Vaccinia 2 o methyltransferase

Manufactured by New England Biolabs

Vaccinia 2'O-methyltransferase is an enzyme that catalyzes the methylation of the 2'-hydroxyl group of the terminal nucleoside in viral RNA molecules. This enzymatic activity is important for the capping and stabilization of viral mRNA, which is a crucial step in the viral replication process.

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Lab products found in correlation

Vaccinia capping enzyme, by New England Biolabs (4 mentions) Pyrophosphatase, by Merck Group (1 mentions) Dnase 1, by Promega (1 mentions) Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions) Zymo rna clean and concentrator, by Zymo Research (1 mentions) Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions) Sodium acetate ph 5, by Merck Group (1 mentions) T7 rna polymerase, by New England Biolabs (1 mentions) 0.22 μm filter, by Merck Group (1 mentions) Amicon ultra centrifugal filter, by Merck Group (1 mentions)

4 protocols using vaccinia 2 o methyltransferase

Synthesis and Purification of mRNA for In Vitro Translation

Cited 1 time

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In vitro transcription reactions were performed using PCR products generated with primers encoding a flanking T7 RNA polymerase promoter and a poly-A tail. Reactions were set up, as previously described (66 (link)), with 20 mM Tris-HCl pH 7.5, 35 mM MgCl₂, 2 mM spermidine, 10 mM DTT, 1 u/ml pyrophosphatase (Sigma), 7.5 mM of each NTP, 0.2 u/ml RiboLock RNase Inhibitor (ThermoFisher), 0.1 mg/ml T7 RNA polymerase and 40 ng/μl PCR-generated DNA. After 3 h incubation at 37 °C, 0.1 u/μl DNase I (Promega) was added to the reactions, which were incubated at 37 °C for 30 min to remove the template DNA. RNA was precipitated for 2–3 h at −20 °C after adding 0.5x volume of 7.5 M LiCl/50 mM EDTA, and the resulting pellet was washed with cold 70% ethanol and dissolved with RNase-free water. The mRNA was further purified by using a Zymo RNA Clean and Concentrator (Zymo Research) before use in in vitro translation reactions.
DNA templates were amplified from a plasmid containing the corresponding 5’ UTR and the NanoLuc Luciferase coding sequence. Primers used for this amplification added a 30T sequence at the 3′ end to form a poly(A) tail after transcription. The HBB 5’ UTR containing mRNA was then capped using Vaccinia Capping enzyme (New England Biolabs) and 2′O-methylated using Vaccinia 2′O Methyltransferase (New England Biolabs). The IRES-containing mRNAs were uncapped and polyadenylated.

Aleksashin N.A., Chang S.T, & Cate J.H. (2023). A highly efficient human cell-free translation system. bioRxiv.

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Synthesis and Purification of Modified mRNA

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mRNA was synthesized in vitro by T7 RNAP-mediated transcription at 37 °C using 100% substituted N1-methylpseudouridine-triphosphate and a linearized DNA template, which incorporated the 5′ and 3′ untranslated regions and a polyadenosine tail. Reactions with WT and mutant enzymes were treated similarly. NTPs were included at equimolar concentrations. After transcription, the Cap 1 structure was added to the 5′ end using Vaccinia capping enzyme (New England Biolabs) and Vaccinia 2′O-methyltransferase (New England Biolabs). The mRNA was purified by oligo-dT affinity purification. For mRNAs described as ‘with RP’, ion-paired reversed-phase (RP) chromatography was subsequently used for purification. All mRNAs were buffer exchanged by tangential flow filtration into sodium citrate, pH 6.5, and sterile filtered. The mRNA was kept frozen at –20 °C until further use.

Dousis A., Ravichandran K., Hobert E.M., Moore M.J, & Rabideau A.E. (2022). An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts. Nature Biotechnology, 41(4), 560-568.

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Synthesis and Formulation of mRNA LNPs

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A sequence-optimized mRNA was synthesized by in vitro T7 RNA polymerase (New England Biolabs, Ipswich, MA, USA) mediated transcription with complete replacement of uridine by N1-methyl-pseudouridine. The final mRNA encoded the protein of interest, a 5′ untranslated region (UTR), a 3′ UTR, and a DNA-template encoded polyA tail. After transcription, the Cap 1 structure was added to the 5′ end using Vaccinia Capping Enzyme (New England Biolabs) and Vaccinia 2′ O-methyltransferase (New England Biolabs). Lipid nanoparticles were produced via nanoprecipitation by mixing lipids dissolved in ethanol (ionizable lipid, phospholipid, sterol, and polyethylene glycol lipid) with mRNA diluted in 25 mM sodium acetate (pH 5) (Sigma Aldrich, St. Louis, MO, USA) at a ratio of 3:1 (aqueous: ethanol). Formulations were then dialyzed against PBS (pH 7.4) (Lonza, Basel, Switzerland) in a Slide-A-Lyzer dialysis cassette (ThermoFisher, MA, USA) for ≥18 h at 4 °C. Formulations were concentrated using Amicon ultra centrifugal filters (EMD Millipore, Burlington, MA, USA), passed through a 0.22-μm filter (EMD Millipore, MA, USA), and stored at 4 °C. All formulations were tested for particle size, RNA encapsulation, and endotoxins. Formulations were between 80 nm–100 nm, with >90% encapsulation and <10 EU/mL endotoxin and were deemed acceptable for in vivo study.

Narayanan E., Falcone S., Elbashir S.M., Attarwala H., Hassett K., Seaman M.S., Carfi A, & Himansu S. (2022). Rational Design and In Vivo Characterization of mRNA-Encoded Broadly Neutralizing Antibody Combinations against HIV-1. Antibodies, 11(4), 67.

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In Vitro Synthesis of Capped mRNA

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All mRNA constructs were manufactured in vitro by T7 RNA polymerase-mediated transcription, with complete replacement of uridine by N1-methyl-pseudouridine, as previously described.²⁹ (link) Briefly, the DNA template used in the in vitro reaction contained the immunogen open reading frame flanked by 5′ UTR and 3′ UTR sequences and was terminated by a polyA tail. After transcription, the pre-mRNA was purified by oligo-dT affinity, and the cap 1 structure was added to the 5′ end using Vaccinia capping enzyme (New England Biolabs, Ipswich, MA) and Vaccinia 2′O-methyltransferase (New England Biolabs, Ipswich, MA). The capped mRNA was then purified by reverse-phase purification; buffer exchanged by tangential flow filtration into sodium citrate, pH 6.5, sterile filtered, and kept frozen at −80°C until further use.
mRNA-LNPs were manufactured via nanoprecipitation by mixing the ionizable lipid (heptadecan-9-yl-8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy)hexyl)amino)octanoate), distearoylphosphatidylcholine, cholesterol, and PEG2k-DMG lipids dissolved in ethanol with mRNA diluted in sodium acetate buffer (pH 5.0).¹² (link) mRNA-LNPs were then buffer exchanged into a physiologically relevant buffer system and sterile filtered before storage. All of the formulations were tested for particle size, mRNA encapsulation, and endotoxin levels and were deemed acceptable for in vivo study.

Hassett K.J., Rajlic I.L., Bahl K., White R., Cowens K., Jacquinet E, & Burke K.E. (2023). mRNA vaccine trafficking and resulting protein expression after intramuscular administration. Molecular Therapy. Nucleic Acids, 35(1), 102083.

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