Enzymatic RNA capping method

Abstract

Provided herein are methods for effectively capping RNA in vitro. In some embodiments, a capping reaction can be performed at high temperature using a vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reaction can include a capping enzyme from an amoeba giant virus, such as a Faustovirus, a mimivirus, or a moumouvirus, or a variant thereof. Compositions and kits for practicing the methods are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 890,821, filed on August 23, 2019, which is hereby incorporated by reference in its entirety into this application. Background Art

[0003] Adding a cap to uncapped synthetic RNA is important for efficient expression of proteins in many eukaryotic cells. In addition, uncapped RNA (at least RNA with a 5'-triphosphate) is reported to activate innate immune responses (Pichlmair et al., Science 2006 314:997-1001; Diamond et al., Cytokine & Growth Factor Reviews, 2014 25:543-550). Therefore, adding a cap to synthetic RNA is very desirable in many therapeutic applications (e.g., protein replacement therapy and preventive or therapeutic vaccination).

[0004] Currently, there are two methods used to cap RNA. In the first method, RNA capping enzymes are used to convert synthetic RNA (e.g., in vitro transcribed RNA) into capped RNA. In another method (commonly referred to as "co-transcriptional capping"), cap analogs such as reverse transcription cap analogs (ARCA) and capped dinucleotides are added to the in vitro transcription reaction. In the co-transcription method, the cap is co-transcribedly incorporated into the RNA molecule during in vitro transcription.

[0005] Compared with the co-transcriptional capping method, the enzymatic RNA capping method can obtain a higher yield of capped RNA. However, the enzymatic RNA capping reaction is very inefficient, so either a large amount of enzyme is used or the capped RNA must be purified from the uncapped RNA. In addition, the efficiency of the enzymatic RNA capping reaction (expressed as the percentage of capped RNA after the capping reaction is completed) can vary from one RNA sequence to another, and this difference is usually attributed to RNA structure (see, e.g., Fuchs, RNA. 2016 22: 1454-66).

[0006] Therefore, there remains a need for a more efficient method of adding caps to synthetic RNA. Summary of the invention

[0007] The present disclosure further provides methods for effectively capping RNA in vitro. In some embodiments, the method may include contacting (i) an RNA sample comprising an uncapped target RNA, (ii) an RNA capping enzyme comprising 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 donor at a temperature in the range of 40°C-60°C to form (e.g., effectively form) a capped target RNA. The efficiency determined by the yield (50%) of capped RNA / enzyme concentration (nM) may be increased by at least 2-fold or at least 3-fold compared to the capping efficiency of the enzyme at 37°C. Optionally, the uncapped target RNA may be free of 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 comprising an uncapped target RNA, (ii) a single-stranded RNA capping enzyme having RNA triphosphatase (TPase), guanylate transferase (GTase), and guanine-N7 methyltransferase (N7 MTase) activities, (iii) GTP or modified GTP, (iv) a buffer, and optionally (v) a methyl donor at a temperature in the range of 37°C-60°C to form (e.g., effectively form) a capped target RNA. The single-stranded RNA capping enzyme (e.g., an RNA capping enzyme from a giant virus such as Faustovirus, Mimivirus, or Moumouvirus) may include: (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, the RNA capping enzyme of a giant virus such as Faustovirus, Mimivirus, or Moumouvirus may include 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. The RNA capping enzyme from 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 may be at least 200 nt (e.g., at least 300 nt, at least 500 nt, or at least 1000 nt) in length and may encode a polypeptide, such as a therapeutic protein or a vaccine. The method disclosed herein may be used to more efficiently cap target RNAs (including therapeutic RNAs) having secondary structures. Efficiency may be defined as the yield of capped RNA (50%) / enzyme concentration (nM). In any embodiment, the method may include contacting at a first temperature (e.g., 37°C-60°C) and raising or lowering the temperature (e.g., to 37°C-60°C), wherein the second temperature is different from the first temperature. For example, the method may include contacting at a first temperature of 37°C for 1 to 120 minutes and raising the temperature to 45°C or 50°C for 1 to 120 minutes. In some embodiments, the method may include raising or lowering the temperature to a third temperature of 37°C-60°C for 1 to 120 minutes. In some embodiments, the capping method may produce (e.g., co-transcriptionally produce) more than 70% of cap ORNA in vitro within one hour or less.

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

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

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

[0013] In any embodiment, contacting can include contacting (i), (ii), (iii), (iv), and (v) at a single location (e.g., in 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, GTase and N7 MTase activity, GTP and a buffer. In some embodiments, the temperature of the composition may be in the range of 37°C-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 that is 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 Mumuvirus (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, a single uncapped target RNA may be at least 200 nt (at least 300 nt, at least 500 nt, or at least 1000 nt) in length and may encode a polypeptide, such as a therapeutic protein or a vaccine.

[0015] A kit is also provided. In some embodiments, the kit may include: a single-stranded RNA capping enzyme having TPase, GTase and N7MTase activity, wherein the enzyme is 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. Examples of methods, compositions and kits utilizing methyltransferases may also include SAM in the reaction mixture. 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 can include an amino acid sequence that is 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 moumuvirus (SEQ ID NO: 12).

[0016] In some embodiments, the RNA capping enzyme can have an amino acid sequence at least 90% identical to SEQ ID NO: 20. In some embodiments, the RNA capping enzyme fusion can comprise: (a) an amino acid sequence at least 90% identical to SEQ ID NO: 7, and (b) an amino acid sequence at least 90% identical to positions 1419 to 1587 of SEQ ID NO: 20. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The predicted secondary structures of the model RNA molecules used are shown. RNA1 was designed to have minimal secondary structure at the 5' end. RNA2, RNA3 and RNA4 were designed to exhibit the same predicted minimum free energy of unfolding (MFE) while containing a blunt end, a one-base overhang or a two-base overhang at their 5' end. The secondary structure and the minimum free energy of unfolding at 37°C and 45°C were calculated using the RNAFold web server (University of Vienna, 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, RNA4: SEQ ID NO: 16.

[0018] Figure 2 The RNA capping activity of H3C2 and VCE on RNA1 at different reaction temperatures is shown. The amount of RNA capped with m7Gppp is a measure of the RNA capping activity of these enzymes. Each bar represents the result of the average of 4 independent experiments, where the error bar represents the standard deviation. As shown in the figure, H3C2 and VCE exhibited maximum RNA capping activity at 45°C. Although VCE capping activity dropped sharply at 50°C and higher temperatures, H3C2 still maintained significant capping activity at 50°C, 52°C, and 55°C.

[0019] Figure 3A-3B RNA capping activity as a function of capping enzyme concentration at 37°C, 45°C, 50°C and 55°C is shown. Figure 3A The activity of VCE is shown. Figure 3B The activity of H3C2 is shown. The quantitative comparison of capping activity under each condition is shown in Table 1.

[0020] Figures 4A-4E The capping activity of putative RNA capping enzymes on 150 nt RNA at various temperatures is shown. Lanes 1 to 6 show the capping reaction of in vitro translation products using AMN83561 (H3C2) (lane 1), AIB52055 (lane 2), SME65026 (lane 3), SMH63629 (lane 4), AAV50651 amino acids 1-668 (Mimivirus capping enzyme N-terminal region) (lane 5), or VP_007354410 (Momovirus 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 was: GGGAGUCUUCGCCGAGAGGGCCAUCGCCAGUUGCCGCAACCUGUGGGAAUUUCUCUUCCAGUUUAUCCGGAUGCUCAACGGUGACUUUAAUUCCGGUAUCUUUCUCGAAUUUCUUACCGACUUCAGCGAGCUCGACAAUGCUCUUCCCUA (SEQ ID NO: 17). Figure 4A Capping activity at 37°C is shown. Figure 4B Capping activity at 45°C is shown. Figure 4C Capping activity at 50°C is shown. Figure 4D Capping activity at 55°C is shown. Figure 4E Capping activity at 60°C is shown.

[0021] Figure 5Shown are conservative regions showing 90% or higher sequence identity between Faustovirus D5b, E12, ST1 and LC9 (SEQ ID NO:7-10) capping enzyme amino acid sequences mapped to AMH83561 (H3C2). These conservative regions are named Fausto_CR_01, Fausto_CR_03 and Fausto_CR_05. Indicated are the functional domains of H3C2 capping enzymes (i.e. TPase, GTase and N7MTase).

[0022] Figure 6 Conserved regions showing 90% or greater sequence identity between the Acanthanomeba polyphaga mimivirus (AAV50651, SEQ ID NO: 11) and the Acanthanomeba polyphaga moumouvirus capping enzyme amino acid sequences mapped to YP_007354410 (Acanthanomeba polyphaga moumouvirus capping enzyme; moumou CE (SEQ ID NO: 12) are shown. These conserved regions are designated moumou_CR_03 (SEQ ID NO: 6) and moumou_CR_04 (SEQ ID NO: 7). In this alignment, the functional domains of moumou-CE (i.e., TPase, GTase, and N7MTase) are indicated with dashed lines above the aligned sequences. Conserved regions are highlighted.

[0023] Figure 7 It is shown that RNA capping reactions performed at 45°C promote efficient capping of hairpin RNAs. At 37°C (white bars), both H3C2 and VCE efficiently cap RNA1, which has at least a 5' secondary structure (see Figure 1 ). RNA2, RNA3 and RNA4 have stable 5' secondary structures at 37°C (see Figure 1 ) were not capped or capped to a low degree at 37°C (white bars). However, at 45°C (grey bars), H3C2 efficiently capped all four RNAs. At 45°C, VCE efficiently capped RNA1 and RNA2, while partially capped RNA3 and RNA4 (grey bars).

[0024] Figure 8It was shown that the RNA capping reaction performed at 45°C promoted the effective capping of long RNA. In the presence of 0.5mM GTP and 0.1mMSAM in 10μL 1x RNA capping buffer (50mM Tris-HCl, 5mM KCl, 1mM MgCl2, 1mM DTT, pH 8), increasing concentrations of H3C2 or VCE were incubated with 400nM fluc RNA (1.7kb) at 37°C or 45°C for 30 minutes. The first 24nt of flucRNA was then cut off by oligo-guided RNaseH cleavage. The degree of capping in the cut 24nt fragment was analyzed by 15% urea polyacrylamide gel electrophoresis. The band intensity was quantified and used to calculate the capping score under each condition. The calculated values ​​were plotted against the enzyme concentration and fitted to the modified Hill equation to obtain the enzyme concentration (cap 50 For H3C2, capping at 37°C or 45°C 50 The values ​​were 33.6 nM and 0.96 nM, respectively. For VCE, the cap 50 The values ​​were 103 nM and 2.97 nM, respectively. For both H3C2 and VCE, reactions at 45°C significantly reduced the amount of enzyme required to cap 50% of the substrate RNA compared to reactions at 37°C.

[0025] Fig. 9 It was shown that RNA capping reactions performed at 45°C promoted more efficient capping of long RNAs (except flucRNA) than at 37°C. 100 nM H3C2 or VCE was incubated with 500 nM clucRNA (1.8 kb) or CFTR RNA (4.0 kb) at 37°C or 45°C in the presence of 0.5 mM GTP and 0.1 mM SAM in 10 μL 1x RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8) for 30 minutes. The defined 5' fragments were cleaved using thermostable RNaseH (New England Biolabs, Ipswich, MA) and the 5' fragments were cleaved using XP beads (Beckman Coulter, Indianapolis, IN) and streptavidin (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 fraction of capped RNA was calculated using the mass intensities of the capped and uncapped species.

[0026] Fig.10 High reaction temperatures were shown to enhance the enzymatic activity of vaccinia virus RNA cap 2'O methyltransferase (MTase). 5 μM chemically synthesized cap-0 25nt RNA (m7Gppp25mer) was reacted with 200 μM SAM at the indicated temperature for 30 minutes in the presence of 100 U vaccinia cap 2'O MTase (New England Biolabs, Ipswich, MA) in 20 μL 1x RNA capping buffer. The reaction was stopped by heating at 70°C for 10 minutes. RNA was purified from the reaction components, digested into nucleosides and cap structures, and analyzed by UPLC. Performing the reaction at 45°C and 50°C produced more cap-1 RNA than that produced at 37°C.

[0027] Fig.11 It is shown that the "one-pot" generation of Cap-1 structures at 45°C is more efficient than at 37°C. In this experiment, 5 μM chemically synthesized 5' triphosphate ribonucleotide oligomers (ppp25-mers) were incubated with 200U vaccinia virus cap 2'OMTase at 37°C or 45°C for 30 minutes in the presence of 50nM H3C2 or VCE, 1mM GTP and 0.2mM SAM in 40μL 1x RNA capping buffer. The relative amounts of reactants and products were obtained from direct LC / MS analysis. As shown in the figure, more Cap-1 structure RNA was generated in a one-pot enzymatic reaction at 45°C than at 37°C.

[0028] Fig.12 It is shown that one-step capped RNA synthesis of a 1.7 kb fluc transcript using commercially available enzymes and recommended reaction temperatures does not generate significant amounts of cap-0 (Cap-0) or cap-1 (Cap-1) RNA. Reactions were performed at 37°C for 1 hour using the indicated components at recommended enzyme concentrations (see Example 7 for details). The transcription output after DNase I treatment was measured using the Qubit RNA BR kit. The products of the capped RNA synthesis reaction were analyzed by LC / MS. The fraction of transcripts containing different 5' groups was estimated using the mass intensity of the corresponding mass. Unless otherwise stated, data points are the average of three experiments.

[0029] Fig.13 A one-step method for synthesizing capped fluc transcripts using high concentrations of H3C2 capping enzyme and elevated temperatures is shown. One-step capped RNA synthesis was performed using T7 RNA polymerase and 0.5 μM H3C2 RNA capping enzyme at 37 or 45°C for 1 hour. The fractions of ppp-, pp-, Gppp-, and m7Gppp-capped RNAs were estimated using capillary electrophoresis. Data points are the average of replicate experiments.

[0030] Fig.14 One-step Cap-0 or Cap-1 RNA synthesis using high capping enzyme concentrations at 45 or 50°C is shown. Products of the capped RNA synthesis reaction were analyzed by LC / MS. The fraction of transcripts containing different 5' groups was estimated using the mass intensities of the corresponding masses. Unless otherwise stated, data points are the average of three experiments.

[0031] Fig.15 One-step Cap-0 RNA synthesis using H3C2:T7 fusion protein at 37°C or 45°C is shown. The fusion protein generated 98% Cap-0 transcripts within 1 hour at 45°C. Products of the capped RNA synthesis reaction were analyzed by LC / MS. The fraction of transcripts containing different 5' groups was estimated using the mass intensities of the corresponding masses. Unless otherwise stated, data points are the average of duplicate experiments.

[0032] Fig.16 VCE and H3C2 capping enzymes were shown to effectively cap 1.8kb cluc / A120 transcripts. 500nM unmodified or pseudouridine cluc / A120 transcripts were incubated with 100nM H3C2 or VCE at 37°C for 30min in the presence of 0.5mM GTP and 0.1mM SAM in 10mL 1xRNA capping buffer (50mM Tris-HCl, 5mM KCl, 1mM MgCl2, 1mM DTT, pH 8). The capping reaction was analyzed by RNaseH / complete LC / MS workflow, as described in Example 9. VCE and H3C2 generated 68.4% or 100% m7Gppp-capped cluc / A120 transcripts containing pseudouridine, respectively. Obviously, the capping scores of cluc / A120 transcripts containing pseudouridine were higher for these two capping enzymes compared to their uridine counterparts.

[0033] Fig.17The efficiency of cap-1 formation for larger RNA amounts is shown. A total of 250 pmol (145 μg) of approximately 1800 nt cluc / A120 in vitro transcripts were capped for 1 hour at 45°C in a 500 μL reaction containing 1x 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 vaccinia cap 2'O methyltransferase, and 500 nM H3C2. The efficiency of cap-1 formation was assessed by RNaseH / intact LC / MS, as described in Example 10. Under these reaction conditions, 54.8% of the cluc / A120 transcripts containing pseudouridine were cap-1, while 47.7% of the cluc / A120 transcripts of the uridine counterpart were cap-1. Optimizing the reaction conditions (as described in Example 10) can increase the yield of cap-1 formation. Results are the average of replicate experiments.

[0034] Fig.18 Cap-1cluc / A120 transcripts were shown to be functionally active in vivo. HEK293 cells were transfected with cap-1cluc / A120 transcripts containing either uridine or pseudouridine. Four hours after transfection, the translation efficiency of capped cluc / A120 transcripts was measured as the relative luciferase activity of HEK293 cells transfected with capped cluc / A120 transcripts compared to uncapped cluc / A120 transcripts. Luciferase assays showed that capped transcripts containing rU and pseudouridine exhibited 10-fold or 20-fold higher luciferase activity than uncapped controls, respectively, indicating that RNA capped with H3C2 and vaccinia 2'O methyltransferase at 45°C was functionally active.

[0035] Fig.19It was shown that multiple temperature steps can improve the transcription output and capping efficiency of single-container capped RNA synthesis. Single-container capped RNA synthesis reactions were performed on 1.7 kb fluc transcripts using T7 RNA polymerase in combination with VCE or H3C2 capping enzymes, as described in Example 11, wherein the reaction was incubated at 37°C for 30 min, followed by a second 30-minute incubation at a temperature between 28°C and 50°C. Transcription output was assessed using the DNase I / Qubit method. Capping scores were estimated by RNaseH / Klenow fill-in and urea PAGE methods. Complete LC / MS analysis was performed on samples showing the highest capping scores. For VCE, the m7Gppp transcript formation efficiency peaked at a second reaction temperature of 45°C, while for H3C2, the m7Gppp formation efficiency peaked at a second reaction temperature of 50°C. On the other hand, T7 RNAP transcription output peaked at 45°C when coupled with VCE or H3C2, reflecting low transcriptional activity at the upper and lower ends of the tested temperatures (28°C and 50°C, respectively). Therefore, in this example, the tandem temperature steps of 37°C for 30 min followed by 45°C for 30 min achieved the best balance of transcriptional output and capping efficiency. Modifications to reaction conditions such as reaction time, reaction temperature, and number of temperature steps can further improve the transcription yield and fraction of m7G-capped transcripts. DETAILED DESCRIPTION

[0036] The various aspects of the present disclosure can be further understood according to the embodiments, part titles, drawings, descriptions and examples, none of which should be interpreted as limiting the entire scope of the present disclosure in any way. Therefore, the claims listed below should be interpreted according to the full breadth and spirit of the present disclosure.

[0037] Each of the individual embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any other several embodiments without departing from the scope or spirit of this teaching. Any method of narration can be performed in the order of the events described 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 can be changed.

[0038] 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 the present disclosure belongs. Nevertheless, for clarity and ease of reference, certain terms are defined herein with respect to embodiments of the present disclosure.

[0039] Sources for commonly understood terms and symbols may include standard papers and texts such as Kornberg and Baker, DNA Replication, second edition (WH Freeman, New York, 1992); Lehninger, Biochemistry, second edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, second 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, second edition, John Wiley and Sons, New York (1994), and Hale & Markham, the Harper Collins Dictionary of Biology, Harper Perennial, NY (1991), etc.

[0040] As used herein and in the appended claims, the singular forms "a," "an," and "an" include plural reference unless the context clearly dictates otherwise. For example, the term "protein" refers to one or more proteins, i.e., a single protein and a plurality of proteins. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "only," "only," etc., in connection with the limitation or "negative" limitation of claim elements.

[0041] Numerical ranges include the numbers defining the range. All numbers are understood to encompass the midpoints of the integers above and below the integer, i.e., the number 2 encompasses 1.5-2.5. The number 2.5 encompasses 2.45-2.55, etc. When sample values ​​are provided, unless otherwise specified, for example, each individually may represent a middle value within the numerical range, and together may represent an extreme value within the range.

[0042] In the context of the present disclosure, "buffer" refers to an agent that allows a solution to resist pH changes when an acid or base 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.

[0043] In the context of the present disclosure, "capping" refers to the enzymatic addition of an Nppp-moiety to the 5' end of an RNA, wherein 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 an added tag at the 2 or 3 position of the ribose, and in some embodiments, the tag may be an oligonucleotide, a detectable tag such as a fluorophore, or a capture moiety such as biotin or desthiobiotin, for example, wherein the tag may be optionally attached to the ribose of the nucleotide via a linker. See, for example, WO2015 / 085142. The cap may have a cap-0 structure, a cap-1 structure, or a cap-2 structure (as reviewed in Ramanathan, Nucleic Acids Res. 2016 44: 7511–7526), ​​depending on which enzymes are present in the capping reaction and / or whether SAM is present.

[0044] In the context of the present disclosure, "DNA template" refers to a double-stranded DNA molecule transcribed in an in vitro transcription reaction. The DNA template has a promoter (e.g., T7, T3 or SP6 promoter) that is recognized by RNA polymerase upstream of the transcribed region.

[0045] In the context of the present disclosure, "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 comprise a non-naturally occurring combined polypeptide chain comprising two proteins or two protein domains directly linked to each other by a peptide bond or linked by a peptide linker.

[0046] 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 replicated by a DNA-directed RNA polymerase (usually a bacteriophage polymerase) to produce a product comprising an RNA molecule copied from the template.

