Clean PCR products generated with plasmid templates, purchased from GenScript, were used as the template for mRNA. modRNAs were generated by transcription in vitro with a customized ribonucleoside blend of ARCA; 3′-O-Me-m7G(5′)ppp(5′)G (Trilink Biotechnologies, San Diego, CA, USA); Guanosine-5′-triphosphate (GTP); Adenosine triphosphate (ATP); Cytidine triphosphate (CTP); Uridine-5′-triphosphate (UTP) (in case of synthetic mRNA) (Life Technologies, Carlsbad, CA, USA); and N1-Methylpseudouridine-5′-triphosphate (Trilink Biotechnologies) in the case of synthetic modRNA [24 (link)]. The mRNA was purified either with the MEGA clear kit (Life Technologies) according to the manufacturer’s instructions or using Amicon Ultra-4 Centrifugal Filter Unit 4 mL,10 kDa (Millipore Sigma, Burlington, MA, USA) and treated with Antarctic Phosphatase (NEB). Next, the mRNA was re-purified with the MEGA clear kit. The mRNA was quantified using a Nano Drop spectrometer (Thermo Scientific, Waltham, MA, USA), precipitated with ethanol and ammonium acetate, and re-suspended in 10 mM Tris-HCl and 1 mM EDTA. The open reading frame for the Luc and nGFP modRNA is listed below. Endogenous Luc mRNA is a mixture of mRNA enriched with luciferase gene, obtained by isolation of RNA form 4T1-Luc cell line (ATCC #CRL2539LUC2).
N1 methylpseudouridine 5 triphosphate
Manufactured by TriLink
Sourced in United States
N1-methylpseudouridine-5′-triphosphate is a modified nucleotide triphosphate. It is used as a substrate for in vitro transcription and as a building block for the synthesis of modified RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (7 mentions)
Megaclear kit, by Thermo Fisher Scientific (6 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (5 mentions)
Cytidine triphosphate, by Thermo Fisher Scientific (5 mentions)
3 o me m7g 5 ppp 5 g, by TriLink (4 mentions)
Guanosine triphosphate, by Thermo Fisher Scientific (4 mentions)
Antarctic phosphatase, by New England Biolabs (4 mentions)
Pseudouridine 5 triphosphate, by TriLink (4 mentions)
Cleancap reagent ag, by TriLink (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Nanodrop spectrometer, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by New England Biolabs (2 mentions)
Geneart, by Thermo Fisher Scientific (2 mentions)
Qiaquick pcr purification kit, by Thermo Fisher Scientific (2 mentions)
Phusion u green multiplex master mix, by Thermo Fisher Scientific (2 mentions)
Transcriptaid t7 high yield transcription kit, by Thermo Fisher Scientific (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Cleancap ag, by TriLink (2 mentions)
Nanodrop one uv vis spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Megaclear transcription clean up kit, by Thermo Fisher Scientific (2 mentions)
20 protocols using n1 methylpseudouridine 5 triphosphate
1
Synthetic mRNA Production and Purification
2
SARS-CoV-2 S Protein mRNA Synthesis
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To generate the template for RNA synthesis, the sequence of the SARS-CoV-2 S protein (GenBank: QHD43416.1 ) was codon-optimized and cloned into the pVAX1-based backbone, which features a 5′-UTR, 3′-UTR, and Poly-A tail. To increase the protein stability, 2P mutations at positions 986–987 were introduced. The plasmid DNA was produced in bacteria, purified, and linearized by a single-site restriction enzyme digestion. The template DNA was purified, spectrophotometrically quantified, and in vitro transcribed by T7 RNA polymerase (Cat: M0251, NEB) in the presence of a trinucleotide cap1 analog, m7(3′OMeG) (5′)ppp(5′) (2′OMeA)pG (Cat: N-7113, TriLink), and N1-methylpseudouridine-5′-triphosphate (Cat: N-1081, TriLink) in place of UTP. After the reaction, DNase I (Cat: M0303, NEB) was added to remove the template DNA and the mRNA was purified by LiCl precipitation (Cat: AM9480, ThermoFisher).
