Guanylyl transferase

Manufactured by Aldevron

Guanylyl transferase is an enzyme that catalyzes the addition of a guanine nucleotide to the 5' end of messenger RNA (mRNA) molecules. This process, known as capping, is an essential step in the maturation of mRNA and facilitates its efficient translation into proteins.

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Lab products found in correlation

Dnase 1, by Aldevron (2 mentions) Nanodrop 1000, by Thermo Fisher Scientific (2 mentions) Alkaline phosphatase, by New England Biolabs (1 mentions) Sodium acetate, by Thermo Fisher Scientific (1 mentions) Noti hf, by New England Biolabs (1 mentions) Hiscribe t7 kit, by New England Biolabs (1 mentions) Pyrophosphatase enzymes, by Aldevron (1 mentions) Ribogreen assay, by Thermo Fisher Scientific (1 mentions) Capto core 700 resin, by GE Healthcare (1 mentions) T7 polymerase, by Aldevron (1 mentions) Rnase inhibitor, by Aldevron (1 mentions) S adenosyl methionine, by Aldevron (1 mentions) Rntp mix, by New England Biolabs (1 mentions)

2 protocols using guanylyl transferase

Protocol for Optimized mRNA Production

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Plasmid templates were designed using Geneious software, and codon optimized and ordered from GenScript. Plasmids were linearized with Not‐I HF (New England Biolabs) overnight at 37 °C. Linearized templates were purified by sodium acetate (Thermo Fisher Scientific) precipitation and rehydrated with nuclease‐free water. In vitro transcription was performed overnight at 37 °C using a HiScribe T7 Kit (NEB) following the manufacturer's instructions (complete N1‐methyl‐pseudouridine modification). The resulting RNA was treated with DNase I (Aldevron) for 30 min and purified using lithium chloride precipitation (Thermo Fisher Scientific). The RNA was heat denatured at 65 °C for 10 min before capping with a Cap‐1 structure using guanylyl transferase and 2′‐O‐methyltransferase (Aldevron). mRNA was then purified by lithium chloride precipitation, treated with alkaline phosphatase (NEB), and purified again. mRNA concentration was measured using a Nanodrop. mRNA stock concentrations were 1–3 mg mL⁻¹ and were stored at −80 °C until use. Purified mRNA products were analyzed by gel electrophoresis to ensure purity.

Vanover D., Zurla C., Peck H.E., Orr‐Burks N., Joo J.Y., Murray J., Holladay N., Hobbs R.A., Jung Y., Chaves L.C., Rotolo L., Lifland A.W., Olivier A.K., Li D., Saunders K.O., Sempowski G.D., Crowe JE J.r., Haynes B.F., Lafontaine E.R., Hogan R.J, & Santangelo P.J. (2022). Nebulized mRNA‐Encoded Antibodies Protect Hamsters from SARS‐CoV‐2 Infection. Advanced Science, 9(34), 2202771.

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In Vitro Transcription of saRNA

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Vaccine saRNA was generated by T7 polymerase-mediated in vitro transcription (IVT) using NotIlinearized DNA plasmids as templates. An in-house optimized IVT protocol was used with commercially available rNTP mix (NEB) and commercially available T7 polymerase, RNase inhibitor, and pyrophosphatase enzymes (Aldevron, Fargo, ND). DNA plasmids were digested away (DNase I, Aldevron), and Cap 0 structures were added to the transcripts by treatment with guanylyltransferase (Aldevron), GTP, and S-adenosylmethionine (NEB). RNA was chromatographically purified using Capto Core 700 resin (GE Healthcare, Chicago, IL) followed by diafiltration and concentration through tangential flow filtration using a 750 kDa molecular weight cut-off (MWCO) modified polyethersulfone (mPES) membrane (Repligen, Waltham, MA). Terminal filtration of the saRNA material was done using a 0.22 µm PES filter, and the saRNA materials were stored at -80°C until use/complexation. Agarose gel electrophoresis was used to characterize saRNA size and integrity, and RNA concentration was quantified by UV absorbance (NanoDrop 1000) and RiboGreen assay (Thermo Fisher Scientific, Waltham, MA).

Voigt E.A., Gerhardt A., Hanson D., Battisti P., Reed S., Singh J., Mohamath R., Jennewein M.F., Bakken J., Beaver S., Press C., Soon‐Shiong P., Paddon C.J., Fox C.B., & Casper C. (2022). A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability.

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