[0047] In the context of the present disclosure, "Faustovirus RNA capping enzyme" refers to a single-stranded RNA capping enzyme capable of capping RNA (e.g., having detectable TPase, GTase, and N7 MTase activity), including, for example, an enzyme with 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 an RNA capping enzyme from faustovirus D5b (SEQ ID NO: 7). Unless otherwise expressly stated, for the purposes of the examples and examples disclosed herein, Faustovirus RNA capping enzymes may be interchangeable with each other for similar properties, effects, and / or benefits.

[0048] In the context of the present disclosure, "H3C2 fusion" refers to a bifunctional enzyme capable of synthesizing RNA from a template polynucleotide and capable of capping RNA, the fusion comprising an RNA polymerase (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase) and a Faustovirus RNA capping enzyme, which is arranged at the N-terminus or C-terminus relative to the polymerase. The H3C2 fusion may further comprise a leader and / or a connector (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. H3C2: T7 RNA polymerase fusion is an example of an H3C2 fusion.

[0049] In the context of the present disclosure, "H3C2 variant" refers to H3C2, H3C2 fusions, and enzymes capable of capping RNA, which in each case comprise an amino acid sequence that is 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% identical to SEQ ID NO:7, 8, 9, 10, 11, and / or 12.

[0050] In the context of the present disclosure, "modified nucleotides" (including references to modified NTPs, modified ATPs, modified GTPs, modified CTPs, and modified UTPs) refer to any non-standard nucleosides, nucleotides, or their corresponding phosphorylated versions. Modified nucleotides may include one or more backbone or base modifications. Examples of modified nucleotides include dI, dU, 8-oxo-dG, dX, and THF. Other examples of modified nucleotides include modified nucleotides disclosed in U.S. Patent Publication Nos. US20170056528A1, US20160038612A1, US2015 / 0167017A1, and US20200040026A1. Modified nucleotides may include naturally occurring or non-naturally occurring nucleotides.

[0051] In the context of the present disclosure, "non-naturally occurring" refers to polynucleotides, polypeptides, carbohydrates, lipids, or compositions that do not exist in nature. Such polynucleotides, polypeptides, carbohydrates, lipids, or compositions may differ from naturally occurring polynucleotides, polypeptides, carbohydrates, lipids, or compositions in one or more aspects. For example, a polymer (e.g., a polynucleotide, polypeptide, or carbohydrate) may differ in the type and arrangement of component building blocks (e.g., nucleotide sequence, amino acid sequence, or sugar molecule). A polymer may differ from a naturally occurring polymer in terms of the molecule (or molecules) to which it is attached. For example, a "non-naturally occurring" protein may differ from a naturally occurring protein in terms of its secondary, tertiary, or quaternary structure by having a chemical bond (e.g., covalent bond, including peptide bond, phosphate bond, disulfide bond, ester bond, and ether bond and other bonds) with a polypeptide (e.g., fusion protein), lipid, carbohydrate, or any other molecule. Similarly, a "non-naturally occurring" polynucleotide or nucleic acid may include one or more other modifications (e.g., added tags or other moieties) to the 5'-end, 3'-end, and / or between the 5'-end and the 3'-end (e.g., methylation) of the nucleic acid. A "non-naturally occurring" composition may differ from a naturally occurring composition in one or more of the following respects: (a) having components that are not combined in nature, (b) having components at concentrations not found in nature, (c) omitting one or more components otherwise found in a naturally occurring composition, (d) having a form not found in nature, e.g., dried, lyophilized, crystalline, aqueous, and (e) having one or more other components beyond those found in nature (e.g., buffers, detergents, dyes, solvents, or preservatives). All publications, patents, and patent applications mentioned in this specification are incorporated herein 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.

[0052] In the context of the present disclosure, "RNA sample" or "sample" refers to a composition that may or may not contain a target RNA. For example, an RNA sample may be known to contain or suspected to contain such a target RNA, and / or an RNA sample may be a composition that is assessed for the presence of a target RNA. An RNA sample may contain a naturally occurring target RNA (e.g., extracted from a cell, tissue, or organism), a target RNA produced by in vitro transcription, and / or a chemically synthesized RNA.

[0053] In the context of the present disclosure, "single-stranded RNA capping enzyme" refers to a capping enzyme in which a single polypeptide chain includes TPase, GTase and N7 MTase activities. Faustovirus, Mimivirus and Mumuvirus capping enzymes are examples of single-stranded RNA capping enzymes. H3C2 fusion is another example of a single-stranded RNA capping enzyme. VCE is a heterodimer and is therefore not a single-stranded RNA capping enzyme.

[0054] In the context of the present disclosure, a "single uncapped RNA target species" refers to a mixture of target RNA molecules having substantially identical sequences. Transcripts produced by in vitro transcription and RNA oligonucleotides produced by solid phase synthesis are examples of single uncapped RNA target species. It is well known that a certain amount of RNA products in such a mixture may be truncated. A single uncapped RNA target species may sometimes contain modified nucleotides (e.g., non-standard nucleotides not found in nature). Preparations of RNA from cells contain a complex mixture of naturally occurring RNA molecules with different sequences; such preparations contain not only the targeted uncapped RNA species, but also a variety of non-target RNAs. In some embodiments, the targeted uncapped RNA species is a single RNA species.

[0055] In the context of the present disclosure, "target RNA" refers to a polyribonucleotide of interest. The polyribonucleotide can be or comprise a therapeutic RNA or a precursor thereof (e.g., an uncapped precursor of a capped therapeutic RNA). The target RNA can be produced by cell transcription or in vitro transcription. The target RNA can be present in a mixture, e.g., in an in vitro transcription reaction mixture, a cell, or a cell lysate. The target RNA can be uncapped. If desired or necessary, the target RNA can be contacted with a decapping enzyme, e.g., as a co-treatment of capping or a pre-treatment before capping.

[0056] In the context of the present disclosure, "uncapped" refers to RNA that (a) does not have a cap and (b) can serve as a substrate for a capping enzyme. Uncapped RNA typically has a triphosphorylated or diphosphorylated 5' end. In vitro transcribed RNA has a triphosphate group at the 5' end.

[0057] In the context of the present disclosure, "variant" refers to a protein having an amino acid sequence that is different from a naturally occurring amino acid sequence (i.e., has less than 100% identity with the amino acid sequence of a 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.

[0058] Various in vitro RNA capping methods are provided herein. Some embodiments of the method are based in part on the discovery that the VCE of SEQ ID NO: 1 is significantly more active at 45°C than at 37°C (see below Figure 2 and Tables 1 and 2)—particularly for RNAs with secondary structure. For example, when used to add caps to in vitro transcribed RNA encoding a protein, less than one-thirtieth (1 / 30) of the amount of enzyme was used at 45°C as compared to at 37°C to produce the same amount of capped product (see Table 6). In addition, incubation at 45°C allows the VCE to efficiently add caps to the 5' end (e.g., the 5' end predicted to base pair with another sequence in the molecule to produce a blunt end or a 3' or 5' overhang of 1 or 2 nucleotides, such as Figure 1 The uncapped RNA oligonucleotide has a secondary structure (as shown in ). This RNA is capped very inefficiently with VCE at 37°C (see Fig. 9 ). Thus, in some embodiments, the method may include incubating a reaction mixture comprising RNA, VCE or variants thereof, GTP or modified GTP, SAM, and a buffer at a temperature in the range of 42°C-47°C (e.g., 44°C-46°C) to effectively add a cap structure to an uncapped RNA target. These embodiments of the method are particularly useful for RNAs having or predicted to have secondary structure (e.g., RNAs longer than 200 nt transcribed in vitro, and RNA oligonucleotides having secondary structure at the 5' end).

[0059] Other embodiments of the method are 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 and referred to herein by the abbreviation "H3C2") is able to cap RNA significantly more efficiently than VCE at almost all temperatures tested (see below). Figure 2and Tables 1 and 6), and like VCE, can be used to efficiently add caps to in vitro transcribed RNA at 45°C. For example, less than one-thirtieth (1 / 30) the amount of faustovirus D5b (H3C2) can be used at 45°C to produce the same amount of capped product as at 37°C (see Table 6). In addition, incubation at 45°C reactions allows faustovirus D5b (H3C2) to efficiently add caps to uncapped RNA oligonucleotides that have secondary structure at the 5' end. Such RNA is capped very inefficiently with faustovirus D5b (H3C2) at 37°C (see Fig. 9 ). Capping enzymes from other giant viruses from amoeba, including faustovirusST1, faustovirusLC9, faustovirusE12, mimivirus, and mumuvirus, were also tested and found to be active at higher temperatures. Thus, in some embodiments, the method may include incubating a reaction mixture comprising a sample containing RNA, a single-stranded capping enzyme, GTP or modified GTP, and a buffer at a temperature in the range of 37°C-60°C (e.g., 37°C-42°C, 42°C-47°C, 47°C-52°C, or 52°C-60°C) to effectively add a cap structure to an uncapped RNA target in vitro. These embodiments of the method are particularly useful for RNAs having or predicted to have secondary structure, such as RNAs transcribed in vitro that are longer than 200 nt, and RNA oligonucleotides having secondary structure at the 5' end.

[0060] More efficient capping of RNA substrates can support: using less enzyme added to the capping reaction for capping, producing more capped RNA (as a percentage of the 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.

[0061] The disclosed reaction conditions can be varied and include, without limitation, reaction temperature, reaction duration, concentrations of reaction components (e.g., SAM, inorganic pyrophosphatase, NTP, transcript 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 terms of the fraction of cap-0 transcripts and transcriptional output. In addition, inclusion of a cap 2'O methyltransferase, such as vaccinia cap 2'O methyltransferase, in the reaction can efficiently generate cap-1 transcripts. Depending on which enzyme is used and the other components in the reaction mixture, the disclosed methods can be used to prepare RNAs having a Gppp cap, a 7-methylguanylate cap (i.e., an m7Gppp cap or "cap-0"), or RNAs having an m7Gppp cap with an additive modification in 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 there is no SAM in the reaction mixture, RNA with a Gppp cap may be produced. If the reaction mixture contains SAM in addition to the capping enzyme, cap-0 RNA may be produced. If the reaction mixture contains other enzymes in addition to SAM, for example, cap 2'OMTase, cap-1 and / or cap-2 RNA may be produced. In addition to the components explicitly described above, the reaction mixture may contain other components.

[0062] In some embodiments, the uncapped RNA in the reaction mixture can 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 can be a length in the range of 10-500 bases, e.g., 20-200 bases). In other embodiments, the uncapped RNA in the reaction mixture can be prepared in a cell-free in vitro transcription (IVT) reaction, wherein a double-stranded DNA template containing an RNA polymerase promoter (e.g., T7, T3, or SP6 promoter) upstream of the transcribed region is replicated by a DNA-guided RNA polymerase (usually a bacteriophage polymerase) to produce a product comprising RNA molecules replicated from the template. In any embodiment, the capped RNA sample in the present method comprises RNA (synthetic oligonucleotides or transcripts) of a single species. In addition, the reaction mixture can be RNase-free and can optionally comprise one or more RNase inhibitors. The RNA in the sample can comprise non-natural nucleotide sequences, and in some embodiments, can comprise non-naturally occurring nucleotides. In some embodiments, the in vitro transcription reaction may employ thermostable variants of T7, T3, and SP6 RNA polymerases (see, e.g., PCT / US2017 / 013179 and U.S. Application Serial No. 15 / 594,090). In these embodiments, RNA may be transcribed at a temperature greater than 44°C (e.g., at least 45°C, at least 50°C, at least 55°C, or at least 60°C, up to a temperature of about 70°C or 75°C) to reduce the immunogenicity of the RNA (see, e.g., WO2018 / 236617). In some cases, uncapped RNA may be capped immediately after it is prepared, e.g., by adding RNA capping enzymes and GTP / modified GTP to the reaction as needed after the in vitro transcription reaction has run its course. In some embodiments, RNA prepared in an in vitro transcription reaction may be purified prior to capping.

[0063] In some embodiments, the RNA in the sample can be a therapeutic RNA. In these embodiments, the product of the present method can be used without purifying the capped RNA from the uncapped RNA. In these embodiments, the product of this reaction can be combined with a pharmaceutically acceptable excipient to produce a preparation, wherein a "pharmaceutically acceptable excipient" is any solvent compatible with administering to a living mammalian organism by transdermal, oral, intravenous, or other administration means used in the art. Examples of pharmaceutically acceptable excipients include, for example, those described in US 2017 / 0119740. The preparation can be administered in vivo, for example, to an object, and examples of the object include humans or any non-human animals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horses, or primates). Depending on the object, RNA can be directly introduced into cells by injecting RNA (modified or unmodified), or RNA can be indirectly introduced into cells by the surrounding culture medium. Administration can be performed by standardized methods. RNA can be naked or formulated in a form suitable for administration to an object (e.g., a human). The formulation may include liquid formulations (solutions, suspensions, dispersions), topical formulations (gels, ointments, drops, creams), liposomal formulations (such as those described in US 9,629,804 B2; US 2012 / 0251618 A1; WO 2014 / 152211; US ​​2016 / 0038432 A1). The cells into which the RNA product is introduced may be in vitro (i.e., cells cultured in vitro on a synthetic medium). Therefore, the RNA product can be transfected into the cells. The cells into which the RNA product is introduced may be in vivo (cells that are part of a mammal). Therefore, the introduction can be accomplished by administering the RNA product to a subject in vivo. The cells into which the RNA product is introduced may exist ex vivo (cells that are part of a tissue, for example, soft tissue that has been removed from a mammal or separated from the blood of a mammal).

[0064] In order to prepare mRNA for therapeutic applications, it may be desirable or necessary to synthesize a large number of uniformly capped mRNA transcripts in a cost-effective and streamlined manner that can be scalably supported for multi-gram synthesis. Current methods for preparing mRNA use expensive mRNA cap analogs in in vitro transcription reactions. Alternatively, a separate reaction is required to produce 5'-triphosphate RNA by in vitro transcription and then use an enzyme to cap the mRNA, which is a more complex process and more difficult to scale. The preparation process can be simplified by synthesizing capped RNA in one step in vitro using T7 RNA polymerase and a capping enzyme. A single container reaction with these two enzymes can reduce or eliminate the excessive cost of synthesizing mRNA cap analogs and can reduce the complexity of expanding this workflow to support multi-gram (and more) synthesis.

[0065] method

[0066] Provided herein are methods for efficiently producing capped RNA. In some embodiments, the method includes a thermoactive Hi-T7 RNA polymerase and an H3C2 RNA capping enzyme and an unexpectedly thermoactive vaccinia mRNA cap 2'O methyltransferase. Surprisingly, reaction temperatures much higher than 37°C enable the synthesis of single-reaction capped mRNA. In some embodiments, RNA transcription and capping activity can be performed in a single reaction at higher temperatures (e.g., 40°C to 60°C). For example, an RNA polymerase and a separate capping enzyme with or without a vaccinia cap 2'O methyltransferase can be used simultaneously for the synthesis reaction of capped RNA. The combination of RNA transcription and capping activity can be achieved, for example, by a fusion protein comprising H3C2, a single subunit RNA capping enzyme, and a T7 RNA polymerase or a Hi-T7 RNA polymerase. The methods described herein can achieve high levels of cap-0 or cap-1 RNA synthesis.

[0067] In some embodiments, the capping method may include contacting an RNA polymerase (e.g., T7 RNA polymerase, thermoactive 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), NTP, 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), 2'O methylated NTP, and / or combinations thereof. In some embodiments, the capping method may include contacting a capping enzyme (e.g., VCE, H3C2, H3C2 fusion), a target RNA, and guanosine triphosphate (GTP), and / or modified GTP. In some embodiments, the capping method can include contacting a capping enzyme (e.g., VCE, H3C2, H3C2 fusion), a target RNA, and guanosine triphosphate (GTP) and / or modified GTP, an O-methyltransferase (e.g., heat-activated vaccinia mRNA cap 2' O-methyltransferase) in the presence or absence of SAM.

[0068] According to some embodiments, contacting can be performed, for example, in a single location (e.g., in one step) on any surface (e.g., a plate or bead) or in any container (e.g., a tube, flask, vial, column, container, bioreactor or other space). In some embodiments, contacting can include contacting some or all of the subject elements in any desired order or simultaneously. In some embodiments, contacting can include contacting some or all of the subject elements in the presence of a buffer. For example, contacting can include contacting a capping enzyme (e.g., VCE, H3C2, H3C2 fusion), a target RNA, a GTP, and a buffer in any order or simultaneously in the presence or absence of a SAM. In some embodiments, the contacting may further include contacting at a temperature of 40°C to 60°C (e.g., at 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 period of 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 embodiment disclosed herein, the contacting 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 different from the first temperature, wherein, optionally, at least one of the first temperature or the second temperature is not less than 40°C. The first temperature may be selected to produce, stabilize and / or maintain a desired amount of uncapped target RNA, and the second temperature may be selected to produce, stabilize and / or maintain a desired amount of capped target RNA. Each temperature may be maintained for any desired time period (e.g., 1 minute to 120 minutes). For example, the first temperature may be 37°C for a first period of 1 to 30 minutes, and the second temperature may 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, contact may include placing one item in direct contact with another item, placing the two items together on a surface or in a container, close enough so that they can physically and / or chemically interact with each other with or without further stirring or mixing. Contact may include placing one item in direct contact or proximity to another item and / or maintaining an initial behavior that allows or facilitates the conditions for the two items to contact each other.

[0069] In some embodiments, the method may include synthesizing RNA in vitro (e.g., guided by a template or not guided by a template) to produce an uncapped target RNA and capping the target RNA (e.g., in a one-step reaction). Synthesizing RNA from a template may include, for example, contacting an RNA polymerase (e.g., T7 RNA polymerase, thermoactive Hi-T7 RNA polymerase, and / or H3C2 fusion) with a template (e.g., an RNA template or a DNA template) and one or more NTPs for producing an uncapped target RNA, a buffer, and optionally with or without 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), 2'O-methylated NTPs, and / or combinations thereof. Capping the target RNA may include, for example, contacting H3C2 (e.g., H3C2 or 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 may include contacting the target RNA with the 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 an existing cap and / or ensure that it is not capped.

[0070] 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-activated vaccinia mRNA cap 2' O-methyltransferase), SAM, guanosine triphosphate (GTP), a modified GTP, and / or a buffer. For example, capping can include contacting a capping enzyme, a target RNA, a GTP (optionally with or without a modified GTP), and optionally a buffer. Capping can include contacting a capping enzyme, a target RNA, a GTP (optionally with or without a modified GTP), an O-methyltransferase, a SAM, and optionally a buffer in a single container (e.g., in one step). Capping can include capping a pre-existing target RNA and / or synthesizing and capping a target RNA in a single container (e.g., in one step), for example, by contacting 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 including or excluding one or more modified NTPs), a capping enzyme (e.g., VCE, H3C2, H3C2 fusion), and / or a buffer.

[0071] Composition

[0072] Various compositions related to the disclosed methods are also provided. In some embodiments, the composition (e.g., a reaction mixture) may include a 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-stranded RNA capping enzyme, GTP, SAM, and a buffer having TPase, GTase, and N7 MTase activities. The temperature of the composition may be within the range of 37°C-60°C, for example, 37°C-42°C, 42°C-47°C, 47°C-52°C, or 52°C-60°C. In some embodiments, the composition may not have RNase activity because RNase is inhibited, inactivated, or missing. For example, an RNase-free composition may include one or more RNase inhibitors. In some embodiments, the composition may further include a DNA template operably connected to a promoter, a phage polymerase that initiates transcription at the promoter, and NTPs for transcribing RNA. In some embodiments, the composition may include a cap 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. According to some embodiments, the composition may comprise an H3C2 fusion, which comprises (a) a Faustovirus RNA capping enzyme (e.g., an H3C2 RNA capping enzyme) and an RNA polymerase or (b) an RNA polymerase and an H3C2 RNA capping enzyme in the order of N-terminus to C-terminus, wherein the H3C2 fusion may optionally further comprise a leader region (e.g., operably positioned within the fusion) and / or a linker positioned between H3C2 and the RNA polymerase. In some embodiments, any or all of the components mentioned in this paragraph may be combined or otherwise contained in a single container. Components, such as components in a single container, may be present in a dry (e.g., anhydrous and / or lyophilized) form. The container containing one or more of the components mentioned may also contain an aqueous medium.

[0073] Reagent test kit

[0074] As described above, the present disclosure also provides a kit for practicing the subject method. In some embodiments, the kit may include any one or more of the components listed above. For example, the kit may include a single-stranded RNA capping enzyme with TPase, GTase and N7 MTase activity, wherein the enzyme is in a storage buffer and a reaction buffer (e.g., it can be a 5× or 10× concentrated reaction buffer). In some embodiments, the kit may include a phage polymerase and NTP for transcribing RNA from a DNA template. The kit may additionally include SAM and cap 2'OMTase. As will be apparent from the above discussion, the enzyme may include (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.

[0075] The various components of the kit may be present in separate containers, or certain compatible components may be pre-combined into a single container as desired. In addition to the above components, the subject kit may also include instructions for using the components of the kit to practice the subject method, i.e., instructions for capping RNA.

