beads were incubated in 10 μL of nuclease-free water to elute the bound RNA. Next, the eluted RNA was 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 proprietary software. Peak areas corresponding to m7Gppp-RNase H cleavage transcripts, unmethylated Gppp-RNase H cleavage transcripts, uncapped pp-RNase H cleavage transcripts, and ppp-RNase H cleavage transcripts were quantified and used to calculate the proportion of capping RNA synthesis reactions in ppp transcripts, pp transcripts, Gppp transcripts, and m7Gppp transcripts.

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

[0084] Figure 14 shows the results of one-step transcription using currently available reaction conditions. Comparing bar 1 and bar 2, the presence of VCE did not significantly affect the transcription output of T7 RNA polymerase and did not result in detectable m7GpppG(Cap-0) transcripts. A small proportion (5.4%) of unmethylated G capping RNA, an undesirable capping reaction intermediate, was detected. Approximately 50% of the transcripts also contained a 5' diphosphate group, another capping reaction intermediate. As shown in bar 3, when vaccinia virus cap 2'O methyltransferase was incorporated into the reaction, the transcription output was not significantly affected, and m7GpppGm(Cap-1) transcripts were detected. Approximately 50% of the transcripts contained a 5' diphosphate group, and 15% contained unmethylated G capping intermediates. When Hi-T7 RNA polymerase was used instead of T7 RNA polymerase, similar results to the T7 RNA polymerase reaction were observed (the presence of VCE did not significantly affect Hi-T7 RNA polymerase transcription and did not yield detectable levels of Cap-0 transcript). Addition of vaccinia virus cap 2'O methyltransferase to the reactant did not yield Cap-1 transcript. These results suggest that the currently standard enzyme reagents and reaction conditions for in vitro mRNA synthesis (i.e., mRNA capping following RNA synthesis) do not support the generation of capping mRNA in a single reactant in vitro (i.e., one-step capping RNA synthesis).

[0085] H3C2, a B. Faustovirus RNA capping enzyme, efficiently caps transcripts in a one-step in vitro reaction. Since the reagents and conditions of Example 7A were ineffective in generating capped transcripts in a one-step in vitro reaction, new enzymes and reaction conditions were investigated for their ability to perform one-step in vitro capped RNA synthesis. 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 yielded 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 the transcripts had m7Gppp caps, while only 1.4% had unmethylated G caps. At both temperatures, the transcription output was 50-60% of that without H3C2 capping enzyme. Therefore, 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 extending the range of enzyme reagents and reaction temperatures were investigated by performing one-step capped RNA synthesis of Cap-0 at 45°C and 50°C using different combinations of T7 RNA polymerase, Hi-T7 RNA polymerase, VCE, H3C2, and vaccinia virus 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 together with 5 U / μL T7 RNA polymerase or Hi-T7 RNA polymerase in their respective reaction buffers, with or without 5 U / μL of vaccinia virus mRNA cap 2'O methyltransferase. The reaction was carried out for 1 hour at 45 or 50°C.

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

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

[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 an H3C2 RNA capping enzyme linked by a presumed flexible linker sequence and a T7 RNA polymerase (SEQ ID NO: 20). The fusion protein was encoded by a DNA sequence (SEQ ID NO: 21) expressed by E. coli under the control of either the T7 promoter or the tac promoter, and purified using standard chromatography.

[0093] A one-step capped RNA synthesis reaction for a 1.7kb fluc transcript was carried out in 1×T7 RNA polymerase buffer supplemented with 19mM MgCl2 and 5mM each of ATP, UTP, GTP, and CTP, and 0.1mM SAM, in the presence of 0.5mM H3C2:T7 RNA polymerase fusion, as indicated in Example 7. For comparison, reactions containing 5U / μL T7 RNA polymerase with or without 0.5mM H3C2 capping enzyme were performed. The reaction was run 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 the reaction was carried out at 37°C, the H3C2:T7 fusion protein yielded 75% m7Gppp capped (Cap-0) transcript and 20% unmethylated G-capped transcript (Bar 1). Under the same conditions, the individual enzymes, H3C2 alone and T7 RNA polymerase alone, yielded 12% Cap-0 transcript and 27% unmethylated G-capped transcript (Bar 2). The transcription output decreased from 0.45 mg / μL when only T7 RNA polymerase was present (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 transcript and 5% unmethylated G-capped transcript (Bar 4), compared to 60% Cap-0 transcript and 6% unmethylated G-capped transcript (Bar 5) when using the individual enzymes. Similar to the reaction performed at 37°C, the transcription output decreased from 0.31 mg / μL (Bar 6) when only T7 RNA polymerase 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 described reaction conditions, this embodiment supports efficient (e.g., with a high efficiency of 98%), in vitro one-step Cap-0 RNA synthesis using an H3C2:T7 RNA polymerase fusion protein.