3
Efficient Synthesis of Customized modRNA
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All modRNA was generated our laboratory by in vitro transcription of plasmid templates (GeneArt, Thermo Fisher Scientific). The full list of open reading frame sequences used to make modRNA for this study can be found in Table S1 . The transcription step involved a customized ribonucleotide blend of anti-reverse cap analog; 30-O-Me-m7G(50) ppp(50)G (6 mM, TriLink Biotechnologies); guanosine triphosphate (1.5 mM, Life Technologies); adenosine triphosphate (7.5 mM, Life Technologies); cytidine triphosphate (7.5 mM, Life Technologies) and N1-methylpseudouridine-5-triphosphate (7.5 mM, TriLink Biotechnologies). Next, modRNA was purified with the Megaclear kit (Life Technologies) and treated with Antarctic Phosphatase (New England Biolabs). To eliminate any remaining impurities, modRNA was re-purified with the Megaclear kit and quantified using a Nanodrop spectrometer (Thermo Scientific). Lastly, modRNA was precipitated with ethanol and ammonium acetate and resuspended in 10 mM Tris-HCl and 1 mM EDTA.
4
In Vitro Synthesis of Modified mRNA
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Clean PCR products generated with plasmid templates (GeneArt; Thermo Fisher Scientific) were used as the template for mRNA. modRNAs were generated by transcription in vitro with a customized ribonucleoside blend of anti-reverse cap analog, 3′-O-Me-m7G(5′)ppp(5′)G (6 mM; TriLink Biotechnologies), guanosine triphosphate (1.5 mM; Life Technologies), adenosine triphosphate (7.5 mM; Life Technologies), cytidine triphosphate (7.5 mM; Life Technologies), and N1-Methyl-Pseudouridine-5'-Triphosphate (7.5 mM; TriLink Biotechnologies). The mRNA was purified with the MEGAclear kit (Life Technologies) and treated with Antarctic Phosphatase (New England Biolabs). It was then repurified with the MEGAclear kit. The mRNA was quantified on a NanoDrop spectrometer (Thermo Scientific), precipitated with ethanol and ammonium acetate, and resuspended in 10 mM Tris-HCl and 1 mM EDTA.
5
Production of mRNA with Modified Nucleotides
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Plasmid templates for in vitro transcription carry an inactivated T7 promoter, 5′ UTR, Kozak sequence, coding sequences and 3′ UTR. Transcription templates were PCR amplified from these plasmids using Phusion U Green Multiplex Master Mix (Thermo Fisher Scientific) with primers that correct the T7 promoter and add a 119-nt poly(A) tail to the 3′ UTR. After purification of the product with QIAquick PCR Purification Kit (Thermo Fisher Scientific), PE2 and hMLH1dn mRNAs were transcribed from these templates using HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs) with full replacement of UTP with N1-methylpseudouridine-5′-triphosphate (TriLink Biotechnologies) and co-transcriptional capping by CleanCap Reagant AG (TriLink Biotechnologies). mRNA products were precipitated in 2.5 M lithium chloride, washed twice with 70% ethanol, dissolved in nuclease-free water and stored at −80 °C.
6
In vitro Transcription of Modified RNA
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In vitro transcription reactions were carried out in 40 μL volumes with 10 pmol of DNA template, using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher, K0441). In some syntheses, pseudouridine-5’-triphosphate (TriLink Biotechnologies, N-1019) or N1-methylpseudouridine-5’-triphosphate (TriLink Biotechnologies, N-1081) were used to replace regular UTP. Reactions were incubated for 3 hours at 37°C, followed by degradation of DNA template with 2 μL of DNase I at 37°C for 30 min. RNA samples were purified with 1.8x volume of AMPure XP beads (Beckman Coulter) mixed with 40% PEG-8000 (ratio of 7:3), following manufacturer’s instructions. Concentrations were measured by absorbance at 260 nm on Nanodrop 100 or 8000 spectrophotometers.