[0076] Example

[0077] All reagents were obtained from New England Biolabs (Ipswich, MA) and / or indicated suppliers.

[0078] Example 1: RNA capping activity of H3C2 and VCE at high temperature

[0079] In the presence of 0.5 mM GTP and 0.1 mM SAM in 10 μL 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8.0), 10 nM purified VCE or H3C2 RNA capping enzyme was mixed with 500 nM RNA1 ( Figure 1 ) was incubated for 30 minutes. RNA1 contains a fluorescent group (FAM) at its 3' end. The reaction was quenched by adding 1 μL 50 mM EDTA followed by heat inactivation at 70°C for 10 minutes. The quenched reaction was analyzed by capillary electrophoresis using an Applied Biosystem 3730×l Genetic Analyzer. The peak areas corresponding to m7Gppp-, unmethylated Gppp-, uncapped pp-, and ppp-RNA1 were quantified and used to calculate the fraction of m7Gppp- at the end of the 30-minute capping reaction. Figure 2In the figure, each bar represents the mean of 4 independent experiments. The error bars represent the standard deviation of 4 replicates. It is obvious that at low enzyme concentration (10 nM), both H3C2 and VCE showed the highest capping activity at 45 °C, while H3C2 maintained higher capping activity than VCE at 50 °C and 55 °C.

[0080] To better quantify the RNA capping activity of VCE and H3C2 at different reaction temperatures, dilutions of the enzymes were incubated at the same reaction conditions of 37°C, 45°C, 50°C, or 55°C for 30 min. The calculated values ​​of the fraction m7Gppp-RNA1 were plotted against the enzyme concentration and fitted to the derivative of the Hill equation to obtain the enzyme concentration that achieved 50% capping (Cap 50). Figure 3A-3B As shown in Table 1, 3.06nM, 0.94nM, 4.08nM, and 30.00nM VCE were required to convert 50% of 0.5μm ppp-RNA1 to m7Gppp-RNA1 at 37°C, 45°C, 50°C, or 55°C, respectively, within 30 minutes. In addition, at 50°C and below, 400nM or more VCE capped >90% of 0.5μm RNA within 30 minutes. For H3C2, 0.69nM, 0.67nM, 2.15nM, or 21.3nM of the enzyme can convert 50% of ppp-RNA1 to m7Gppp-RNA1 within 30 minutes at 37°C, 45°C, 50°C, or 55°C, respectively. In addition, 50nM H3C2 effectively capped >90% of ppp-RNA1 at up to 55°C within 30 minutes.

[0081] Table 1. RNA capping activity of VCE and H3C2 at various temperatures expressed as Cap50 values ​​in nM (A) or milliunits / μl reaction (B).

[0082]

[0083]

[0084]

[0085] Example 2: Identification and testing of H3C2 orthologs

[0086] In vitro, the faustovirus D5b ( Purified RNA capping enzyme H3C2 from faustovirus (Accession No. AMN83561) was active up to 60°C. Several putative orthologs in other faustovirus strains were identified by sequence homology. Gene products of putative RNA capping enzymes from other faustovirus strains and the related giant viruses Acanthamoeba mimivirus and Acanthamoeba mimivirus were found to be active in RNA capping up to 55-60°C. ORFs of the identified orthologs were synthesized and inserted into a T7 expression vector such that expression of the target gene was under the control of the T7 promoter. Expression was performed using the PCR product according to the manufacturer's recommendations. The T7 expression cassette was amplified by PCR using High Fidelity 2× Master Mix (New England Biolabs, Ipswich, MA) according to the manufacturer's instructions. The amplified T7 expression cassette was purified using a PCR & DNA cleanup kit (New England Biolabs, Ipswich, MA) according to the manufacturer's instructions. Purified T7 expression cassettes were subjected to in vitro transcription / translation using an in vitro protein synthesis kit (New England Biolabs, Ipswich, MA). Gene products containing the indicated Genbank accession numbers ( Figures 4A-4E ) PURExpress reaction products were used for RNA capping assay. Briefly, 2 μL PURExpress product, 0.4 μM 150 nt in vitro transcript RNA, 4 μM GTP, 0.1 mM SAM, and trace amount of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8.0) were assembled on ice. 32 50 μL RNA capping reaction consisting of 10 μL 2× RNA loading dye (New England Biolabs, Ipswich, MA) was added to the reaction and analyzed by denaturing polyacrylamide electrophoresis and autoradiography. As shown, the radiographic signal migrated at the position of the substrate RNA indicating capping activity. Figures 4A-4EAs shown, putative RNA capping enzymes from faustovirus ST1 (Genbank Accession No. SME65026; lane 3) and faustovirus LC9 (SMH63629; lane 4), the N-terminal portion of the Mimivirus capping enzyme (amino acids 1-668 of Genbank Accession No. AAV50651; Benarroch et al., 2008; lane 5), and the Mumu virus capping enzyme (Genbank Accession No. YP_007354410; lane 6) capped 150-mer RNAs at temperatures up to 55°C, while RNA capping enzymes from Faustovirus D5b (Genbank Accession No. AMN83561; H3C2; lane 1), Faustovirus E12 (Genbank Accession No. AIB52055; lane 2), and purified H3C2 (lane 7) capped 150-mer RNAs at temperatures up to 60°C.

[0087] Example 3: Sequence Analysis

[0088] Sequence alignment analysis revealed that among the four Faustovirus RNA capping enzymes active up to 55°C, three regions of 60 or 111 amino acids in length exhibited 90% or greater amino acid sequence identity ( Figure 5). The conserved region Fausto_CR_01 spans 60 amino acids within the TPase domain of H3C2. Fausto_CR_03 spans 111 amino acids at the N-terminus of the guanosine N7 methyltransferase domain. Fausto_CR_05 spans 60 amino acids on the C-terminal side of the guanosine N7 methyltransferase domain of H3C2. The amino acid sequence of Fausto_CR_01 (which corresponds to amino acids 43-102 of the H3C2 sequence) is FDKLKPDGEITTTMRVSNADGMAREITFGGGVKTGEMFVKKQNICVFDVVDIFSYKVAVS (SEQ ID NO: 2). The amino acid sequence of Fausto_CR_03 (which corresponds to amino acids 537-647 of the H3C2 sequence) is GFYGNNYKIASDIYLNYIDVFNFDDLWKYNPGYFEKNKSDIYIAPNKYRRYLIKSLFNKYIKNAKWVIDAAAGRGADLHLYKAECVENLLAIDIDPTAISELIRRRNEITG (SEQ ID NO: 3). The amino acid sequence of Fausto_CR_05 (which corresponds to amino acids 778-873 of the H3C2 sequence) is KYSIKRLYDSDKLTKTGQKIAVLLPMSG EMKEEPLCNIKNIISMARKMGLDLVESANFSV (SEQ ID NO: 4).

[0089] The following table summarizes the sequence identity between the amino acid sequences of Faustovirus capping enzymes.

[0090] Table 2

[0091]

[0092] Table 3

[0093]

[0094] Figure 6 An alignment of various Faustovirus RNA capping enzyme sequences is shown, showing the conserved domains as well as the sequences of the TPase, GTase, and MTase regions.

[0095] Sequence alignment analysis also revealed two 67 amino acid or 111 amino acid long regions that were active up to 55°C and exhibited 90% or greater sequence identity between the RNA capping enzymes of A. polyphagous and A. polyphagous viruses ( Figure 7The conserved region moumou_CR_03 (which corresponds to amino acids 576-642 of the moumou virus sequence) spans 67 amino acids between the GTase and guanosine N7 methyltransferase domains. Moumou_CR_04 (which corresponds to amino acids 672-782 of the moumou virus 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 (SEQ ID NO: 5). The amino acid sequence of moumou_CR_04 is YYQKNTSNAAGMRAFNNFIKSNMITTYCKDGDKVLDIGCGRGGDLIKFIHAGIEEYVGIDIDNNGLYVINDSAFNRYKNLKKTIKNIPPMTFINADARGLFNLEAQEKILP (SEQ ID NO: 6).

[0096] The following table summarizes the sequence identity between the amino acid sequences of the Mimivirus and Momovirus RNA capping enzymes.

[0097] Table 4

[0098]

[0099] Table 5

[0100]

[0101] Figure 8 An alignment of mimivirus and moumouvirus sequences is shown, showing the sequences of the conserved domains as well as the TPase, GTase, and MTase regions.

[0102] Example 4: RNA capping reaction at 45°C improves the capping efficiency of short model hairpin RNA

[0103] Figure 1 The short RNAs shown were designed as described herein to assess capping efficiency. The control RNA (RNA1) was designed with a shorter hairpin structure and an unstructured 5' end, with an MFE of -1.5 kcal / mol at 37°C or an MFE of -0.73 kcal / mol at 45°C ( Figure 1RNA2, RNA3, and RNA4 were designed to form stable hairpin structures with a theoretical minimum free energy of unfolding (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 a blunt end, a one-base, or a two-base overhang at the 5' end of each minimum free energy structure ( 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 substrate in the presence of 0.5 mM GTP and 0.1 mM SAM in 10 μL 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 min. At 37°C, H3C2 capped 100% of RNA1, which was predicted to have an unstructured 5' end; and capped 22% of RNA4, which was predicted to have a two-base overhang structure at the 5' end ( Fig. 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 capped only 80% of RNA1. At 45°C, VCE capped 100% of RNA1 and RNA2 and approximately 60% of RNA3 and RNA4.

[0104] Example 5: RNA capping reaction at 45°C improves the capping efficiency of long RNA

[0105] A 1766 nt RNA containing the firefly luciferase gene (flucfluc) was generated by in vitro transcription with T7 RNA polymerase. 400 nM of flucRNA was incubated with the indicated concentrations of H3C2 or VCE at 37°C or 45°C for 30 minutes in the presence of 0.5 mM GTP and 0.1 mM SAM in 10 μL 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8). A target oligomer (TO) designed to direct RNaseH to cleave a 25 nt 5' fragment was then added to each capping reaction at a final concentration of 2.5 μM, and the capping reaction was then heated at 80°C for 30 s to inactivate the capping enzyme and then slowly cooled to room temperature. Thermostable RNaseH (New England Biolabs, Ipswich, MA) was added to each reaction at a final concentration of 0.5 U / μL and then incubated at 37°C for 1 hour. The RNase H reaction was then analyzed by 15% urea polyacrylamide gel electrophoresis. The gel was stained with ELISA Gold (Thermo Fisher Scientific, Waltham, MA) (according to the manufacturer's instructions) and then stained with The Cy2 channel was scanned using an RGB (GE Healthcare, Marlborough, MA) scanner. The intensities of the capped and uncapped bands were normalized to the intensity of TO in the same lane. The normalized values ​​were used to calculate the fraction of RNA that was capped under each condition. The calculated values ​​were plotted against the enzyme concentration and fitted to the derivative of the Hill equation to obtain the enzyme concentration that achieved 50% capping (Cap50). For H3C2, the Cap50 values ​​at 37°C or 45°C were 33.6 nM and 0.96 nM, respectively. For VCE, the Cap50 values ​​at 37°C or 45°C were 103 nM and 2.97 nM, respectively (hereinafter referred to as "Cap50"). Fig.10 and Table 6). For both enzymes, when the capping reaction was performed at 45°C, much less enzyme was required to achieve 50% capping.

[0106] Table 6. RNA capping activity of VCE and H3C2 on 1.7 kb fluc in vitro transcripts at 37°C and 45°C, expressed as Cap50 values.

[0107]

[0108] The activity of these enzymes can be expressed in milliunits / μL reaction as follows:

[0109]

[0110] To show that temperature-enhanced capping efficiency can be achieved on other long RNA molecules, the capping efficiency of H3C2 and VCE was studied by mass spectrometry readout using in vitro transcripts of cypridina luciferase (cluc; 1823nt) and cystic fibrosis transmembrane receptor (CFTR; 4712nt). Briefly, 0.5 μM of cluc or CFTR in vitro transcripts were incubated with 100 nM of H3C2 or VCE at 37°C or 45°C for 30 minutes in the presence of 0.1 mM SAM and 0.5 mM GTP in a reaction volume of 100 μL. The reaction was stopped by heating at 80°C for 30 seconds in the presence of 2.5 μM of the target oligomer (composed 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 / s. The reaction was then subjected to RNaseH cleavage by incubation with a final concentration of 0.5 U / μL of thermostable RNaseH (New England Biolabs, Ipswich, MA) at 37°C for 1 h. XP beads for size selection and streptavidin magnetic beads for target selection to purify the 5' fragments of the RNaseH cleavage reaction. Briefly, 100 μL of the RNaseH cleavage reaction was added to 200 μL of XP beads and incubated at room temperature for 5 minutes. The beads were then placed in a magnetic field and the clear supernatant was retrieved and added to the pre-cleared 200 μL bead suspension. XP beads. After incubation at room temperature for 5 minutes, the beads were placed in a magnetic field. The clear supernatant was added to the 200 μL In the pre-cleaned beads of MyOne Streptavidin C1. After washing 4 times with 200 μL wash buffer (5mMTris, pH 7.5, 0.5mM EDTA, 1M NaCl), the bound RNA was eluted by incubating the clarified beads with 50 μL biotin elution buffer (1mM biotin, 5mM Tris, pH 7.5, 0.1M NaCl, 0.1M NaCl) at 37°C for 1 hour. The eluted RNA was then analyzed by LC / MS by an external contractor, Novatia LLC, to determine the relative amount of the target mass. The degree of RNA capping was assessed by the mass intensity ratio of the capped and uncapped RNA species.

[0111] like Fig.11 As shown in , both H3C2 and VCE exhibited more capping activity on cluc RNA and CFTR RNA at 45°C than at 37°C.

[0112] Example 6: Efficient one-pot enzymatic synthesis of cap-1 structures on RNA triphosphates at elevated temperatures

[0113] To verify whether vaccinia virus cap 2'OMTase is active at elevated temperatures, a reaction containing 5 μM chemically synthesized cap-O RNA1 (25 nt) and 20 μL 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, followed by the addition of 100 U of vaccinia virus cap 2'OMTase (New England Biolabs, Ipswich, Massachusetts). The reaction was allowed to proceed at the preheated temperature for 30 minutes, and then stopped by heating at 70°C for 10 minutes. RNA was purified from the reaction components using an oligo clean-up and concentration kit (Oligo Clean-up and Concentration Kit) (Norgen Biotek, Thorold, Canada). The purified RNA was then digested into nucleosides and cap structures by incubation with 2 μL of nucleotide digestion mix (New England Biolabs, Ipswich, MA) in a 20 μL reaction containing nucleoside digestion mix reaction buffer (50 mM sodium acetate, pH 5.4, 1 mM ZnCl2) at 37°C for 1 hour. The purified RNA was then analyzed using an Agilent 1290 Infinity II UHPLC (Agilent Technologies, Santa Clara, CA) on a Waters XSelect TM Nucleoside digestion reactions were analyzed on an HSS T3 XP column (Waters Corporation, Milford, MA) (2.1 × 100 mm, 2.5 μm) with a gradient mobile phase consisting of methanol and 10 mM potassium phosphate buffer (pH 7.0). Fig.12 As shown in , more cap-0 RNA was methylated at 45°C and 50°C than at 37°C.

[0114] To demonstrate one-pot enzymatic synthesis of cap-1 structures on 5' triphosphate RNA at elevated temperatures, 5 μM chemically synthesized 5' triphosphate RNA1 was incubated with 200 U of vaccinia virus cap 2'OMTase (final concentration 5 U / μL) 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 in a 40 μL reaction containing 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8.0). The reactions were directly analyzed by LC / MS to determine the relative amount of mass and mass. When combined with 50 nM VCE, although 5 U / μL of 2'OMTase generated only about 50% of cap-01 RNA at 37°C, it generated 100% of cap-1 RNA at 45°C ( Fig.13). When combined with 50 nM H3C2, 5 U / μL of vaccinia 2'OMTase generated approximately 90% of cap-1 RNA at 37°C and 100% of cap-1 RNA at 45°C ( Fig.13 ). Thus, carrying out the reaction at 45°C in the presence of VCE roughly doubled the yield of cap-1 RNA, whereas carrying out the reaction at 45°C in the presence of H3C2 increased the yield of cap-1 RNA.

[0115] Example 7: One step using separate RNA polymerases, RNA capping enzymes and Cap 2'O methyltransferase Cap-0 Cap-1 Cap-0 and cap-1 RNA synthesis

[0116] A. One-step synthesis of capped RNA using vaccinia capping enzyme is inefficient

[0117] As indicated for transcription and capping of the 1.7 kb fluc transcript (SEQ ID NO: 18), 5 U / μl of T7 RNA polymerase (New England Biolabs; Catalog No. M0251), Hi-T7 RNA polymerase (New England Biolabs; Catalog No. M0658), 1 U / μl of vaccinia capping enzyme (New England Biolabs; Catalog No. M2080), and 5 U / μl of vaccinia mRNA cap 2' O-methyltransferase (New England Biolabs; Catalog No. M0366) were used according to the manufacturer's recommendations for the corresponding reactions. When using T7 RNA polymerase, the reaction contained 1×T7 RNA polymerase buffer (40 mM Tris-HCl, pH 7.9, 10 mM NaCl, 1 mM DTT, 2 mM spermidine), or when using Hi-T7 RNA polymerase, the reaction contained 1×Hi-T7 RNA polymerase buffer (40 mM Tris-HCl, pH 7.9, 60 mM NaCl, 1 mM DTT, 2 mM spermidine). The reaction was supplemented with 19 mM MgCl2 and 5 mM each of ATP, UTP, GTP, CTP, and 0.5 mM SAM. The reaction was allowed to proceed for 1 hour at 37°C, which is the recommended reaction temperature and duration for T7 RNA polymerase and vaccinia capping enzyme.

[0118] To assess the yield of in vitro transcription, after 1 hour of incubation, for each reaction, 1 μL of the reaction mixture 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. The total RNA concentration of the DNase I reaction was then analyzed using the Qubit RNA Broad Range Kit (Thermo Fisher) according to the manufacturer's instructions. To verify the size and integrity of the transcripts, DNase I reactions were analyzed using 2% E-Gel (Thermo Fisher) and images were taken using a Typhoon RGB scanner (GE Healthcare). The results are shown in Fig.14 The output of the transcription is shown in .

[0119] To analyze the extent of RNA capping, the one-step capped RNA synthesis reaction was analyzed by RNaseH cleavage followed by complete LC / MS analysis. In brief, the one-step capped RNA synthesis reaction was stopped by heating at 80°C for 30s in the presence of 2.5 μM of the target oligomer (TO-1) (composed of 5' deoxynucleotides and 3' ribonucleotides) and the TEG-desthiobiotin group (SEQ ID NO: 19). The reaction was cooled to 25°C at a rate of 0.1°C / s. The reaction was then RNaseH cleaved by incubation at 37°C with a final concentration of 0.5U / μL of thermostable RNaseH (New England Biolabs; catalog number M0523) for 1h. In order to facilitate the analysis of the 5' group using capillary electrophoresis, FAM-labeled nucleotides were added to the 3' end of the RNaseH cleavage fragment. Briefly, 5 μL of the RNaseH reaction was recovered and added to 5 μL of a solution containing 2×NEBuffer 2, 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 for 1 hour at 37°C. In some cases, a small portion of the Klenow reaction was analyzed on urea PAGE to verify the success of capping. Briefly, 2 μL of the Klenow reaction was recovered and added to 8 μL of 2× RNA loading dye (New England Biolabs). The mixture was then analyzed by urea-15% polyacrylamide gel electrophoresis. Gel images were acquired using the Cy2 channel using a Typhoon RGB scanner (GE Healthcare).

[0120] To analyze the 5' group status of the coupled transcription / capping reaction products using capillary electrophoresis or mass spectrometry, the 5' group status of the coupled transcription / capping reaction products was then determined by using XP beads (Thermo Fisher) and subsequent selection using streptavidin magnetic beads to purify Klenow reaction products (RNase H-cleavage products still annealed to the desthiobiotinylated target oligomer). Briefly, 45 μL of nuclease-free water was added to 5 μL of RNase H cleavage reaction, which was then added to 100 μL of The sample was added to the purification beads (New England Biolabs) and incubated at room temperature for 5 minutes. The beads were then placed next to a magnet at room temperature for 2 minutes. The clear supernatant was recovered and added to the pre-cleared 100 μL bead suspension. After incubation at room temperature for 5 min, the beads were placed next to a magnet at room temperature for 2 min. The clear supernatant was added to the 50 μL MyOne Streptavidin C1 (Thermo Fisher) pre-cleared beads. After washing 4 times with 50 μL low salt wash buffer (5 mM Tris, pH 7.5, 0.5 mM EDTA, 60 mM NaCl), bound RNA was eluted by incubating the cleared beads in 10 μL nuclease-free water. 120LIZ dye size standard (Applied Biosystems) was used to analyze the eluted RNA by capillary electrophoresis on an Applied Biosystems 3130xl Genetic Analyzer (16 capillary array) or an Applied Biosystems 3730xl Genetic Analyzer (96 capillary array). The reaction products were analyzed using PeakScanner software (Thermo Fisher Scientific) and an in-house software suite. The peak areas corresponding to transcripts cleaved by m7Gppp-, unmethylated Gppp-, uncapped pp-, and ppp-RNaseH were quantified and used to calculate the fractions of ppp-, pp-, Gppp-, and m7Gppp- transcripts in the capped RNA synthesis reaction.