[0096] [Example 9] The RNA capping enzyme efficiently caps in vitro transcripts containing pseudouridine. Approximately 1800 nt of RNA encoding Cypridina luciferase protein (cluc / A120), accompanied by 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 the unmodified transcript or the pseudouridine cluc / A120 transcript was incubated with 100 nM of H3C2 or VCE in 10 mL of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8) at 37°C for 30 minutes in the presence of 0.5 mM GTP and 0.1 mM SAM. Next, a targeting oligonucleotide (TO), designed to direct the RNase to cleave the 5' fragment, was added to each capping reaction product to achieve a final TO concentration of 2.5 mM. Then, each reaction product was heated at 80°C for 30 seconds to anneal the TO with the transcript and inactivate the capping enzyme, and then slowly cooled to room temperature. Thermally stable RNase H (New England Biolabs) was added to each reaction product 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%, respectively, of the cluc / A120 transcript containing pseudouridine using m7Gppp capping. In particular, all of the capping enzymes cap pseudouridine-containing cluc / A120 transcripts in a larger proportion 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 of cluc / A120 in vitro transcript was capped in 500 μL of a reaction mixture containing 1× RNA capping buffer (50 mM Tris-HCl, pH 8.0, 5 mM KCl, 1 mM MgCl2, 1 mM DTT), 0.5 mM GTP, 0.1 mM SAM, 10 U / μL of vaccinia virus cap 2'O methyltransferase (NEB M0366), and 500 nM H3C2, at 45°C for 1 hour. The efficiency of Cap-1 formation was evaluated 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 in 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 transcript using the Lipofectamine MessengerMax transfection reagent (ThermoFisher). Luciferase activity was assayed in 10 μL of culture medium supernatant 4 hours after transfection using the BioLux® Cypridina Luciferase Assay Kit (New England Biolabs Inc., Ipswich). Luminescence was measured using a Berthold Technologies Centro LB960 microplate illuminometer. As shown in Figure 17, the scale-up capping reaction achieved 47.7% and 54.8% Cap-1 formation on cluc / A120 containing rU and pseudouridine, respectively, which is consistent with the results in Example 5 and Figure 11.The results of the luciferase assay in the triple assay showed that the translation efficiency from purified capping transcripts containing rU and pseudouridine exhibited high luciferase activity (Figure 18). Therefore, modifications to the reaction conditions, such as reaction volume, enzyme and reaction component concentrations, reaction time, and reaction temperature, should be noted to improve the Cap-1 formation efficiency and the resulting Cap-1 RNA translation efficiency when the amount of RNA is large.

[0098] [Example 11] Single-container capping RNA synthesis with multiple temperature steps As indicated in Example 7, a single-container capping RNA synthesis reaction was carried out for a 1.7kb fluc transcript, i.e., 5U / µl of T7 RNA polymerase (New England Biolabs; model number: M0251) was incubated at 37°C for 30 minutes with or without 500nM of vaccinia virus capping enzyme (New England Biolabs; model number: M2080) or H3C2 capping enzyme, followed by further incubation at 28°C, 32°C, 37°C, 45°C, or 50°C for 30 minutes. The reaction product contained a 1×T7 RNA polymerase buffer (40 mM Tris-HCl, pH 7.9, 10 mM NaCl, 1 mM DTT, 2 mM spermidine) and was supplemented with 19 mM MgCl2 and 5 mM each of ATP, UTP, GTP, and CTP, and 0.5 mM SAM. The transcription output was analyzed by DNase I / Qubit quantification as described in Example 7. The capping efficiency of the reaction product was assessed by Klenow's FAM-dCTP fill-in and urea-PAGE following RNase H cleavage as indicated in Example 7. The selected reaction products 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, while for H3C2, the m7Gppp formation efficiency peaked when the second reaction temperature was 50°C. The transcriptional output induced by T7 RNAP, on the other hand, peaked at 45°C when paired with VCE or H3C2, reflecting low transcriptional activity at the lower and upper limits of the test temperature (28°C and 50°C, respectively). Therefore, the tandem temperature steps of 30 minutes at 37°C followed by 30 minutes at 45°C provided the optimal balance between transcriptional output and capping efficiency in this embodiment. Modifications to reaction conditions, such as reaction time, reaction temperature, and number of temperature steps, may further improve the transcription yield and the proportion of m7G capped transcripts.