[0121] To determine the relative amounts of m7GpppGm-(cap-1), m7G-(cap-0), non-methyl-G-capped and uncapped RNAs using mass spectrometry, intact masses of the relevant species were determined using LC / MS performed by an external contractor, Novatia LLC (Newtown, PA) or in-house facilities. The extent of RNA capping was assessed by the ratio of mass intensities of the relevant RNase H cleavage products.

[0122] Fig.14Results for one-step transcription using current commercially available reaction conditions are shown. Comparing bars 1 and 2, the presence of VCE did not significantly affect the transcriptional output of T7 RNA polymerase, but no detectable m7GpppG-(cap-0) transcripts were generated. A small fraction (5.4%) of unmethylated-G capped RNA was detected, an undesirable capping reaction intermediate. In addition, approximately 50% of the transcripts contained a 5' diphosphate group, another capping reaction intermediate. As shown in bar 3, when vaccinia cap 2'O methyltransferase was included in the reaction, the transcriptional output was not significantly affected, and no m7GpppGm-(cap-1) transcripts were detected. Approximately 50% of the transcripts had a 5' diphosphate group, and approximately 15% of the transcripts had a non-methylated-G capping intermediate. When Hi-T7 RNA polymerase was used instead of T7 RNA polymerase, results similar to those of the T7 RNA polymerase reaction were observed—the presence of VCE had no significant effect on Hi-T7 RNA polymerase transcription, but no detectable levels of cap-0 transcripts were generated. Addition of vaccinia cap-2'O methyltransferase to the reaction did not produce any cap-1 transcripts. From these results, it appears that the enzyme reagents and reaction conditions that are currently standard for in vitro mRNA synthesis (i.e., RNA synthesis followed by mRNA capping) do not support the in vitro generation of capped mRNA in a single reaction (i.e., one-step capped RNA synthesis).

[0123] B. Faustovirus RNA capping enzyme H3C2 efficiently caps transcripts in a one-step reaction in vitro

[0124] Since the reagents and conditions of Example 7A were ineffective for generating capped transcripts in an in vitro one-step reaction, the new enzymes and reaction conditions were tested for their ability to synthesize in vitro one-step capped RNA synthesis. Fig.13 Shown are the results of one-step capped RNA synthesis at 37°C or 45°C using high concentrations of H3C2 capping enzyme.

[0125] At 37°C, 0.5 μM H3C2 capping enzyme generated 12% m7Gppp-capped transcripts and up to 24.1% unmethylated-G capped transcripts measured by capillary electrophoresis. On the other hand, at 45°C, up to 61% of transcripts had m7Gppp-caps, while only 1.4% of transcripts had unmethylated-G caps. At both temperatures, the transcription output was 50-60% of that without H3C2 capping enzyme. Thus, increasing H3C2 concentration and reaction temperature increased the yield of cap-0 transcripts in one-step capped RNA synthesis using T7 RNA polymerase and H3C2 RNA capping enzyme.

[0126] The effect of further expanding the range of enzyme reagents and reaction temperatures was tested by performing one-step capped RNA synthesis at 45°C and 50°C Cap-0 using different combinations of T7 RNA polymerase, Hi-T7 RNA polymerase, VCE, H3C2, and vaccinia cap 2'O methyltransferase. Specifically, one-step capped RNA synthesis reactions were performed as described in Example 7A using 0.5 μM VCE or H3C2 capping enzyme in combination with 5 U / μL T7 RNA polymerase or Hi-T7 RNA polymerase as indicated in their respective reaction buffers in the presence or absence of 5 U / μL vaccinia mRNA cap 2'O methyltransferase. Reactions were allowed to proceed at 45 or 50°C for 1 hour.

[0127] Fig.14The results of this expanded measurement are summarized. 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 capped with m7Gppp (cap-0), while 8% of transcripts were capped with unmethylated-G (bar 2). In the presence of H3C2 and cap 2'O methyltransferase, 87% of transcripts had m7GpppGm caps (cap-1), 6% of transcripts had unmethylated-G caps, and cap-0 was not detected (bar 3). When VCE was used, 76% of transcripts had cap-0 structures, and 24% of transcripts had unmethylated-G cap structures (bar 4). In the presence of both VCE and cap 2'O methyltransferase, 55% of transcripts had cap-1 structures, 36% of transcripts had cap-0 structures, and 8% of transcripts had unmethylated-G cap structures. At 45°C, RNA yield was not significantly affected when H3C2 or VCE was present in the reaction. However, when Cap 2'O methyltransferase and either capping enzyme were present, RNA yield decreased from 0.14 μg / μL to 0.09 μg / μL. In capped RNA synthesis reactions using Hi-T7 RNA polymerase at 45°C (bars 6-10), a smaller portion of transcripts were capped compared to reactions using T7 RNA polymerase (bars 1-5). H3C2 capping enzyme and VCE generated 45% (bar 7) and 50% of Cap-0 transcripts (bar 9), respectively. H3C2 combined with Cap 2'O methyltransferase generated 44% Cap-1 transcripts, 39% unmethylated-G capped transcripts, and undetectable Cap-0 transcripts (bar 8). VCE combined with Cap 2'O methyltransferase generated 19% Cap-1 transcripts, 22% Cap-0 transcripts, and 42% unmethylated-G capped transcripts (bar 10). Interestingly, the transcriptional 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 capped RNA synthesis reactions were performed at 50°C using Hi-T7 RNA polymerase, H3C2 capping enzyme generated 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 Cap-1 structures, 2% of transcripts contained Cap-0 structures, and 26% of transcripts contained unmethylated-G cap structures (bar 13). When VCE was used, 55% of transcripts had unmethylated-G capped structures, and no cap-0 transcripts were detected. When both VCE and cap 2'O methyltransferase were present, 23% of transcripts had cap-0 structures, and 31% of transcripts had unmethylated-G capped structures.

[0128] In summary, this example provides one-step reaction conditions that utilize H3C2 RNA capping enzyme in combination with T7 RNA polymerase or Hi-T7 RNA polymerase to generate in vitro cap-0 RNA with up to 80% efficiency (Table 7). This example also describes one-step reaction conditions that can generate cap-1 RNA with 70-80% efficiency using H3C2 RNA capping enzyme, vaccinia mRNA cap 2'O methyltransferase in combination with T7 RNA polymerase or Hi-T7 RNA polymerase (Table 8).

[0129] Table 7

[0130]

[0131] Table 8

[0132]

[0133] Example 8: One-step Cap-O RNA synthesis using H3C2:T7 RNA polymerase fusion protein

[0134] Fusion constructs were prepared to produce a protein consisting of H3C2 RNA capping enzyme and T7 RNA polymerase connected by a putative flexible linker sequence (SEQ ID NO: 20). The fusion protein was encoded by a DNA sequence (SEQ ID NO: 21) under the control of a T7 promoter or a tac promoter, expressed in E. coli, and purified using standard chromatography techniques.

[0135] In the presence of 0.5 μM H3C2: T7 RNA polymerase fusion, a one-step capped RNA synthesis reaction was performed as indicated in Example 7 for 1.7 kbfluc transcripts in 1×T7 RNA polymerase buffer supplemented with 19 mM MgCl2 and 5 mM each of ATP, UTP, GTP, CTP, and 0.1 mM SAM. For comparison, reactions containing 5 U / μL T7 RNA polymerase with 0.5 μM H3C2 capping enzyme or 5 U / μL T7 RNA polymerase but without 0.5 μM H3C2 capping enzyme were performed. The reactions were performed at 37 or 45°C for 1 hour. As indicated in Example 7, RNA yields and the fractions of capped and uncapped transcripts were measured and analyzed.

[0136] The results are as follows Fig.15As shown in. When reacted at 37°C, the H3C2:T7 fusion protein generated 75% of m7Gppp capped (cap-0) transcripts and 20% of unmethylated-G capped transcripts (bar 1). Under the same conditions, the separate enzymes H3C2 and T7 RNA polymerase only generated 12% of cap-0 transcripts and 27% of unmethylated-G capped transcripts (bar 2). The transcription output decreased from 0.45 μg / μL when only T7 RNA polymerase was present (bar 3) to 0.39 μg / μL when H3C2 was added, and further decreased to 0.19 μg / μL when the H3C2:T7 fusion was used for transcription. At 45°C, the H3C2:T7 RNA polymerase fusion generated 95% cap-0 transcripts and 5% unmethylated-G capped transcripts (bar 4), compared to 60% cap-0 and 6% unmethylated-G capped transcripts when using the enzyme alone (bar 5). Similar to reactions performed at 37°C, transcription output decreased from 0.31 μg / μL when T7 RNA polymerase alone was present (bar 6) to 0.18 μg / μL when H3C2 was added, or to 0.12 μg / μL when transcription was performed using the H3C2:T7 fusion.

[0137] Thus, under the described reaction conditions, this example demonstrates efficient (eg, up to 98%) one-step in vitro cap-O RNA synthesis using the H3C2:T7 RNA polymerase fusion protein.

[0138] Example 9: RNA capping enzymes efficiently cap in vitro transcripts containing pseudouridine

[0139] Approximately 1800 nt RNA encoding the Cypridina luciferase protein with a 120 nt poly(A) tail (cluc / A120) was synthesized by in vitro transcription using T7 RNA polymerase (New England Biolabs) in the presence of pseudouridine triphosphate (Trilink Biotechnologies) or unmodified uridine triphosphate (New England Biolabs). 500 nM unmodified or pseudouridine cluc / A120 transcripts were incubated with 100 nM H3C2 or VCE at 37°C for 30 minutes in the presence of 0.5 mM GTP and 0.1 mM SAM in 10 mL 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, pH 8). Target oligomers (TOs) designed to direct RNase H to cleave the 5' fragment were then added to each capping reaction to achieve a final TO concentration of 2.5 μM. Each reaction was then heated at 80°C for 30 s to anneal the TO to the transcript and inactivate the capping enzyme and slowly cooled to room temperature. Thermostable RNaseH (New England Biolabs) was added to each reaction at a final concentration of 0.5 U / mL and incubated at 37°C for 1 h. The reaction products were then purified and analyzed by complete LC / MS as described in Example 7. Fig.16 As shown in , VCE and H3C2 capped 68.4% or 100% of the cluc / A120 transcripts containing pseudouridine, respectively. It is noteworthy that these two capping enzymes capped a higher fraction of cluc / A120 transcripts containing pseudouridine compared to their uridine counterparts.

[0140] Example 10: Expanded Cap-1 Capping and Functional Assays

[0141] A total of 250 pmol (145 μg) of approximately 1800 nt cluc / A120 in vitro transcripts were capped at 45°C for 1 h in a 500 μL reaction 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 vaccinia cap 2'O methyltransferase (NEB M0366) and 500 nM H3C2. The efficiency of cap-1 formation was assessed by RNaseH / intact LC / MS as described in Example 7. RNA was purified with acidic phenol and chloroform, then precipitated with ethanol and rehydrated in nuclease-free water. RNA concentration was estimated using the Qubit RNA (Broad Range) Kit (Thermo Fisher). The translation efficiency of capped cluc / A120 transcripts was measured as the relative luciferase activity from HEK293 cells transfected with capped cluc / A120 transcripts relative to uncapped cluc / A120 transcripts. HEK293 cells were transfected with 250 ng of purified cluc / A120 transcripts using Lipofectamine MessengerMax transfection reagent (Thermo Fisher). Four hours after transfection, cells were transfected with Luciferase activity was determined on 10 μL of culture supernatant using the Lupinus luciferase assay kit (New England Biolabs, Ipswich). Luminescence was measured using a Centro LB960 microplate luminometer from Berthold technologies. Fig.17 As shown in , the expanded capping reaction achieved 47.7% and 54.8% cap-1 formation on cluc / A120 containing rU and pseudouridine, respectively, which is consistent with Examples 5 and Fig.11 The results of luciferase assay (triplicates) showed that the translation efficiency of purified capped transcripts containing rU and pseudouridine showed high luciferase activity ( Fig.18 ), indicating that RNA capped with H3C2 at 45° C. is functionally active. It should be noted that modifications to reaction conditions such as reaction volume, concentrations of enzymes and reaction components, reaction time, and reaction temperature can improve the efficiency of cap-1 formation of bulk RNA and the efficiency of translation of the resulting cap-1 RNA.

[0142] Example 11: Single-Vessel Capped RNA Synthesis Using Multiple Temperature Steps

[0143] As indicated in Example 7, a single-vessel capped RNA synthesis reaction was performed on the 1.7 kb fluc transcript, i.e., 5 U / ul of T7 RNA polymerase (New England Biolabs; Catalog No. M0251) was incubated with or without 500 nM vaccinia capping enzyme (New England Biolabs; Catalog No. M2080) or H3C2 capping enzyme at 37°C for 30 minutes, and then incubated at 28°C, 32°C, 37°C, 45°C, or 50°C for another 30 minutes. The reaction contained 1×T7 RNA polymerase buffer (40 mM Tris-HCl, pH 7.9, 10 mM NaCl, 1 mM DTT, 2 mM spermidine) and supplemented with 19 mM MgCl2 and 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 RNaseH cleavage followed by Klenow fill-in and urea PAGE with FAM-dCTP as indicated in Example 7. Selected reactions were further analyzed by full mass spectrometry. Fig.19 As shown in , for VCE, the m7Gppp formation efficiency peaked at the second reaction temperature of 45°C, while for H3C2, the m7Gppp formation efficiency peaked at the second reaction temperature of 50°C. On the other hand, T7 RNAP transcription output peaked at 45°C when combined with VCE or H3C2, reflecting low transcriptional activity at the lower and upper ends of the tested temperatures (28°C and 50°C, respectively). Therefore, in this example, a tandem temperature step of 37°C for 30 minutes followed by 45°C for 30 minutes produced the best balance of transcriptional output and capping efficiency. Modifications to reaction conditions such as reaction time, reaction temperature, and number of temperature steps can further improve the transcription yield and fraction of m7G-capped transcripts. Sequence Listing <110> New England Biolabs <120> Enzymatic RNA capping method <130> NEB-423 <150> 62 / 890,821 <151> 2019-08-23 <160> twenty one <170> PatentIn Version 3.5 <210> 1 <211> 844 <212> PRT <213> Vaccinia virus <220> <221> misc_feature <223> FL VCE capping enzyme; >YP_232988.1 Large subunit of mRNA capping enzyme <400> 1 Met Asp Ala Asn Val Val Ser Ser Ser Thr Ile Ala Thr Tyr Ile Asp 1 5 10 15 Ala Leu Ala Lys Asn Ala Ser Glu Leu Glu Gln Arg Ser Thr Ala Tyr 20 25 30 Glu Ile Asn Asn Glu Leu Glu Leu Val Phe Ile Lys Pro Pro Leu Ile 35 40 45 Thr Leu Thr Asn Val Val Asn Ile Ser Thr Ile Gln Glu Ser Phe Ile 50 55 60 Arg Phe Thr Val Thr Asn Lys Glu Gly Val Lys Ile Arg Thr Lys Ile 65 70 75 80 Pro Leu Ser Lys Val His Gly Leu Asp Val Lys Asn Val Gln Leu Val 85 90 95 Asp Ala Ile Asp Asn Ile Val Trp Glu Lys Lys Ser Leu Val Thr Glu 100 105 110 Asn Arg Leu His Lys Glu Cys Leu Leu Arg Leu Ser Thr Glu Glu Arg 115 120 125 His Ile Phe Leu Asp Tyr Lys Lys Tyr Gly Ser Ser Ile Arg Leu Glu 130 135 140 Leu Val Asn Leu Ile Gln Ala Lys Thr Lys Asn Phe Thr Ile Asp Phe 145 150 155 160 Lys Leu Lys Tyr Phe Leu Gly Ser Gly Ala Gln Ser Lys Ser Ser Leu 165 170 175 Leu His Ala Ile Asn His Pro Lys Ser Arg Pro Asn Thr Ser Leu Glu 180 185 190 Ile Glu Phe Thr Pro Arg Asp Asn Glu Thr Val Pro Tyr Asp Glu Leu 195 200 205 Ile Lys Glu Leu Thr Thr Leu Ser Arg His Ile Phe Met Ala Ser Pro 210 215 220 Glu Asn Val Ile Leu Ser Pro Pro Ile Asn Ala Pro Ile Lys Thr Phe 225 230 235 240 Met Leu Pro Lys Gln Asp Ile Val Gly Leu Asp Leu Glu Asn Leu Tyr 245 250 255 Ala Val Thr Lys Thr Asp Gly Ile Pro Ile Thr Ile Arg Val Thr Ser 260 265 270 Asn Gly Leu Tyr Cys Tyr Phe Thr His Leu Gly Tyr Ile Ile Arg Tyr 275 280 285 Pro Val Lys Arg Ile Ile Asp Ser Glu Val Val Val Phe Gly Glu Ala 290 295 300 Val Lys Asp Lys Asn Trp Thr Val Tyr Leu Ile Lys Leu Ile Glu Pro 305 310 315 320 Val Asn Ala Ile Asn Asp Arg Leu Glu Glu Ser Lys Tyr Val Glu Ser 325 330 335 Light Leu Val Asp Ile Cys Asp Arg Ile Val Phe Light Ser Light Light Tyr 340 345 350 Glu Gly Pro Phe Thr Thr Thr Ser Glu Val Val Asp Met Leu Ser Thr 355 360 365 Tyr Leu Pro Lys Gln Pro Glu Gly Val Ile Leu Phe Tyr Ser Lys Gly 370 375 380 Pro Lys Ser Asn Ile Asp Phe Lys Ile Lys Lys Glu Asn Thr Ile Asp 385 390 395 400 Gln Thr Ala Asn Val Val Phe Arg Tyr Met Ser Ser Glu Pro Ile Ile 405 410 415 Phe Gly Glu Ser Ser Ile Phe Val Glu Tyr Lys Lys Phe Ser Asn Asp 420 425 430 Lys Gly Phe Pro Lys Glu Tyr Gly Ser Gly Lys Ile Val Leu Tyr Asn 435 440 445 Gly Val Asn Tyr Leu Asn Asn Ile Tyr Cys Leu Glu Tyr Ile Asn Thr 450 455 460 His Asn Glu Val Gly Ile Lys Ser Val Val Val Pro Ile Lys Phe Ile 465 470 475 480 Ala Glu Phe Leu Val Asn Gly Glu Ile Leu Lys Pro Arg Ile Asp Lys 485 490 495 Thr Met Lys Tyr Ile Asn Ser Glu Asp Tyr Tyr Gly Asn Gln His Asn 500 505 510 Ile Ile Val Glu His Leu Arg Asp Gln Ser Ile Lys Ile Gly Asp Ile 515 520 525 Phe Asn Glu Asp Lys Leu Ser Asp Val Gly His Gln Tyr Ala Asn Asn 530 535 540 Asp Lys Phe Arg Leu Asn Pro Glu Val Ser Tyr Phe Thr Asn Lys Arg 545 550 555 560 Thr Arg Gly Pro Leu Gly Ile Leu Ser Asn Tyr Val Lys Thr Leu Leu 565 570 575 Ile Ser Met Tyr Cys Ser Lys Thr Phe Leu Asp Asp Ser Asn Lys Arg 580 585 590 Lys Val Leu Ala Ile Asp Phe Gly Asn Gly Ala Asp Leu Glu Lys Tyr 595 600 605 Phe Tyr Gly Glu Ile Ala Leu Leu Val Ala Thr Asp Pro Asp Ala Asp 610 615 620 Ala Ile Ala Arg Gly Asn Glu Arg Tyr Asn Lys Leu Asn Ser Gly Ile 625 630 635 640 Lys Thr Lys Tyr Tyr Lys Phe Asp Tyr Ile Gln Glu Thr Ile Arg Ser 645 650 655 Asp Thr Phe Val Ser Ser Val Arg Glu Val Phe Tyr Phe Gly Lys Phe 660 665 670 Asn Ile Ile Asp Trp Gln Phe Ala Ile His Tyr Ser Phe His Pro Arg 675 680 685 His Tyr Ala Thr Val Met Asn Asn Leu Ser Glu Leu Thr Ala Ser Gly 690 695 700 Gly Lys Val Leu Ile Thr Thr Met Asp Gly Asp Lys Leu Ser Lys Leu 705 710 715 720 Thr Asp Lys Lys Thr Phe Ile Ile His Lys Asn Leu Pro Ser Ser Glu 725 730 735 Asn Tyr Met Ser Val Glu Lys Ile Ala Asp Asp Arg Ile Val Val Tyr 740 745 750 Asn Pro Ser Thr Met Ser Thr Pro Met Thr Glu Tyr Ile Ile Lys Lys 755 760 765 Asn Asp Ile Val Arg Val Phe Asn Glu Tyr Gly Phe Val Leu Val Asp 770 775 780 Asn Val Asp Phe Ala Thr Ile Ile Glu Arg Ser Lys Lys Phe Ile Asn 785 790 795 800 Gly Ala Ser Thr Met Glu Asp Arg Pro Ser Thr Arg Asn Phe Phe Glu 805 810 815 Leu Asn Arg Gly Ala Ile Lys Cys Glu Gly Leu Asp Val Glu Asp Leu 820 825 830 Leu Ser Tyr Tyr Val Val Tyr Val Phe Ser Lys Arg 835 840 <210> 2 <211> 60 <212> PRT <213> Faustovirus <220> <221> MISC_FEATURE <223> Fausto_CR_01 <400> 2 Phe Asp Lys Leu Lys Pro Asp Gly Glu Ile Thr Thr Thr Met Arg Val 1 5 10 15 Ser Asn Ala Asp Gly Met Ala Arg Glu Ile Thr Phe Gly Gly Gly Val 20 25 30 Lys Thr Gly Glu Met Phe Val Lys Lys Gln Asn Ile Cys Val Phe Asp 35 40 45 Val Val Asp Ile Phe Ser Tyr Lys Val Ala Val Ser 50 55 60 <210> 3 <211> 111 <212> PRT <213> Faustovirus <220> <221> misc_feature <223> Fausto_CR_03 <400> 3 Gly Phe Tyr Gly Asn Asn Tyr Lys Ile Ala Ser Asp Ile Tyr Leu Asn 1 5 10 15 Tyr Ile Asp Val Phe Asn Phe Asp Asp Leu Trp Lys Tyr Asn Pro Gly 20 25 30 Tyr Phe Glu Lys Asn Lys Ser Asp Ile Tyr Ile Ala Pro Asn Lys Tyr 35 40 45 Arg Arg Tyr Leu Ile Lys Ser Leu Phe Asn Lys Tyr Ile Lys Asn Ala 50 55 60 Lys Trp Val Ile Asp Ala Ala Ala Gly Arg Gly Ala Asp Leu His Leu 65 70 75 80 Tyr Lys Ala Glu Cys Val Glu Asn Leu Leu Ala Ile Asp Ile Asp Pro 85 90 95 Thr Ala Ile Ser Glu Leu Ile Arg Arg Arg Asn Glu Ile Thr Gly 100 105 110 <210> 4 <211> 60 <212> PRT <213> Faustovirus <220> <221> MISC_FEATURE <223> Fausto_CR_05 <400> 4 Lys Tyr Ser Ile Lys Arg Leu Tyr Asp Ser Asp Lys Leu Thr Lys Thr 1 5 10 15 Gly Gln Lys Ile Ala Val Leu Leu Pro Met Ser Gly Glu Met Lys Glu 20 25 30 Glu Pro Leu Cys Asn Ile Lys Asn Ile Ile Ser Met Ala Arg Lys Met 35 40 45 Gly Leu Asp Leu Val Glu Ser Ala Asn Phe Ser Val 50 55 60 <210> 5 <211> 67 <212> PRT <213> Mimivirus <220> <221> MISC_FEATURE <223> 5: mimi_CR_03 <400> 5 Ile Asn Asp Asn Thr Val Val Glu Phe Ile Phe Asp Asn Phe Lys Ile 1 5 10 15 Asp Met Asp Asp Pro Tyr Lys Trp Ile Pro Ile Arg Thr Arg Tyr Asp 20 25 30 Lys Thr Glu Ser Val Gln Lys Tyr His Lys Lys Tyr Gly Asn Asn Leu 35 40 45 His Ile Ala Asn Arg Ile Trp Lys Thr Ile Thr Asn Pro Ile Thr Glu 50 55 60 Asp Ile Ile 65 <210> 6 <211> 111 <212> PRT <213> Mimivirus <220> <221> various_features <223> mimi_CR_04 <400> 6 Tyr Tyr Gln Lys Asn Thr Ser Asn Ala Ala Gly Met Arg Ala Phe Asn 1 5 10 15 Asn Phe Ile Lys Ser Asn Met Ile Thr Thr Tyr Cys Lys Asp Gly Asp 20 25 30 Lys Val Leu Asp Ile Gly Cys Gly Arg Gly Gly Asp Leu Ile Lys Phe 35 40 45 Ile His Ala Gly Ile Glu Glu Tyr Val Gly Ile Asp Ile Asp Asn Asn 50 55 60 Gly Leu Tyr Val Ile Asn Asp Ser Ala Phe Asn Arg Tyr Lys Asn Leu 65 70 75 80 Lys Lys Thr Ile Lys Asn Ile Pro Pro Met Thr Phe Ile Asn Ala Asp 85 90 95 Ala Arg Gly Leu Phe Asn Leu Glu Ala Gln Glu Lys Ile Leu Pro 100 105 110 <210> 7 <211> 873 <212> PRT <213> Faustovirus <220> <221> Various features <223> D5b RNA capping enzyme of Float virus Genbank accession number AMN83561 <400> 7 Met Ala Lys Arg Leu Gln Arg Cys Gln Asp Val Asn Gln Val Cys Glu 1 5 10 15 Ile Tyr Asn Ser Lys Gly Gly Ile Gly Glu Leu Glu Leu Arg Phe Asp 20 25 30 Lys Leu Pro Gln Asn Leu Phe Ala Gly Val Phe Asp Lys Leu Lys Pro 35 40 45 Asp Gly Glu Ile Gln Thr Thr Met Arg Val Ser Asn Arg Asp Gly Val 50 55 60 Ala Arg Glu Ile Thr Phe Gly Gly Gly Val Lys Thr Asn Glu Ile Phe 65 70 75 80 Val Lys Lys Gln Asn Ile Cys Val Phe Asp Val Val Asp Ile Phe Ser 85 90 95 Tyr Lys Val Ala Val Ser Thr Glu Glu Thr Val Val Glu Lys Pro Thr 100 105 110 Met Glu Thr Thr Ala Gly Val Arg Phe Lys Ile Arg Leu Ser Val Glu 115 120 125 Asp Val Val Lys Asp Trp Arg Ile Asp Leu Thr Ala Val Lys Thr Ala 130 135 140 Glu Leu Gly Lys Ile Ala Gln His Thr Ala Ser Ile Val Gln Arg Thr 145 150 155 160 Phe Pro Asp Asn Leu Leu Lys Leu Thr Gly Ala Glu Val Ala Lys Leu 165 170 175 Ala Ala Asp Ser Tyr Glu Leu Glu Leu Glu Tyr Thr Gly Lys Ser Pro 180 185 190 Ala Thr Asn Glu Lys Val Asn Val Ala Ala Lys Tyr Ala Val Glu Leu 195 200 205 Leu Ser Ser Val Arg Asn Ala Asn Ser Thr Ala Ala Ala Ser Phe Gly 210 215 220 Glu Ser Val Ser Asp Leu Cys Arg Val Ala Lys Ile Ile His Thr His 225 230 235 240 Glu Tyr Ala Asn Val Val Cys Arg Thr Pro Ser Phe Lys Met Leu Leu 245 250 255 Pro Gln Val Val Ser Leu Thr Lys Ser Ser Tyr Tyr Gly Gly Leu Tyr 260 265 270 Pro Pro Glu Asn Leu Trp Leu Ala Gly Lys Thr Asp Gly Val Arg Ala 275 280 285 Leu Val Val Cys Glu Asp Gly Val Ala Lys Val Ile Thr Ala Glu Ser 290 295 300 Val Asp Ile Thr His Gly Val Cys Ser Ala Thr Thr Ile Leu Asp Cys 305 310 315 320 Glu Leu Asn Val Asp Ala Lys Ile Leu Tyr Val Phe Asp Val Ile Ile 325 330 335 Ser Asn Asn Thr Gln Val Tyr Thr Gln Pro Phe Ser Thr Arg Ile Thr 340 345 350 Thr Asp Ile Ser Asp Ile Lys Ile Asp Gly Tyr Lys Ile Glu Met Lys 355 360 365 Pro Phe Val Lys Val Val Lys Ala Asp Glu Ala Thr Phe Lys Ser Ala 370 375 380 Tyr Lys Ala Pro His Asn Glu Gly Leu Ile Met Ile Glu Asp Gly Ala 385 390 395 400 Ala Tyr Ala Ala Thr Lys Thr Tyr Lys Trp Lys Pro Leu Ser His Asn 405 410 415 Thr Ile Asp Phe Leu Ile Lys Ala Cys Pro Lys Gln Leu Ile Asn Val 420 425 430 Asp Pro Tyr Lys Pro Arg Ala Gly Tyr Lys Leu Trp Leu Leu Phe Thr 435 440 445 Thr Ile Ser Leu Asp Gln Gln Arg Glu Leu Gly Ile Glu Phe Ile Pro 450 455 460 Ala Trp Lys Ile Leu Phe Thr Asp Ile Asn Met Phe Gly Ser Arg Val 465 470 475 480 Pro Ile Gln Phe Gln Pro Ala Ile Asn Pro Leu Ala Tyr Val Cys Tyr 485 490 495 Leu Pro Glu Asp Val Asn Val Asn Asp Gly Asp Ile Val Glu Met Arg 500 505 510 Ala Val Asp Gly Tyr Asp Thr Ile Pro Lys Trp Glu Leu Val Arg Ser 515 520 525 Arg Asn Asp Arg Lys Asn Glu Pro Gly Phe Tyr Gly Asn Asn Tyr Lys 530 535 540 Ile Ala Ser Asp Ile Tyr Leu Asn Tyr Ile Asp Val Phe His Phe Glu 545 550 555 560 Asp Leu Tyr Lys Tyr Asn Pro Gly Tyr Phe Glu Lys Asn Lys Ser Asp 565 570 575 Ile Tyr Val Ala Pro Asn Lys Tyr Arg Arg Tyr Leu Ile Lys Ser Leu 580 585 590 Phe Gly Arg Tyr Leu Arg Asp Ala Lys Trp Val Ile Asp Ala Ala Ala 595 600 605 Gly Arg Gly Ala Asp Leu His Leu Tyr Lys Ala Glu Cys Val Glu His 610 615 620 Leu Leu Ala Ile Asp Ile Asp Pro Thr Ala Ile Ser Glu Leu Val Arg 625 630 635 640 Arg Arg Asn Glu Ile Thr Gly Tyr Asn Lys Ser His Arg Gly Gly Arg 645 650 655 Asn Met His Ser His Arg Gly Gln Ser His Cys Ala Lys Ser Thr Ser 660 665 670 Leu His Ala Leu Val Ala Asp Leu Arg Glu Asn Pro Asp Val Leu Ile 675 680 685 Pro Lys Ile Ile Gln Ser Arg Pro His Glu Arg Cys Tyr Asp Ala Ile 690 695 700 Val Ile Asn Phe Ala Ile His Tyr Leu Cys Asp Thr Asp Glu His Ile 705 710 715 720 Arg Asp Phe Leu Ile Thr Val Ser Arg Leu Leu Ala Pro Asn Gly Val 725 730 735 Phe Ile Phe Thr Thr Met Asp Gly Glu Ser Ile Val Lys Leu Leu Ala 740 745 750 Asp His Lys Val Arg Pro Gly Glu Ala Trp Thr Ile His Thr Gly Asp 755 760 765 Val Asn Ser Pro Asp Ser Thr Val Pro Lys Tyr Ser Ile Arg Arg Leu 770 775 780 Tyr Asp Ser Asp Lys Leu Thr Lys Thr Gly Gln Gln Ile Glu Val Leu 785 790 795 800 Leu Pro Met Ser Gly Glu Met Lys Ala Glu Pro Leu Cys Asn Ile Lys 805 810 815 Asn Ile Ile Ser Met Ala Arg Lys Met Gly Leu Asp Leu Val Glu Ser 820 825 830 Ala Asn Phe Ser Val Leu Tyr Glu Ala Tyr Ala Arg Asp Tyr Pro Asp 835 840 845 Ile Tyr Ala Arg Met Thr Pro Asp Asp Lys Leu Tyr Asn Asp Leu His 850 855 860 Thr Tyr Ala Val Phe Lys Arg Lys Lys 865 870 <210> 8 <211> 890 <212> PRT <213> Faustovirus <220> <221> Various features <223> E12 RNA capping enzyme of Float virus; Genbank accession number AIB52055 <400> 8 Met Arg Arg Val Phe Asn Ser Ala Lys Lys Gln Gln Arg Cys Asp Ser 1 5 10 15 Val Glu Gln Val Cys Glu Phe Tyr Asn Ala Asp Asn Lys Thr Asn Glu 20 25 30 Leu Glu Leu Arg Phe Asp Lys Leu Asn Arg Glu Leu Phe Val Val Leu 35 40 45 Phe Asp Lys Leu Lys Pro Asp Gly Glu Ile Thr Thr Thr Met Arg Val 50 55 60 Ser Asn Ala Asp Gly Met Ala Arg Glu Ile Thr Phe Gly Gly Gly Val 65 70 75 80 Lys Thr Gly Glu Met Phe Val Lys Lys Gln Asn Ile Cys Val Phe Asp 85 90 95 Val Val Asp Val Phe Ser Tyr Lys Val Ala Val Ser Ser Glu Asp Glu 100 105 110 Ile Lys Asp Lys Pro Lys Met Asp Thr Asn Ala Ser Val Arg Phe Lys 115 120 125 Ile Arg Leu Ser Cys Asp Thr Leu Ile Pro Asp Trp Arg Ile Asp Leu 130 135 140 Thr Ala Val Lys Val Ala Asp Leu Gly Lys Ile Ala Gln His Thr Ser 145 150 155 160 Thr Val Val Leu Gln Thr Phe Pro Glu Asn Leu Leu Arg Met Lys Gly 165 170 175 Ala Glu Val Ala Ala Leu Ala Thr Asn Ser Tyr Glu Leu Glu Leu Glu 180 185 190 Tyr Ile Gly Lys Ser Ala Ala Ser Lys Glu Lys Val Leu Ala Ala Ala 195 200 205 Glu Tyr Ala Met Glu Leu Leu Thr Asn Ser Arg Asn Ala Ile Ser Pro 210 215 220 Ala Ala Ala Thr Leu Gly Glu Ser Val Ser Asp Ile Cys Arg Ile Ala 225 230 235 240 Lys Leu Ile His Pro Ala Glu Tyr Ala Asn Val Ile Cys Arg Thr Pro 245 250 255 Ser Phe Lys Asn Leu Leu Pro Gln Val Ile Ser Leu Thr Lys Ser Ser 260 265 270 Tyr Tyr Gly Gly Ile Tyr Pro Pro Val Asp Met Tyr Ile Ala Gly Lys 275 280 285 Thr Asp Gly Val Arg Ala Leu Val Leu Cys Glu Asn Gly Val Ala Lys 290 295 300 Ile Ile Thr Ala Thr Thr Val Asp Thr Thr Thr Val Gly Asn Thr Pro 305 310 315 320 Ile Thr Ile Leu Asp Cys Glu Leu Ser Thr Ser Gly His Asn Gly Ala 325 330 335 Thr Asp Asn Lys His Leu Tyr Ile Phe Asp Val Ile Met Asn Arg Gly 340 345 350 Val His Ser His Arg Glu Gly Phe Asn Lys Arg Ile Asp Ile Asp Leu 355 360 365 Ser Asp Leu Thr Pro Ala Gly Tyr Thr Leu Glu Leu Lys Pro Phe Thr 370 375 380 Lys Leu Val Asp Ala Ala Ser Val Asn Glu Thr Thr Phe Lys Ser Val 385 390 395 400 Phe Lys Pro Pro His Asn Glu Gly Leu Val Leu Val Glu Ser Gly Pro 405 410 415 Pro Tyr Ala Leu Thr Lys Thr Tyr Lys Trp Lys Pro Ile Thr His Asn 420 425 430 Thr Ile Asp Phe Leu Ile Lys Ala Cys Pro Lys Gln Leu Ile Asn Val 435 440 445 Asp Pro Phe Lys Pro Arg Asn Gly His Asp Leu Trp Leu Leu Phe Thr 450 455 460 Thr Ile Ser Leu Asp Gln Gln Arg Glu Leu Gly Ile Asp Leu Ile Pro 465 470 475 480 Ala Trp Lys Leu Leu Phe Thr Asp Val Asn Leu Phe Gly Asn Lys Ile 485 490 495 Pro Ile Gln Phe Met Pro Ala Ile Asn Pro Leu Ala Tyr Ile Cys Tyr 500 505 510 Leu Pro Ser Ser Thr Gly Val Asn Asp Gly Asp Ile Val Glu Met Arg 515 520 525 Ala Val Asp Gly Phe Asp Gly Ile Pro Thr Trp Glu Leu Val Arg Thr 530 535 540 Arg Pro Asp Arg Lys Asp Glu Arg Gly Phe Tyr Gly Asn Asn Tyr Lys 545 550 555 560 Ile Ala Ser Asp Ile Tyr Leu Asn Tyr Ile Asp Val Phe Asn Phe Asp 565 570 575 Asp Leu Trp Lys Tyr Asn Pro Gly Tyr Phe Glu Lys Asn Lys Ser Asp 580 585 590 Ile Tyr Ile Ala Pro Asn Lys Tyr Arg Arg Tyr Leu Ile Lys Ser Leu 595 600 605 Phe Asn Lys Tyr Ile Lys Asn Ala Lys Trp Val Ile Asp Ala Ala Ala 610 615 620 Gly Arg Gly Ala Asp Leu His Leu Tyr Lys Gln Glu Cys Val Glu Asn 625 630 635 640 Leu Leu Ala Ile Asp Ile Asp Pro Thr Ala Ile Ser Glu Leu Ile Arg 645 650 655 Arg Arg Asn Glu Ile Thr Gly Trp Gln Gln Arg Gly Arg Gly Gly Asn 660 665 670 Thr Arg His Asn Ala Arg His Asn Thr His Cys Ala Ser Ser Thr Ser 675 680 685 Leu His Ala Leu Val Ala Asp Leu Arg Thr Glu Pro Asn Met Leu Ile 690 695 700 Pro Lys Ile Ile Gln Ser Arg Pro Pro Glu Arg Gly Tyr Asp Ala Ile 705 710 715 720 Val Ile Asn Phe Ala Ile His Tyr Leu Cys Glu Thr Asp Asp Tyr Ile 725 730 735 Arg Asn Phe Leu Ile Thr Val Ser Arg Leu Leu Ala Pro Asp Gly Val 740 745 750 Phe Ile Phe Thr Thr Met Asp Gly Glu Ala Ile Val Asn Leu Leu Ala 755 760 765 Glu His Lys Val Ala Pro Gly Ala Ser Trp Val Val His Thr Asp Gly 770 775 780 Asn Ala Asn Ala Thr Asp Ala Asn Val Val Lys Tyr Ser Ile Lys Arg 785 790 795 800 Leu Tyr Asp Ser Asp Lys Leu Thr Lys Thr Gly Gln Lys Ile Ala Val 805 810 815 Leu Leu Pro Met Ser Gly Glu Met Arg Glu Glu Pro Leu Cys Asn Ile 820 825 830 Lys Asn Ile Val Ser Met Ala Arg Lys Met Gly Leu Asp Leu Val Glu 835 840 845 Ser Ala Asn Phe Ser Val Leu Tyr Gly Ala Tyr Ala Lys Asp Tyr Pro 850 855 860 Glu Ile Tyr Ala Lys Leu Thr Pro Asp Asp Lys Leu Tyr Asn Asp Leu 865 870 875 880 His Ala Phe Ala Val Phe Lys Arg Lys Lys 885 890 <210> 9 <211> 894 <212> PRT <213> Faustovirus <220> <221> Various features <223> ST1 RNA capping enzyme of SARS-CoV; Genbank accession number SME65026 <400> 9 Met Arg Arg Val Ser Asn Ser Ala Lys Lys Gln Gln Arg Cys Asp Ser 1 5 10 15 Val Glu Gln Val Cys Glu Phe Tyr Asn Ala Asp Asn Lys Thr Asn Glu 20 25 30 Leu Glu Leu Arg Phe Asp Lys Leu Asn Arg Glu Leu Phe Val Ala Leu 35 40 45 Phe Asp Lys Leu Lys Pro Asp Gly Glu Ile Thr Thr Thr Met Arg Val 50 55 60 Ser Asn Ala Asp Gly Met Ala Arg Glu Ile Thr Phe Gly Gly Gly Val 65 70 75 80 Lys Thr Gly Glu Met Phe Val Lys Lys Gln Asn Ile Cys Val Phe Asp 85 90 95 Val Val Asp Val Phe Ser Tyr Lys Val Ala Val Ser Ser Glu Asp Glu 100 105 110 Val Lys Asp Lys Pro Lys Met Asp Thr Asn Ala Ser Val Arg Phe Lys 115 120 125 Ile Arg Leu Ser Cys Asp Thr Leu Ile Pro Asp Trp Arg Ile Asp Leu 130 135 140 Thr Ala Val Lys Val Ala Asp Leu Gly Lys Ile Ala Gln His Thr Ser 145 150 155 160 Thr Val Val Leu Gln Thr Phe Pro Glu Asn Leu Leu Arg Met Lys Gly 165 170 175 Ala Glu Val Ala Ala Leu Ala Thr Asn Ser Tyr Glu Leu Glu Leu Glu 180 185 190 Tyr Ile Gly Lys Ser Thr Ala Gly Lys Glu Lys Val Leu Lys Ala Ala 195 200 205 Glu Tyr Ala Ile Glu Leu Leu Thr Asn Leu Arg Asn Ala Val Ser Pro 210 215 220 Val Ala Ala Thr Leu Gly Glu Ser Val Ser Asp Ile Cys Arg Ile Ala 225 230 235 240 Lys Leu Ile His Pro Ala Glu Tyr Ala Asn Val Ile Cys Arg Thr Pro 245 250 255 Ser Phe Lys Asn Leu Leu Pro Gln Val Ile Ser Leu Thr Lys Ser Ser 260 265 270 Tyr Tyr Gly Gly Ile Tyr Pro Pro Val Asp Met Tyr Ile Thr Gly Lys 275 280 285 Thr Asp Gly Val