Claims

1. (i) Polynucleotides; (ii) A single-chain RNA capping enzyme having RNA triphosphatase (TPase) activity, guanylyltransferase (GTase) activity, and guanine-N7 methyltransferase (N7 MTase) activity, and containing an amino acid sequence that is at least 90% identical to (a) SEQ ID NO: 8, (b) SEQ ID NO: 9, and / or (c) SEQ ID NO: 10; (iii) Guanosine triphosphate (GTP); (iv) buffering agent; and (v) Methyl group donor A composition for in vitro RNA capping, including [the specified element].

2. The composition according to claim 1, having a temperature in the range of 23°C to 60°C.

3. The composition according to claim 1, which does not contain an RNase and optionally contains (vi) one or more RNase inhibitors.

4. The composition according to claim 1, wherein the single-chain RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to (a) SEQ ID NO: 2, (b) SEQ ID NO: 3, and / or (c) SEQ ID NO:

4.

5. The composition according to claim 1, wherein the polynucleotide comprises a DNA template, and the composition further comprises a bacteriophage dipolymerase and ribonucleotide triphosphate for transcribing the DNA template to form an uncapped target RNA.

6. (vii) The composition according to claim 1, comprising S-adenosylmethionine (SAM) and (viiii) the cap 2'O methyltransferase enzyme.

7. The composition according to claim 1, wherein the polynucleotide comprises an uncapped target, and the uncapped target RNA comprises one or more pseudouridines and / or one or more m1-pseudolidines.

8. The composition according to claim 1, further comprising one or more detergents, dyes, solvents, and / or preservatives.

9. The composition according to claim 1, wherein the single-chain RNA capping enzyme comprises an amino acid sequence that is at least 95% identical to (a) SEQ ID NO: 8, (b) SEQ ID NO: 9, and / or (c) SEQ ID NO:

10.

10. A single-strand RNA capping enzyme present in the storage buffer having RNA triphosphatase (TPase) activity, guanylyltransferase (GTase) activity, and guanine-N7 methyltransferase (N7 MTase) activity, and having an amino acid sequence that is at least 90% identical to (a) SEQ ID NO: 8, (b) SEQ ID NO: 9, and / or (c) SEQ ID NO: 10; and Reaction buffer A kit for in vitro RNA capping, including [specific components / tools].

11. The kit according to claim 10, further comprising a bacteriophage dipolymerase and a ribonucleotide for transcribing a template polynucleotide encoding a target RNA.

12. The kit according to claim 10, further comprising S-adenosylmethionine (SAM), cap 2'O methyltransferase enzyme (2'OMTase), or both SAM and 2'OMTase.

13. The kit according to claim 10, wherein the single-chain RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to (a) SEQ ID NO: 2, (b) SEQ ID NO: 3, and / or (c) SEQ ID NO:

4.

14. The kit according to claim 10, further comprising one or more cleaning agents, dyes, solvents, and / or preservatives.

15. The kit according to claim 10, wherein the single-strand RNA capping enzyme comprises an amino acid sequence that is at least 95% identical to (a) SEQ ID NO: 8, (b) SEQ ID NO: 9, and / or (c) SEQ ID NO:

10.

16. A method for efficiently capping RNA in vitro, (i) RNA samples containing uncapped target RNA; (ii) A single-strand RNA capping enzyme having RNA triphosphatase (TPase) activity, guanylyltransferase (GTase) activity, and guanine-N7 methyltransferase (N7 MTase) activity, and having an amino acid sequence that is at least 90% identical to (a) SEQ ID NO: 8, (b) SEQ ID NO: 9, and / or (c) SEQ ID NO: 10; (iii) Guanosine triphosphate (GTP) or modified GTP (iv) buffering agent, and (v) Methyl group donor The step involves contacting the materials at a temperature of 23°C to 60°C to form capped target RNA. A method that includes this.

17. The method according to claim 16, wherein the single-stranded RNA capping enzyme comprises an amino acid sequence that is at least 90% identical to (a) SEQ ID NO: 2, (b) SEQ ID NO: 3, and / or (c) SEQ ID NO:

4.

18. The method according to claim 16, further comprising detecting capped target RNA.

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