Arg Ala Leu Val Leu Cys Glu Asn Gly Val Ala Lys 290 295 300 Ile Ile Thr Ala Thr Thr Val Asp Thr Thr Thr Val Gly Asp Thr Pro 305 310 315 320 Ile Thr Ile Leu Asp Cys Glu Leu Ser Thr Ser Gly His Asn Gly Ala 325 330 335 Thr Asp Asn Lys His Leu Tyr Val Phe Asp Val Ile Met Asn Arg Gly 340 345 350 Ala His Ser His Arg Asp Gly Phe Asn Lys Arg Ile Asp Ile Asp Leu 355 360 365 Ser Asp Leu Thr Pro Ala Gly Tyr Thr Leu Glu Leu Lys Pro Phe Thr 370 375 380 Lys Val Ala Asp Ala Ala Ser Val Asn Glu Thr Thr Phe Lys Ser Val 385 390 395 400 Phe Lys Pro Pro His Asn Glu Gly Leu Val Leu Val Glu Ser Gly Pro 405 410 415 Pro Tyr Ala Leu Thr Lys Thr Tyr Lys Trp Lys Pro Ile Thr His Asn 420 425 430 Thr Ile Asp Phe Leu Ile Lys Ala Cys Pro Lys Gln Leu Ile Asn Val 435 440 445 Asp Pro Phe Lys Pro Arg Asn Gly His Asp Leu Trp Leu Leu Phe Thr 450 455 460 Thr Ile Ser Leu Asp Gln Gln Arg Glu Leu Gly Ile Asp Leu Ile Pro 465 470 475 480 Ala Trp Lys Leu Leu Phe Thr Asp Val Asn Leu Phe Gly Asn Lys Ile 485 490 495 Pro Ile Gln Phe Met Pro Ala Ile Asn Pro Leu Ala Tyr Ile Cys Tyr 500 505 510 Leu Pro Ser Ser Thr Gly Val Asn Asp Gly Asp Ile Val Glu Met Arg 515 520 525 Ala Val Asp Gly Phe Asp Gly Ile Pro Thr Trp Glu Leu Val Arg Thr 530 535 540 Arg Pro Asp Arg Lys Asp Glu Arg Gly Phe Tyr Gly Asn Asn Tyr Lys 545 550 555 560 Ile Ala Ser Asp Ile Tyr Leu Asn Tyr Ile Asp Val Phe Asn Phe Asp 565 570 575 Asp Leu Trp Lys Tyr Asn Pro Gly Tyr Phe Glu Lys Asn Lys Ser Asp 580 585 590 Ile Tyr Ile Ala Pro Asn Lys Tyr Arg Arg Tyr Leu Ile Lys Ser Leu 595 600 605 Phe Asn Lys Tyr Ile Lys Asn Ala Lys Trp Val Ile Asp Ala Ala Ala 610 615 620 Gly Arg Gly Ala Asp Leu His Leu Tyr Lys Gln Glu Cys Val Glu Asn 625 630 635 640 Leu Leu Ala Ile Asp Ile Asp Pro Thr Ala Ile Ser Glu Leu Ile Arg 645 650 655 Arg Arg Asn Glu Ile Thr Gly Trp Gln Gln Arg Gly Arg Gly Gly Asn 660 665 670 Met His His Asn Ser Arg His Asn Thr Arg His Asn Thr His Cys Ala 675 680 685 Ser Ser Thr Ser Leu His Ala Leu Val Ala Asp Leu Arg Thr Glu Pro 690 695 700 Asn Met Leu Ile Pro Lys Ile Ile Gln Ser Arg Pro Pro Glu Arg Gly 705 710 715 720 Tyr Asp Ala Leu Val Ile Asn Phe Ala Ile His Tyr Leu Cys Glu Thr 725 730 735 Asp Asp Tyr Ile Arg Asn Phe Leu Ile Thr Val Ser Arg Leu Leu Ala 740 745 750 His Asp Gly Val Phe Ile Phe Thr Thr Met Asp Gly Glu Ala Ile Val 755 760 765 Asn Leu Leu Thr Glu His Lys Val Ala Pro Gly Ala Ser Trp Val Val 770 775 780 His Thr Asp Gly Asn Thr Asn Ala Thr Asp Ala Asn Val Val Lys Tyr 785 790 795 800 Ser Ile Lys Arg Leu Tyr Asp Ser Asp Lys Leu Thr Lys Thr Gly Gln 805 810 815 Lys Ile Ala Val Leu Leu Pro Met Ser Gly Glu Met Arg Glu Glu Pro 820 825 830 Leu Cys Asn Ile Lys Asn Ile Val Ser Met Ala Arg Lys Met Gly Leu 835 840 845 Asp Leu Val Glu Ser Ala Asn Phe Ser Val Leu Tyr Gly Ala Tyr Ala 850 855 860 Lys Asp Tyr Pro Glu Ile Tyr Ala Lys Leu Thr Pro Asp Asp Lys Leu 865 870 875 880 Tyr Asn Asp Leu His Ala Phe Ala Val Phe Lys Arg Lys Lys 885 890 <210> 10 <211> 901 <212> PRT <213> Faustovirus <220> <221> Various features <223> ST1 RNA capping enzyme of SARS-CoV; Genbank accession number SMH63629 <400> 10 Leu Glu Tyr Gln Tyr Tyr Lys Tyr Ile Leu Val Asn Ile Met Ser Arg 1 5 10 15 Arg Leu Gln Arg Cys Arg Asp Val Asp Gln Val Cys Glu Tyr Tyr Asn 20 25 30 Ala Lys Gly Ala Ile Gly Glu Leu Glu Leu Arg Phe Asp Lys Leu Thr 35 40 45 Pro Asp Leu Phe Ala His Val Phe Asp Lys Leu Lys Pro Asp Gly Glu 50 55 60 Ile Ser Thr Thr Met Arg Val Ser Asn Ser Asp Gly Thr Ala Arg Glu 65 70 75 80 Ile Thr Phe Gly Gly Gly Val Lys Thr Gly Glu Thr Phe Val Arg Lys 85 90 95 Gln Asn Ile Cys Val Phe Asp Val Val Asp Ile Phe Ser Tyr Lys Val 100 105 110 Ala Val Ser Thr Glu Glu Thr Leu Val Asp Lys Pro Ala Met Glu Lys 115 120 125 Asp Ala Ser Val Arg Phe Lys Ile Arg Met Ser Val Glu Gly Ala Val 130 135 140 Pro Asn Trp Arg Ile Asp Leu Thr Ala Val Lys Thr Ala Glu Leu Gly 145 150 155 160 Lys Ile Ala Gln His Thr Ala Ser Leu Val Leu Gln Thr Phe Pro Pro 165 170 175 Asn Leu Leu Lys Met Ser Gly Ala Glu Val Ala Lys Leu Ala Asn Asn 180 185 190 Ser Tyr Glu Leu Glu Leu Glu Tyr Ile Gly Lys Thr Pro Ala Thr Lys 195 200 205 Glu Arg Val Asp Ala Ala Ala Lys Tyr Ala Val Asp Leu Leu Ala Gly 210 215 220 Ile Lys Asn Ala Asn Ser Ala Val Gly Ala Val Leu Gly Glu Ser Ile 225 230 235 240 Ser Asp Ile Cys Arg Val Ala Lys Val Ile His Thr Pro Asp Tyr Ala 245 250 255 Thr Val Val Cys Arg Asn Pro Ser Phe Lys Met Leu Leu Pro Gln Val 260 265 270 Ile Ser Leu Thr Lys Ser Ser Tyr Tyr Gly Gly Ile Tyr Pro Pro Glu 275 280 285 Gly Met Tyr Val Ala Gly Lys Thr Asp Gly Val Arg Ala Leu Val Leu 290 295 300 Cys Glu Asp Gly Val Ala Lys Val Ile Thr Ala Glu Ser Val Asp Ile 305 310 315 320 Thr Thr Gly Thr Cys Thr Gly Thr Thr Ile Leu Asp Cys Glu Leu Ser 325 330 335 Thr Gly Lys Ser Gly Ala Thr Leu His Val Phe Asp Ile Ile Met His 340 345 350 Asn Ser Lys Pro Ile His Ser Gln Pro Phe Ser Thr Arg Ile Ala Thr 355 360 365 Asp Ile Ser Asp Val Lys Ile Pro Glu Tyr Lys Ile Ala Ile Lys Pro 370 375 380 Phe Val Lys Ile Gln Ala Thr Ala Leu Glu Ala Ala Phe Lys Glu Val 385 390 395 400 Tyr Lys Ala Pro His Asn Glu Gly Leu Ile Leu Ile Met Asp Gly Asn 405 410 415 Asp Tyr Ala Met Thr Lys Thr Tyr Lys Trp Lys Pro Leu Ser His Asn 420 425 430 Thr Ile Asp Phe Leu Ile Lys Ala Cys Pro Lys Gln Leu Leu Asn Ile 435 440 445 Asp Pro Tyr Lys Pro Arg Pro Gly His Lys Leu Trp Leu Leu Phe Thr 450 455 460 Thr Ile Ser Leu Asp Gln Gln Arg Glu Leu Gly Ile Glu Phe Ile Pro 465 470 475 480 Ala Trp Lys Met Leu Phe Thr Asp Ile Asn Leu Phe Gly Ser Arg Val 485,490,495 Pro Ile Gln Phe Met Pro Ala Ile Asn Pro Leu Ala Tyr Ile Cys Tyr 500 505 510 Leu Pro Glu Ala Thr Cys Ala Asn Gly Asp Ala Ile Asn Asp Gly 515,520,525 Asp Ile Val Glu Met Arg Ala Val Asp Gly Phe Asp Thr Val Pro Lys 530 535 540 Trp Glu Pro Ile Arg Val Arg Ser Asp Arg Lys Asp Glu Pro Gly Phe 545 550 555 560 Tyr Gly Asn Asn Tyr Lys Ile Only Ser Asp With Tyr Leu Asn Tyr Ile 565,570,575 Asp Ile Phe Gln Phe Glu Asp Leu Tyr Lys Tyr Asn Pro Gly Tyr Phe 580,585,590 Glue Asn Lys Ser Asp Ile Tyr Val Ala Pro Asn Lys Tyr Arg Arg 595,600,605 Phe Leu And Lys Asn And Phe Ser Lys Tyr Leu Lys Ala Lys Trp 610 615 620 Val Ile Asp Ala Ala Ala Gly Arg Gly Ala Asp Leu His Leu Tyr Lys 625 630 635 640 Ala Glu Cys Val Glu Asn Leu Leu Ala Ile Asp Ile Asp Pro Thr Ala 645 650 655 Ile Ser Glu Leu Val Arg Arg Arg Asn Glu Ile Thr Gly Tyr Asn Arg 660 665 670 Gly His Arg Gly His Arg Gly Gly Ser Met Arg Ala His Met Gly Ala 675 680 685 Ser His His Gly Ala Gln Asn Cys Ala Lys Ser Thr Thr Leu His Ala 690 695 700 Leu Val Ala Asp Leu Arg Thr Asp Pro Asp Val Leu Ile Pro Lys Ile 705 710 715 720 Ile Gln Ser Arg Pro Pro Glu Arg Gly Tyr Asp Ala Ile Val Ile Asn 725 730 735 Phe Ala Ile His Tyr Leu Cys Asp Thr Asp Glu His Ile Arg Asp Phe 740 745 750 Leu Ile Thr Val Ser Arg Leu Leu Ala Pro Asn Gly Ile Phe Met Phe 755 760 765 Thr Thr Met Asp Gly Glu Ser Ile Val Lys Leu Leu Glu Thr His Lys 770 775 780 Val Lys Ser Gly Glu Ser Trp Thr Val His Thr Gly Ala Asp Asp Pro 785 790 795 800 Glu Ala Gly Val Ile Lys Tyr Ser Ile Arg Arg Leu Tyr Asp Ser Asp 805 810 815 Lys Leu Thr Lys Thr Gly Gln Gln Ile Ala Val Leu Leu Pro Met Ser 820 825 830 Gly Glu Met Lys Thr Glu Pro Leu Cys Asn Ile Lys Asn Ile Ile Ser 835 840 845 Ile Ala Arg Lys Met Gly Leu Asp Leu Val Glu Ser Ala Asp Phe Ser 850 855 860 Val Met Tyr Asp Ala Phe Ala Arg Ala Tyr Pro Glu Ile Ser Ala Arg 865 870 875 880 Leu Thr Pro Asp Asp Lys Leu Tyr Asn Asp Leu His Ser Tyr Ala Val 885 890 895 Phe Lys Arg Lys Lys 900 <210> 11 <211> 1170 <212> PRT <213> Acanthochete polyphaga <220> <221> Various features <223> Mimivirus RNA capping enzyme; Genbank accession number AAV50651 <400> 11 Met Gly Thr Lys Leu Lys Lys Ser Asn Asn Asp Ile Thr Ile Phe Ser 1 5 10 15 Glu Asn Glu Tyr Asn Glu Ile Val Glu Met Leu Arg Asp Tyr Ser Asn 20 25 30 Gly Asp Asn Leu Glu Phe Glu Val Ser Phe Lys Asn Ile Asn Tyr Pro 35 40 45 Asn Phe Met Arg Ile Thr Glu His Tyr Ile Asn Ile Thr Pro Glu Asn 50 55 60 Lys Ile Glu Ser Asn Asn Tyr Leu Asp Ile Ser Leu Ile Phe Pro Asp 65 70 75 80 Lys Asn Val Tyr Arg Val Ser Leu Phe Asn Gln Glu Gln Ile Gly Glu 85 90 95 Phe Ile Thr Lys Phe Ser Lys Ala Ser Ser Asn Asp Ile Ser Arg Tyr 100 105 110 Ile Val Ser Leu Asp Pro Ser Asp Asp Ile Glu Ile Val Tyr Lys Asn 115 120 125 Arg Gly Ser Gly Lys Leu Ile Gly Ile Asp Asn Trp Ala Ile Thr Ile 130 135 140 Lys Ser Thr Glu Glu Ile Pro Leu Val Ala Gly Lys Ser Lys Ile Ser 145 150 155 160 Lys Pro Lys Ile Thr Gly Ser Glu Arg Ile Met Tyr Arg Tyr Lys Thr 165 170 175 Arg Tyr Ser Phe Thr Ile Asn Lys Asn Ser Arg Ile Asp Ile Thr Asp 180 185 190 Val Lys Ser Ser Pro Ile Ile Trp Lys Leu Met Thr Val Pro Ser Asn 195 200 205 Tyr Glu Leu Glu Leu Glu Leu Ile Asn Lys Ile Asp Ile Asn Thr Leu 210 215 220 Glu Ser Glu Leu Leu Asn Val Phe Met Ile Ile Gln Asp Thr Lys Ile 225 230 235 240 Pro Ile Ser Lys Ala Glu Ser Asp Thr Val Val Glu Glu Tyr Arg Asn 245 250 255 Leu Leu Asn Val Arg Gln Thr Asn Asn Leu Asp Ser Arg Asn Val Ile 260 265 270 Ser Val Asn Ser Asn His Ile Ile Asn Phe Ile Pro Asn Arg Tyr Ala 275 280 285 Val Thr Asp Lys Ala Asp Gly Glu Arg Tyr Phe Leu Phe Ser Leu Asn 290 295 300 Ser Gly Ile Tyr Leu Leu Ser Ile Asn Leu Thr Val Lys Lys Leu Asn 305 310 315 320 Ile Pro Val Leu Glu Lys Arg Tyr Gln Asn Met Leu Ile Asp Gly Glu 325 330 335 Tyr Ile Lys Thr Thr Gly His Asp Leu Phe Met Val Phe Asp Val Ile 340 345 350 Phe Ala Glu Gly Thr Asp Tyr Arg Tyr Asp Asn Thr Tyr Ser Leu Pro 355 360 365 Lys Arg Ile Ile Ile Ile Asn Asn Ile Ile Asp Lys Cys Phe Gly Asn 370 375 380 Leu Ile Pro Phe Asn Asp Tyr Thr Asp Lys His Asn Asn Leu Glu Leu 385 390 395 400 Asp Ser Ile Lys Thr Tyr Tyr Lys Ser Glu Leu Ser Asn Tyr Trp Lys 405 410 415 Asn Phe Lys Asn Arg Leu Asn Lys Ser Thr Asp Leu Phe Ile Thr Arg 420 425 430 Lys Leu Tyr Leu Val Pro Tyr Gly Ile Asp Ser Ser Glu Ile Phe Met 435 440 445 Tyr Ala Asp Met Ile Trp Lys Leu Tyr Val Tyr Asn Glu Leu Thr Pro 450 455 460 Tyr Gln Leu Asp Gly Ile Ile Tyr Thr Pro Ile Asn Ser Pro Tyr Leu 465 470 475 480 Ile Arg Gly Gly Ile Asp Ala Tyr Asp Thr Ile Pro Met Glu Tyr Lys 485 490 495 Trp Lys Pro Pro Ser Gln Asn Ser Ile Asp Phe Tyr Ile Arg Phe Lys 500 505 510 Lys Asp Val Ser Gly Ala Asp Ala Val Tyr Tyr Asp Asn Ser Val Glu 515 520 525 Arg Ala Glu Gly Lys Pro Tyr Lys Ile Cys Leu Leu Tyr Val Gly Leu 530 535 540 Asn Lys Gln Gly Gln Glu Ile Pro Ile Gln Phe Lys Val Asn Gly Val 545 550 555 560 Glu Gln Thr Ala Asn Ile Tyr Thr Lys Asp Gly Glu Ala Thr Asp Ile 565 570 575 Asn Gly Asn Ala Ile Asn Asp Asn Thr Val Val Glu Phe Val Phe Asp 580 585 590 Thr Leu Lys Ile Asp Met Asp Asp Ser Tyr Lys Trp Ile Pro Ile Arg 595 600 605 Thr Arg Tyr Asp Lys Thr Glu Ser Val Gln Lys Tyr His Lys Arg Tyr 610 615 620 Gly Asn Asn Leu Gln Ile Ala Asn Arg Ile Trp Lys Thr Ile Thr Asn 625 630 635 640 Pro Ile Thr Glu Asp Ile Ile Ser Ser Leu Gly Asp Pro Thr Thr Phe 645 650 655 Asn Lys Glu Ile Thr Leu Leu Ser Asp Phe Arg Asp Thr Lys Tyr Asn 660 665 670 Lys Gln Ala Leu Thr Tyr Tyr Gln Lys Asn Thr Ser Asn Ala Ala Gly 675 680 685 Met Arg Ala Phe Asn Asn Trp Ile Lys Ser Asn Met Ile Thr Thr Tyr 690 695 700 Cys Arg Asp Gly Ser Lys Val Leu Asp Ile Gly Cys Gly Arg Gly Gly 705 710 715 720 Asp Leu Ile Lys Phe Ile Asn Ala Gly Val Glu Phe Tyr Val Gly Ile 725 730 735 Asp Ile Asp Asn Asn Gly Leu Tyr Val Ile Asn Asp Ser Ala Asn Asn 740 745 750 Arg Tyr Lys Asn Leu Lys Lys Thr Ile Gln Asn Ile Pro Pro Met Tyr 755 760 765 Phe Ile Asn Ala Asp Ala Arg Gly Leu Phe Thr Leu Glu Ala Gln Glu 770 775 780 Lys Ile Leu Pro Gly Met Pro Asp Phe Asn Lys Ser Leu Ile Asn Lys 785 790 795 800 Tyr Leu Val Gly Asn Lys Tyr Asp Thr Ile Asn Cys Gln Phe Thr Ile 805 810 815 His Tyr Tyr Leu Ser Asp Glu Leu Ser Trp Asn Asn Phe Cys Lys Asn 820 825 830 Ile Asn Asn Gln Leu Lys Asp Asn Gly Tyr Leu Leu Ile Thr Ser Phe 835 840 845 Asp Gly Asn Leu Ile His Asn Lys Leu Lys Gly Lys Gln Lys Leu Ser 850 855 860 Ser Ser Tyr Thr Asp Asn Arg Gly Asn Lys Asn Ile Phe Phe Glu Ile 865 870 875 880 Asn Lys Ile Tyr Ser Asp Thr Asp Lys Val Gly Leu Gly Met Ala Ile 885 890 895 Asp Leu Tyr Asn Ser Leu Ile Ser Asn Pro Gly Thr Tyr Ile Arg Glu 900 905 910 Tyr Leu Val Phe Pro Glu Phe Leu Glu Lys Ser Leu Lys Glu Lys Cys 915 920 925 Gly Leu Glu Leu Val Glu Ser Asp Leu Phe Tyr Asn Ile Phe Asn Thr 930 935 940 Tyr Lys Asn Tyr Phe Lys Lys Thr Tyr Asn Glu Tyr Gly Met Thr Asp 945 950 955 960 Val Ser Ser Lys Lys His Ser Glu Ile Arg Glu Phe Tyr Leu Ser Leu 965 970 975 Glu Gly Asn Ala Asn Asn Asp Ile Glu Ile Asp Ile Ala Arg Ala Ser 980 985 990 Phe Lys Leu Ala Met Leu Asn Arg Tyr Tyr Val Phe Arg Lys Thr Ser 995 1000 1005 Thr Ile Asn Ile Thr Glu Pro Ser Arg Ile Val Asn Glu Leu Asn 1010 1015 1020 Asn Arg Ile Asp Leu Gly Lys Phe Ile Met Pro Tyr Phe Arg Thr 1025 1030 1035 Asn Asn Met Phe Ile Asp Leu Asp Asn Val Asp Thr Asp Ile Asn 1040 1045 1050 Arg Val Tyr Arg Asn Ile Arg Asn Lys Tyr Arg Thr Thr Arg Pro 1055 1060 1065 His Val Tyr Leu Ile Lys His Asn Ile Asn Glu Asn Arg Leu Glu 1070 1075 1080 Asp Ile Tyr Leu Ser Asn Asn Lys Leu Asp Phe Ser Lys Ile Lys 1085 1090 1095 Asn Gly Ser Asp Pro Lys Val Leu Leu Ile Tyr Lys Ser Pro Asp 1100 1105 1110 Lys Gln Phe Tyr Pro Leu Tyr Tyr Gln Asn Tyr Gln Ser Met Pro 1115 1120 1125 Phe Asp Leu Asp Gln Ile Tyr Leu Pro Asp Lys Lys Lys Tyr Leu 1130 1135 1140 Leu Asp Ser Asp Arg Ile Ile Asn Asp Leu Asn Ile Leu Ile Asn 1145 1150 1155 Leu Thr Glu Lys Ile Lys Asn Ile Pro Gln Leu Ser 1160 1165 1170 <210> 12 <211> 1162 <212> PRT <213> Acanthochete polyphaga <220> <221> Various features <223> Moumouvirus RNA capping enzyme; Genbank accession number YP_007354410 <400> 12 Met Val Thr Lys Asn Lys Ser Glu Asn Ile Arg Asp Ile Leu Gly Ser 1 5 10 15 Asp Asn Val Ser Arg Val Glu Glu Met Ile Asn Asn Phe Arg Lys Asn 20 25 30 Arg Asn Thr Glu Phe Glu Ile Ser Val Arg Lys Ile Asn Tyr Ser Asn 35 40 45 Tyr Ile Arg Ile Ser Glu Tyr Tyr Val Asn Thr Ser Ser Asp Ile Gln 50 55 60 Gln Val Thr Ser Leu Asp Ile Ser Ile Ile Leu Glu Asp Gly Asn Thr 65 70 75 80 Tyr Arg Val Ser Phe Leu Asn Glu Asn Leu Ile Asn Asp Phe Leu Ser 85 90 95 Lys Tyr Ser Asn Met Lys Tyr Gly Asp Ile Val Lys Tyr Ile Leu Ala 100 105 110 Leu Asn Pro Asn Asp Asp Ile Glu Ile Ile Tyr Lys Asn Arg Gly Ser 115 120 125 Ala Asp Arg Leu Ser Ile Glu Asp Leu Asn Leu Val Val Lys Leu Thr 130 135 140 Glu Glu Val Pro Val Leu Asn Asn Thr Thr Lys Pro Lys Leu Ser Gly 145 150 155 160 Arg Glu Lys Ile Leu Tyr Arg Tyr Lys Asn Arg Tyr Ser Phe Ile Ile 165 170 175 Asp Asp Ile Thr Arg Ile Asp Ile Thr Asp Val Lys Glu Thr Pro Asn 180 185 190 Ile Trp Glu Leu Ser Arg Lys Ile Ser Asn Tyr Glu Ile Glu Leu Glu 195 200 205 Phe Thr Asn Asn Lys Ile Lys Ser Asn Gln Val Phe Glu Lys Ile Phe 210 215 220 Asp Leu Leu Arg Ile Val Gln Asn Thr Glu Ile Pro Ile Gly Ile Arg 225 230 235 240 Glu Ser Lys Gln Val Ile Thr Asp Tyr Gln Asn Leu Leu Asn Leu Arg 245 250 255 Ser Ser Asn His Leu Asp Ser Arg Asn Val Val Ser Ile Glu Thr Gln 260 265 270 His Ile Val Lys Phe Val Pro Asn Arg Tyr Ala Ile Thr Asp Lys Ala 275 280 285 Asp Gly Glu Arg Tyr Phe Leu Phe Ser Thr Pro Asn Gly Val Tyr Leu 290 295 300 Leu Ser Thr Asn Leu Thr Val Lys Lys Val Asn Ile Pro Val Leu Gln 305 310 315 320 Lys Asp Phe Gln Asn Met Leu Leu Asp Gly Glu Leu Ile Asp Ile Asp 325 330 335 Gly Lys Glu Leu Phe Met Val Phe Asp Val Val Tyr His Asn Gly Ile 340 345 350 Asp Tyr Arg Tyr Asp Thr Asn Tyr Thr Leu Thr His Arg Ile Ile Ile 355 360 365 Ile Asn Asp Ile Ile Asp Lys Ala Phe Asn Asn Leu Ile Pro Phe Thr 370 375 380 Asp Tyr Thr Asp Lys Tyr Asn Asn Leu Glu Leu Asp Lys Ile Lys Glu 385 390 395 400 Phe Tyr Ser Asn Glu Ile Lys Thr Tyr Trp Lys Asn Phe Ser Lys Lys 405 410 415 Leu Lys Asn Tyr Ser Gly Leu Phe Ile Ser Arg Lys Leu Tyr Phe Val 420 425 430 Pro Tyr Gly Ile Asp Ser Ser Glu Val Phe Met Tyr Ala Asp Leu Val 435 440 445 Trp Lys Leu Cys Val Tyr Asp Gln Leu Thr Pro Tyr Lys Leu Asp Gly 450 455 460 Ile Ile Tyr Thr Pro Ile Ala Ser Pro Tyr Met Ile Lys Thr Ser Ala 465 470 475 480 Asn Glu Leu Asp Ser Val Pro Met Glu Tyr Lys Trp Lys Pro Pro Ser 485 490 495 Gln Asn Ser Ile Asp Phe Tyr Val Lys Phe Asp Lys Asp Ala Arg Gly 500 505 510 Asn Glu Ala Ile Tyr Tyr Asp Asn Ala Val Val Arg Gly Glu Gly Arg 515 520 525 Pro Tyr Lys Val Cys Gly Leu Phe Val Gly Leu Asn Lys Gly Gly Glu 530 535 540 Glu Lys Pro Ile Ala Phe Lys Val Ala Gly Val Glu Gln Lys Ala Phe 545 550 555 560 Ile Tyr Leu Thr Asn Asp Glu Ala Leu Asp Leu Ser Gly Asn Val Ile 565 570 575 Asn Asp Asn Thr Val Val Glu Phe Ile Phe Asp Asn Phe Lys Thr Asp 580 585 590 Met Asp Asp Pro Tyr Lys Trp Ile Pro Val Arg Thr Arg Tyr Asp Lys 595 600 605 Thr Glu Ser Val Gln Lys Tyr Arg Lys Lys Tyr Gly Asn Asn Leu His 610 615 620 Ile Ala Thr Arg Ile Trp Arg Thr Ile Thr Asn Pro Val Thr Glu Glu 625 630 635 640 Ile Ile Ala Ala Leu Gly Asn Ala Ser Thr Phe Glu Lys Glu Met Ser 645 650 655 Lys Leu Val Lys Met Asn Glu Ser Tyr Asn Lys Gln Ser Phe Ser Tyr 660 665 670 Tyr Gln Lys Asn Thr Ser Asn Ala Ile Gly Met Arg Ala Phe Asn Asn 675 680 685 Phe Ile Lys Ser Asn Met Ile Thr Thr Tyr Cys Lys Asp Lys Asp Ser 690 695 700 Val Leu Asp Ile Gly Cys Gly Arg Gly Gly Asp Leu Ile Lys Phe Ile 705 710 715 720 His Ala Asn Ile Arg Glu Tyr Val Gly Leu Asp Ile Asp Asn Asn Gly 725 730 735 Leu Tyr Val Ile Asn Asp Ser Ala Phe Asn Arg Tyr Lys Asn Leu Lys 740 745 750 Lys Thr Asn Lys Asn Val Pro Pro Met Thr Phe Ile Asn Ala Asp Ala 755 760 765 Arg Gly Leu Phe Asn Val Glu Ala Gln Glu Lys Ile Leu Pro Asn Met 770 775 780 Ser Glu Ser Asn Lys Lys Leu Ile Asn Asn Tyr Leu Ser Ser Asn Lys 785 790 795 800 Lys Tyr Asp Ala Ile Asn Cys Gln Phe Thr Ile His Tyr Tyr Leu Ser 805 810 815 Asp Asp Ile Ser Trp Asn Asn Phe Cys Gln Asn Ile Asn Asn His Ile 820 825 830 Lys Asp Asn Gly Tyr Leu Leu Ile Thr Cys Phe Asp Gly Gln Leu Ile 835 840 845 Tyr Asp Lys Leu Lys Gly Lys Gln Lys Tyr Ser Ser Ser Tyr Thr Asp 850 855 860 Asn Phe Gly Lys Lys Asn Ile Phe Phe Glu Ile Asn Lys Ile Tyr Ser 865 870 875 880 Asp Glu Glu Ile Lys Pro Val Gly Met Ala Ile Asp Ile Tyr Asn Ser 885,890,895 Leu Is Asn Pro Gly Thr Tyr Gln Arg Glu Tyr Leu Val Phe Pro 900 905 910 Asp Phe Leu Gln Lys Ser Leu Lys Asp Gln Cys Gly Leu Glu Leu Val 915,920,925 Glu Thr Asp Met Phe Tyr Asn Ile Phe Asn Leu Tyr Arg Asn Tyr Phe 930,935,940 Thr Ile Asn Asn Gly Thr Phe Served Thr Gly Glu Ile Served Lys Arg 945 950 955 960 Tyr Asn Glu Ile Lys Asp Phe Tyr Leu Ala Leu Glu Gly Lys Ser Ser 965,970,975 Ser Val Thr Glu Ser Asp Ile Ala Phe Ala Ser Phe Lys Leu Ala Met 980,985,990 Leu Asn Arg Tyr Tyr Ile Phe Lys Lys Thr Val Ile Asn Ile Thr 995 1000 1005 Glu Pro Ser His Ile Val Ser Gly Val Asn Lys Lys Thr Asp Leu 1010 1015 1020 Gly Lys Val Leu Met Pro Tyr Phe Ile Thr Asn Asn Met Ile Ile 1025 1030 1035 Asp Tyr Ser Leu Gly Asn Asn Asp Val Asn Lys Ile Tyr His Phe 1040 1045 1050 Ile Arg Lys Lys Tyr Ser Pro Ile Lys Pro Ser Val Tyr Leu Val 1055 1060 1065 Arg His Asn Ile Ile Asp Asn Pro Met Asp Gly Ile Thr Phe Ser 1070 1075 1080 Arg Asn Lys Leu Glu Phe Ile Lys Ile Lys Asn Gly Thr Asp Pro 1085 1090 1095 Lys Val Leu Leu Ile Tyr Lys Ser Pro Glu Lys Ile Phe Tyr Pro 1100 1105 1110 Phe Tyr Tyr Gln Arg Leu Glu Asn His Asp Tyr Ser Glu Asp Tyr 1115 1120 1125 Leu Lys Asn Asn Ile Tyr Leu Lys Asp Asn Gly Thr Tyr Leu Leu 1130 1135 1140 Asp Ser Asn Lys Ile Ile Asn Asp Leu Asn Met Leu Val Asn Ile 1145 1150 1155 Ser Gly Lys Val 1160 <210> 13 <211> 25 <212> RNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Various features <223> RNA1 <400> 13 guagaacuuc gucgaguacg cucaa 25 <210> 14 <211> twenty three <212> RNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Various features <223> RNA2 <400> 14 gcaagucuuc gucgaguacu ugc 23 <210> 15 <211> twenty four <212> RNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Various features <223> RNA3 <400> 15 ggcaagucuu cgucgaguac uugc 24 <210> 16 <211> 25 <212> RNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Various features <223> RNA4 <400> 16 gggcaagucuucgucgaguacuugc 25 <210> 17 <211> 150 <212> RNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 17 gggagucuuc gccgagaggg ccaucgccag uugccgcaac cugugggaau uucucuucca 60 guuuauccgg augcucaacg gugacuuuaa uuccgguauc uuucucgaau uucuuaccga 120 cuucagcgag cucgacaaug cucuucccua 150 <210> 18 <211> 1753 <212> RNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Various features <223> Sequence of the Fluc transcript <400> 18 gggucuagaa auaauuuugu uuaacuuuaa gaaggagaua uaaccaugaa aaucgaagaa 60 gguaaagguc accaucacca ucaccacgga uccauggaag acgccaaaaa cauaaagaaa 120 ggcccggcgc cauucuaucc ucuagaggau ggaaccgcug gagagcaacu gcauaaggcu 180 240 aacauacgu acgcggaaua cuucgaaaug uccguucggu uggcagaagc uaugaaacga 300 uaugggcuga auacaaauca cagaaucguc guaugcagug aaaacucucu ucaauucuuu 360 augccggugu ugggcgcguu auuuaucgga guugcaguug cgcccgcagaa cgacauuuau 420 aaugaacgug aauugcucaa caguaugaac auuucgcagc cuaccguagu guuuguuucc 480 aaaaaaggggu ugcaaaaaau uuugaacgug caaaaaaaau uaccaauauau ccagaaaauuu 540 auuaucaugg auucuaaac ggauuaccag ggauuucagu cgauguacac guucgucaca 600 ucucaucuac cucccgguuu uaaugaauac gauuuguac cagaguccuu ugaucgugac 660 aaaacaauug cacugauaau gaauuccucu ggaucuacug gguuaccuaa ggguguggcc 720 cuuccgcaua gaacugccug cgucagauuc ucgcaugcca gagauccuau uuuuggcaau 780 caaaucauuc cggauacugc gauuuuaagu guuguuccau uccaucacgg uuuuggaaug 840 uuuacuacac ucgguaauuu gauaugugga uuucgagucg ucuuaaugua uagauuugaa 900 960 acccuauuuu caucuucgc caaaagcacu cugauugaca aauacgauuu aucuaauuua 1020 cacgaaauug cuucuggggg cgcaccucuu ucgaaagaag ucgggggaagc gguugcaaaa 1080 cgcuuccauc uuccagggau acgacaagga uaugggcuca cugagacuac aucagcuauu 1140 cugauuacac ccgaggggga ugauaaaccg ggcgcggucg guaaaguugu uccauuuuuu 1200 gaagcgaagg uugugguacu gguaaccggg aaaacgcugg gcguuaauca gagaggcgaa 1260 uuaugugaca gaggaccuau gauuaugucc gguuaugua acaauccgga agcgaccaac 1320 gccuugauug acaaggaugg auggcuacau ucugggagaca uagcuuacug ggacgaagac 1380 gaacacuucu ucauaguuga ccgcuugaag ucuuuaauua aauacaaagg auucaggug 1440 gcccccgcug aauugggaauc gauauuguua caacacccca acaucuucga cgcgggcgug 1500 cgaggucuuc ccgacgauga cgccggugaa cuucccgccg ccguuguugu uuuggagcac 1560 ggaaagacga ugacggaaaa agaaucgug gauuacgucg ccagucaagu aacaaccgcg 1620 aaaaaguugc gcggaggagu uguguuugug gacgaaguac cgaaaggucu uaccggaaaa 1680 cucgacgcaa gaaaaaucag agagauccuc auaaaggcca agaagggcgg aaaguccaaa 1740 cucgaguaag guu 1753 <210> 19 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Various features <223> Targeting oligos for fluc transcripts <220> <221> Various features <222> (44)..(44) <223> C is TEG-desthiobiotin <400> 19 gttaaarcra rarararuru rarurururc rurargrarc rcrc 44 <210> 20 <211> 1778 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Various features <223> Amino acid sequence of H3C2:T7 RNA polymerase fusion protein <400> 20 Met Lys Ile Glu Glu His His His His His His His Gly Ala Ser 1 5 10 15 Thr Ala Lys Arg Leu Gln Arg Cys Gln Asp Val Asn Gln Val Cys Glu 20 25 30 Ile Tyr Asn Ser Lys Gly Gly Ile Gly Glu Leu Glu Leu Arg Phe Asp 35 40 45 Lys Leu Pro Gln Asn Leu Phe Ala Gly Val Phe Asp Lys Leu Lys Pro 50 55 60 Asp Gly Glu Ile Gln Thr Thr Met Arg Val Ser Asn Arg Asp Gly Val 65 70 75 80 Ala Arg Glu Ile Thr Phe Gly Gly Gly Val Lys Thr Asn Glu Ile Phe 85 90 95 Val Lys Lys Gln Asn Ile Cys Val Phe Asp Val Val Asp Ile Phe Ser 100 105 110 Tyr Lys Val Ala Val Ser Thr Glu Glu Thr Val Val Glu Lys Pro Thr 115 120 125 Met Glu Thr Thr Ala Gly Val Arg Phe Lys Ile Arg Leu Ser Val Glu 130 135 140 Asp Val Val Lys Asp Trp Arg Ile Asp Leu Thr Ala Val Lys Thr Ala 145 150 155 160 Glu Leu Gly Lys Ile Ala Gln His Thr Ala Ser Ile Val Gln Arg Thr 165 170 175 Phe Pro Asp Asn Leu Leu Lys Leu Thr Gly Ala Glu Val Ala Lys Leu 180 185 190 Ala Ala Asp Ser Tyr Glu Leu Glu Leu Glu Tyr Thr Gly Lys Ser Pro 195 200 205 Ala Thr Asn Glu Lys Val Asn Val Ala Ala Lys Tyr Ala Val Glu Leu 210 215 220 Leu Ser Ser Val Arg Asn Ala Asn Ser Thr Ala Ala Ala Ser Phe Gly 225 230 235 240 Glu Ser Val Ser Asp Leu Cys Arg Val Ala Lys Ile Ile His Thr His 245 250 255 Glu Tyr Ala Asn Val Val Cys Arg Thr Pro Ser Phe Lys Met Leu Leu 260 265 270 Pro Gln Val Val Ser Leu Thr Lys Ser Ser Tyr Tyr Gly Gly Leu Tyr 275 280 285 Pro Pro Glu Asn Leu Trp Leu Ala Gly Lys Thr Asp Gly Val Arg Ala 290 295 300 Leu Val Val Cys Glu Asp Gly Val Ala Lys Val Ile Thr Ala Glu Ser 305 310 315 320 Val Asp Ile Thr His Gly Val Cys Ser Ala Thr Thr Ile Leu Asp Cys 325 330 335 Glu Leu Asn Val Asp Ala Lys Ile Leu Tyr Val Phe Asp Val Ile Ile 340 345 350 Ser Asn Asn Thr Gln Val Tyr Thr Gln Pro Phe Ser Thr Arg Ile Thr 355 360 365 Thr Asp Ile Ser Asp Ile Lys Ile Asp Gly Tyr Lys Ile Glu Met Lys 370 375 380 Pro Phe Val Lys Val Val Lys Ala Asp Glu Ala Thr Phe Lys Ser Ala 385 390 395 400 Tyr Lys Ala Pro His Asn Glu Gly Leu Ile Met Ile Glu Asp Gly Ala 405 410 415 Ala Tyr Ala Ala Thr Lys Thr Tyr Lys Trp Lys Pro Leu Ser His Asn 420 425 430 Thr Ile Asp Phe Leu Ile Lys Ala Cys Pro Lys Gln Leu Ile Asn Val 435 440 445 Asp Pro Tyr Lys Pro Arg Ala Gly Tyr Lys Leu Trp Leu Leu Phe Thr 450 455 460 Thr Ile Ser Leu Asp Gln Gln Arg Glu Leu Gly Ile Glu Phe Ile Pro 465 470 475 480 Ala Trp Lys Ile Leu Phe Thr Asp Ile Asn Met Phe Gly Ser Arg Val 485 490 495 Pro Ile Gln Phe Gln Pro Ala Ile Asn Pro Leu Ala Tyr Val Cys Tyr 500 505 510 Leu Pro Glu Asp Val Asn Val Asn Asp Gly Asp Ile Val Glu Met Arg 515,520,525 Ala Val Asp Gly Tyr Asp Thr Ile Pro Lys Trp Glu Leu Val Arg Ser 530 535 540 Arg Asn Asp Arg Lys Asn Glu Pro Gly Phe Tyr Gly Asn Asn Tyr Lys 545 550 555 560 They Have Only Asp They Have Tyr Leu They Have Tyr They Have Asp Val Phe His Phe Glu 565,570,575 Asp Leu Tyr Lys Tyr Asn Pro Gly Tyr Phe Glu Lys Asn Lys Ser Asp 580,585,590 Ile Tyr Val Ala Pro Asn Lys Tyr Arg Arg Tyr Leu Ile Lys Ser Leu 595,600,605 Phe Gly Arg Tyr Leu Arg Asp Ala Lys Trp Val Ile Asp Ala Ala Ala Ala 610 615 620 Gly Arg Gly Ala Asp Leu His Leu Tyr Lys Ala Glu Cys Val Glu His 625 630 635 640 Leu Leu Wing Asp Wing Asp Pro Thr Wing Ser Glu Leu Val Arg 645,650,655 Arg Arg Asn Glu Ile Thr Gly Tyr Asn Lys Ser His Arg Gly Gly Arg 660 665 670 Asn Met His Ser His Arg Gly Gln Ser His Cys Ala Lys Ser Thr Ser 675 680 685 Leu His Ala Leu Val Ala Asp Leu Arg Glu Asn Pro Asp Val Leu Ile 690 695 700 Pro Lys Ile Ile Gln Ser Arg Pro His Glu Arg Cys Tyr Asp Ala Ile 705 710 715 720 Val Ile Asn Phe Ala Ile His Tyr Leu Cys Asp Thr Asp Glu His Ile 725 730 735 Arg Asp Phe Leu Ile Thr Val Ser Arg Leu Leu Ala Pro Asn Gly Val 740 745 750 Phe Ile Phe Thr Thr Met Asp Gly Glu Ser Ile Val Lys Leu Leu Ala 755 760 765 Asp His Lys Val Arg Pro Gly Glu Ala Trp Thr Ile His Thr Gly Asp 770 775 780 Val Asn Ser Pro Asp Ser Thr Val Pro Lys Tyr Ser Ile Arg Arg Leu 785 790 795 800 Tyr Asp Ser Asp Lys Leu Thr Lys Thr Gly Gln Gln Ile Glu Val Leu 805 810 815 Leu Pro Met Ser Gly Glu Met Lys Ala Glu Pro Leu Cys Asn Ile Lys 820 825 830 Asn Ile Ile Ser Met Ala Arg Lys Met Gly Leu Asp Leu Val Glu Ser 835 840 845 Ala Asn Phe Ser Val Leu Tyr Glu Ala Tyr Ala Arg Asp Tyr Pro Asp 850 855 860 Ile Tyr Ala Arg Met Thr Pro Asp Asp Lys Leu Tyr Asn Asp Leu His 865 870 875 880 Thr Tyr Ala Val Phe Lys Arg Lys Lys Gly Ala Ser Thr Ala Ser Thr 885 890 895 Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu Ala 900 905 910 Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu Ala 915 920 925 Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu Ala 930 935 940 Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val Ala 945 950 955 960 Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys Met 965 970 975 Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg Gly 980 985 990 Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu Ala 995 1000 1005 Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 1010 1015 1020 Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg 1025 1030 1035 Ala Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu 1040 1045 1050 Ala Lys His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg 1055 1060 1065 Val Gly His Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala 1070 1075 1080 Asp Met Leu Ser Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser 1085 1090 1095 Trp His Lys Glu Asp Ser Ile His Val Gly Val Arg Cys Ile Glu 1100 1105 1110 Met Leu Ile Glu Ser Thr Gly Met Val Ser Leu His Arg Gln Asn 1115 1120 1125 Ala Gly Val Val Gly Gln Asp Ser Glu Thr Ile Glu Leu Ala Pro 1130 1135 1140 Glu Tyr Ala Glu Ala Ile Ala Thr Arg Ala Gly Ala Leu Ala Gly 1145 1150 1155 Ile Ser Pro Met Phe Gln Pro Cys Val Val Pro Pro Lys Pro Trp 1160 1165 1170 Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala Asn Gly Arg Arg Pro 1175 1180 1185 Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala Leu Met Arg Tyr 1190 1195 1200 Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile Asn Ile Ala 1205 1210 1215 Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala Val Ala 1220 1225 1230 Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile Pro 1235 1240 1245 Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 1250 1255 1260 Met Asn Pro Glu Ala Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala 1265 1270 1275 Val Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu 1280 1285 1290 Glu Phe Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala 1295 1300 1305 Ile Trp Phe Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala 1310 1315 1320 Val Ser Met Phe Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu 1325 1330 1335 Leu Thr Leu Ala Lys Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr 1340 1345 1350 Trp Leu Lys Ile His Gly Ala Asn Cys Ala Gly Val Asp Lys Val 1355 1360 1365 Pro Phe Pro Glu Arg Ile Lys Phe Ile Glu Glu Asn His Glu Asn 1370 1375 1380 Ile Met Ala Cys Ala Lys Ser Pro Leu Glu Asn Thr Trp Trp Ala 1385 1390 1395 Glu Gln Asp Ser Pro Phe Cys Phe Leu Ala Phe Cys Phe Glu Tyr 1400 1405 1410 Ala Gly Val Gln His His Gly Leu Ser Tyr Asn Cys Ser Leu Pro 1415 1420 1425 Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln His Phe Ser Ala 1430 1435 1440 Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn Leu Leu Pro 1445 1450 1455 Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys Lys Val 1460 1465 1470 Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn Glu 1475 1480 1485 Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 1490 1495 1500 Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr 1505 1510 1515 Gly Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala 1520 1525 1530 Tyr Gly Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp 1535 1540 1545 Thr Ile Gln Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr 1550 1555 1560 Gln Pro Asn Gln Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu 1565 1570 1575 Ser Val Ser Val Thr Val Val Ala Ala Val Glu Ala Met Asn Trp 1580 1585 1590 Leu Lys Ser Ala Ala Lys Leu Leu Ala Ala Glu Val Lys Asp Lys 1595 1600 1605 Lys Thr Gly Glu Ile Leu Arg Lys Arg Cys Ala Val His Trp Val 1610 1615 1620 Thr Pro Asp Gly Phe Pro Val Trp Gln Glu Tyr Lys Lys Pro Ile 1625 1630 1635 Gln Thr Arg Leu Asn Leu Met Phe Leu Gly Gln Phe Arg Leu Gln 1640 1645 1650 Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu Ile Asp Ala His Lys 1655 1660 1665 Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His Ser Gln Asp Gly 1670 1675 1680 Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu Lys Tyr Gly 1685 1690 1695 Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr Ile Pro 1700 1705 1710 Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met Val 1715 1720 1725 Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln 1730 1735 1740 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala 1745 1750 1755 Leu Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser 1760 1765 1770 Asp Phe Ala Phe Ala 1775 <210> twenty one <211> 5337 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Various features <223> Nucleotide sequence of H3C2:T7 RNA polymerase fusion protein <400> twenty one atgaagattg aggagcatca tcatcatcat catcatcatg gcgcgagcac cgcgaagcgt 60 ctgcagcgtt gccaggacgt gaaccaagtg tgcgaaatct acaacagcaa gggtggcatt 120 ggcgagctgg aactgcgttt cgataaactg ccgcagaacc tgttcgcggg cgtttttgat 180 aagctgaaac cggacggcga gattcaaacc accatgcgtg ttagcaaccg tgacggtgtg 240 gcgcgtgaaa tcacctttgg tggcggtgtt aagaccaacg agatcttcgt gaaaacag 300 aacatttgcg ttttcgacgt ggttgatatc tttagctaca aggtggcggt tagcaccgag 360 gaaaccgtgg ttgaaaagcc gaccatggag accaccgcgg gcgtgcgttt taaaattcgt 420 ctgagcgttg aagacgtggt taaggattgg cgtatcgatc tgaccgcggt gaagaccgcg 480 gagctgggta aaatcgcgca gcacaccgcg agcattgttc aacgtacctt cccggataac 540 ctgctgaagc tgaccggtgc ggaagtggcg aaactggcgg cggacagcta cgagctgggaa 600 ctggagtata ccggcaagag cccggcgacc aacgaaaagg tgaacgttgc ggcgaaatac 660 gcggttgagc tgctgagcag cgtgcgtaac gcgaacagca ccgcggcggc gagctttggt 720 gaaagcgtta gcgacctgtg ccgtgtggcg aaaatcattc acacccacaga gtacgcgaac 780 gtggtttgcc gtaccccgag cttcaaaatg ctgctgccgc aggtggttag cctgaccaag 840 agcagctact atggcggtct gtatccgccg gaaaacctgt ggctggcgggg caagaccgat 900 ggcgtgcgtg cgctggttgt gtgcgaagac ggcgtggcga aagttatcac cgcggagagc 960 gttgatatta cccacggtgt gtgcagcgcg accaccattc tggattgcga gctgaacgtt 1020 gacgcgaaga tcctgtacgt gtttgacgtt atcattagca aaacaccca ggtgtatacc 1080 1140 atcgaaatga agccgtttgt taaggtggtt aaggcggacg aggcgacctt caagagcgcg 1200 tataaagcgc cgcacaacga aggcctgatc atgattgaag atggtgcggc gtacgcggcg 1260 accaagacct ataagtggaa accgctgagc caaacacca tcgatttcct gattaaggcg 1320 tgcccgaaac agctgattaa cgtggacccg tacaagccgc gtgcgggtta taaactgtgg 1380 ctgctgttta ccaccatcag cctggatcag caacgtgaac tgggcatcga gttcattccg 1440 gcgtggaaaa tcctgttcac cgacattaac atgtttggta gccgtgtgcc gatccagttt 1500 1560 gacggcgata ttgttgagat gcgtgcggtg gacggttacg atacatccc gaaatgggaa 1620 1680 atcgcgagcg acatttacct gaactatatt gatgttttcc actttgaaga cctgtacaaa 1740 tataacccgg gctacttcga gaacaag agcgatatct acgtggcgcc gaacaagtac 1800 cgtcgttatc tgatcaaaag cctgttcggt cgttacctgc gtgatgcgaa gtgggtgatt 1860 gatgcggcgg cgggtcgtgg cgcggacctg cacctgtata aagcggaatg cgttgagcac 1920 ctgctggcga tcgacattga tccgaccgcg atcagcgaac tggtgcgtcg tcgtaacgag 1980 attaccggct aacaagag ccaccgtggc ggtcgtaaca tgcacagcca ccgtggtcag 2040 agccactgcg cgaaaagcac cagcctgcac gcgctggttg cggatctgcg tgaaaacccg 2100 gacgttctga ttccgaagat cattcaaagc cgtccgcacg agcgttgcta cgatgcgatc 2160 gttattaact ttgcgatcca ctatctgtgc gacaccgatg aacacatccg tgacttcctg 2220 attaccgtga gccgtctgct ggcgccgaac ggtgttttca tctttaccac catggatggt 2280 gaaagcattg tgaagctgct ggcggaccac aaagttcgtc cgggtgaagc gtggaccatc 2340 cacaccggtg atgtgaacag cccggacagc accgttccga aatacagcat tcgtcgtctg 2400 tatgacagcg ataagctgac caaaaccggc cagcaaatcg aggttctgct gccgatgagc 2460 ggtgaaatga aggcggagcc gctgtgcaac attaaaaaaca tcattagcat ggcgcgtaag 2520 atgggtctgg atctggtgga aagcgcgaac tttagcgttc tgtacgaggc gtatgcgcgt 2580 gactacccgg atatctatgc gcgtatgacc ccggacgata agctctacaa cgacctgcac 2640 acctatgcgg tgttcaagcg taagaaggc gcgagcaccg cgagcaccaa caccatcaac 2700 attgcgaaga acgactttag cgatatcgag ctggcggcga ttccgttcaa caccctggcg 2760 2820 atgggcgagg cgcgtttccg taagatgttt gaacgtcaac tgaaagcggg tgaggttgcg 2880 gataacgcgg cggcgaaacc gctgattacc accctgctgc cgaagatgat cgcgcgtatt 2940 aacgactggt tcgaggaagt taaggcgaaa cgtggtaaac gtccgaccgc gttccagttt 3000 ctgcaagaaa tcaaaccgga ggcggtggcg tacatcacca ttaaaaccac cctggcgtgc 3060 ctgaccagcg cggacaacac caccgtgcag gcggttgcga gcgcgattgg tcgtgcgatt 3120 gaagatgagg cgcgttttgg tcgtatccgt gacctggaag cgaagcactt caagaaaaac 3180 gttgaggaac agctgaacaa acgtgtgggc cacgtttata agaaagcgtt catgcaagtg 3240 gttgaggcgg atatgctgag caagggtctg ctgggcggtg aagcgtggag cagctggcac 3300 aaagaggaca gcatccacgt gggcgttcgt tgcatcgaaa tgctgattga gagcaccggt 3360 atggttagcc tgcaccgtca gaacgcgggt gtggttggcc aagatagcga aaccatcgag 3420 ctggcgccgg aatacgcgga ggcgattgcg acccgtgcgg gtgcgctggc gggtatcagc 3480 ccgatgtttc aaccgtgcgt ggttccgccg aagccgtgga ccggtattac cggcggtggc 3540 tactgggcga acggtcgtcg tccgctggcg ctggtgcgta cccacagcaa gaaagcgctg 3600 atgcgttacg aagacgttta tatgccggaa gtgtataagg cgatcaacat tgcgcaaaac 3660 accgcgtgga aaattaacaa gaaagtgctg gcggttgcga acgtgatcac caagtggaaa 3720 cactgcccgg ttgaagatat cccggcgatt gagcgtgagg aactgccgat gaaaccggaa 3780 gacatcgata tgaacccgga agcgctgacc gcgtggaaac gtgcggcggc ggcggtgtac 3840 cgtaaggaca aagcgcgtaa aagccgtcgt attagcctgg aattcatgct ggagcaggcg 3900 aacaagtttg cgaaccacaa agcgatctgg ttcccgtaca acatggattg gcgtggccgt 3960 gtttatgctg tgagcatgtt caacccgcaa ggcaacgaca tgaccaaggg cctgctgacc 4020 ctggcgaagg gtaaaccgat tggcaaggaa ggctactatt ggctgaaaat ccacggtgcg 4080 aactgcgcgg gcgttgataa agtgccgttc ccggaacgta tcaagtttat tgaggaaaac 4140 cacgagaaca tcatggcgtg cgcgaaaagc ccgctggaaa acacctggtg ggcggagcag 4200 gacagcccgt tctgctttct ggcgttctgc tttgagtacg cgggtgttca acaccacggc 4260 ctgagctata actgcagcct gccgctggcg tttgatggta gctgcagcgg catccagcac 4320 ttcagcgcga tgctgcgtga cgaagttggt ggccgtgcgg tgaacctgct gccgagcgag 4380 accgtgcagg atatctacgg tattgttgcg aagaaagtga acgagatcct gcaagcggat 4440 gcgattaacg gcaccgacaa cgaagtggtt accgtgaccg acgagaacac cggtgaaatc 4500 agcgagaagg ttaaactggg taccaaagcg ctggcgggcc agtggctggc gtacggtgtt 4560 acccgtagcg tgaccaagcg tagcgttatg accctggcgt atggtagcaa agaattcggc 4620 tttcgtcagc aagtgctgga ggataccatc caaccggcga ttgacagcgg caagggcctg 4680 atgtttaccc agccgaacca agcggcgggt tacatggcga aactgatctg ggaaagcgtg 4740 agcgttaccg tggttgcggc ggttgaggcg atgaactggc tgaagagcgc ggcgaaactg 4800 ctggcggcgg aagtgaaaga taagaaaacc ggcgagattc tgcgtaagcg ttgcgcggtt 4860 cactgggtga ccccggacgg tttcccggtt tggcaggaat atagaaacc gatccaaacc 4920 cgtctgaacc tgatgttcct gggccagttt cgtctgcaac cgaccatcaa caccaaaaa 4980 gatagcgaaa ttgacgcgca caagcaggag agcggcattg cgccgaactt tgtgcacagc 5040 caacacggta gccacctgcg taaaaccgtg gtttgggcgc acgaaaagta cggcatcgag 5100 agcttcgcgc tgattcacga tagctttggt accattccgg cggatgcggc gaacctgttc 5160 aaggcggttc gtgaaaccat ggtggatacc tacgagagct gcgacgtgct ggcggacttc 5220 tatgatcagt ttgcggatca actgcacgag agccagctgg acaaaatgcc ggcgctgccg 5280 gcgaagggta acctgaacct gcgtgacatt ctggagagcg attttgcgtt tgcgtaa 5337

Claims

1. A method for capping an uncapped target RNA in vitro, comprising: make (i) an RNA sample comprising the uncapped target RNA; (ii) an RNA capping enzyme consisting of the amino acid sequence according to SEQ ID NO: 7 or an RNA capping enzyme fusion consisting of the amino acid sequence according to SEQ ID NO: 20; (iii) guanosine triphosphate (GTP) or modified GTP; (iv) a buffer; and (v) a methyl donor, contacting at a temperature of 37°C-60°C to form capped target RNA; The RNA capping enzyme is a single-stranded RNA capping enzyme having RNA triphosphatase (TPase), guanylate transferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities.

2. The method of claim 1, wherein the uncapped target RNA is at least 200 nt in length and / or wherein the uncapped target RNA comprises one or more pseudouridines.

3. The method of claim 1 or claim 2, wherein contacting further comprises increasing or decreasing the temperature from a first temperature of 37°C-60°C to a second temperature of 37°C-60°C, wherein the second temperature is different from the first temperature.

4. The method of claim 1, wherein (i), (ii), (iii), and (iv) are RNase-free, and contacting optionally further comprises contacting with one or more RNase inhibitors.

5. The method of claim 1, further comprising chemically synthesizing the uncapped target RNA using solid phase oligonucleotide synthesis.

6. The method of claim 1, further comprising producing the uncapped target RNA by contacting a DNA template encoding the uncapped target RNA with a polymerase to synthesize the uncapped target RNA.

7. The method of claim 1, wherein the methyl donor is S-adenosylmethionine and the contacting further comprises contacting (vi) a cap 2'O methyltransferase.

8. The method of claim 1, wherein the uncapped target RNA comprises one or more pseudouridines.

9. The method of claim 1, wherein contacting further comprises contacting (i), (ii), (iii), (iv), and optionally (v) at a single location.

10. The method of claim 1, further comprising cleaving the capped target RNA with RNase H to form RNase H cleaved fragments, adding FAM-labeled nucleotides to the 3' ends of the cleaved fragments, and analyzing the products by capillary electrophoresis.

11. A method for effectively capping RNA in vitro, comprising: Use at a temperature of 25°C-60°C (i) an RNA sample comprising uncapped target RNA; (ii) a single-stranded RNA capping enzyme having RNA triphosphatase (TPase), guanylate transferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities and consisting of the amino acid sequence according to SEQ ID NO: 7, or an RNA capping enzyme fusion consisting of the amino acid sequence according to SEQ ID NO: 20; (iii) guanosine triphosphate (GTP) or modified GTP; and optionally (iv) O-methyltransferase, in the presence or absence of a methyl donor; and / or (v) buffering agents, Contacting to form a capped target RNA. 12 . The method of claim 11 , further comprising cleaving the capped target RNA with RNase H to form RNase H cleaved fragments, adding FAM-labeled nucleotides to the 3′ ends of the cleaved fragments, and analyzing the products by capillary electrophoresis.

13. A method for capping RNA in vitro, comprising: make (i) Uncapped target RNA; (ii) a single-stranded RNA capping enzyme having RNA triphosphatase (TPase), guanylate transferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities and consisting of the amino acid sequence according to SEQ ID NO: 7, or an RNA capping enzyme fusion consisting of the amino acid sequence according to SEQ ID NO: 20; (iii) guanosine triphosphate (GTP); (iv) a buffer; and (v) methyl donor S-adenosylmethionine (SAM); The contact temperature was between 23°C and 60°C for 30 minutes to form capped target RNA.

14. A composition comprising: (i) Uncapped target RNA; (ii) an RNA capping enzyme fusion having RNA triphosphatase (TPase), guanylate transferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities and consisting of an amino acid sequence according to SEQ ID NO: 20; (iii) guanosine triphosphate (GTP); (iv) a buffer; and (v) Methyl donor.

15. The composition of claim 14, wherein the composition has a temperature in the range of 25°C-60°C.

16. The composition of claim 14, wherein the composition is RNase-free and optionally comprises one or more RNase inhibitors.

17. The composition of claim 14, wherein the composition further comprises a DNA template, a bacteriophage polymerase, and ribonucleotide triphosphates for transcribing the DNA template to form the uncapped target RNA.

18. The composition of claim 14, wherein the composition optionally comprises (vi) S-adenosylmethionine (SAM) and (vii) a cap 2'O methyltransferase.

19. The composition of claim 14, wherein the uncapped target RNA comprises one or more pseudouridines.

20. A kit comprising: an RNA capping enzyme fusion having RNA triphosphatase (TPase), guanylate transferase (GTase), and guanine-N7 methyltransferase (N7MTase) activities, wherein the RNA capping enzyme fusion is in a storage buffer and consists of an amino acid sequence according to SEQ ID NO: 20; and Reaction buffer.

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

22. The kit of claim 20, wherein the kit further comprises S-adenosylmethionine (SAM), cap 2'O methyltransferase (2'OMTase), or both SAM and 2'OMTase.

23. An RNA capping enzyme fusion consisting of the amino acid sequence according to SEQ ID NO: 20.

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