7
In vitro Transcription of Modified RNA
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In vitro transcription reactions were carried out in 40 µL volumes with 10 pmol of DNA template, using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher, K0441). In some syntheses, pseudouridine-5′-triphosphate (TriLink Biotechnologies, N-1019) or N1-methylpseudouridine-5’-triphosphate (TriLink Biotechnologies, N-1081) were used to replace regular UTP. Reactions were incubated for 3 h at 37 °C, followed by degradation of DNA template with 2 µL of DNase I at 37 °C for 30 min. RNA samples were purified with 1.8x volume of AMPure XP beads (Beckman Coulter) mixed with 40% PEG-8000 (ratio of 7:3), following the manufacturer’s instructions. Concentrations were measured by absorbance at 260 nm on Nanodrop 100 or 8000 spectrophotometers.
8
Synthesizing Modified mRNA Constructs
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As described previously (Nelson et al., 2021 (link)), plasmids were cloned to encode an inactivated T7 promoter followed by a 5′ untranslated region (UTR), Kozak sequence, coding sequences of PE2 or MLH1dn, and a 3′ UTR. T7 promoter inactivation prevents potential transcription from circular plasmid template during mRNA generation. These components together were PCR amplified with Phusion U Green Multiplex Master Mix (Thermo Fisher Scientific) using primers that correct T7 promoter inactivation and append a 119-nt poly(A) tail to the 3′ UTR. The resulting PCR product was purified with the QIAquick PCR Purification Kit (Thermo Fisher Scientific) and served as a template for subsequent in vitro transcription. PE2 and MLH1dn mRNAs were transcribed from these templates using the HiScribe T7 High-Yield RNA Synthesis Kit (New England BioLabs) with co-transcriptional capping by CleanCap AG (TriLink Biotechnologies) and full replacement of UTP with N1-Methylpseudouridine-5′-triphosphate (TriLink Biotechnologies). Transcribed mRNAs were precipitated in 2.5 M lithium chloride (Thermo Fisher Scientific), washed twice in 70% ethanol, then dissolved in nuclease-free water. The resulting PE2 and MLH1dn mRNA was quantified with a NanoDrop One UV-Vis spectrophotometer (Thermo Fisher Scientific) and was stored at −80°C.
9
In Vitro mRNA Transcription and Purification
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mRNAs with ARCA or CleanCap® with or without uridine modification were in vitro transcribed using the mMESSAGE mMACHINE® T7 Ultra transcription kit (Ambion, AMB13455). Linearized plasmid DNA containing the target gene downstream of a T7 RNA polymerase promoter was used as the template, and synthesis reactions were performed according to the manufacturer's protocol. For mRNAs with CleanCap®, T7 2× NTP/ARCA was substituted with 8 mM CleanCap® Reagent AG (TriLink Biotechnologies, N-7113) and 10 mM of each NTP. Modified uridines used included pseudouridine-5′-triphosphate (TriLink Biotechnologies, N-1019), N1-methyl-pseudouridine-5′-triphosphate (TriLink Biotechnologies, N-1081) or 5-methoxyuridine-5′-triphosphate (TriLink Biotechnologies, N-1093). mRNAs were subsequently purified by the MegaClear Transcription Clean-up kit (Ambion, AM1908) and quantified on the NanoDrop spectrophotometer.
10
In Vitro mRNA Transcription and Purification
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mRNAs with ARCA or CleanCap® with or without uridine modification were in vitro transcribed using the mMESSAGE mMACHINE® T7 Ultra transcription kit (Ambion, AMB13455). Linearized plasmid DNA containing the target gene downstream of a T7 RNA polymerase promoter was used as the template, and synthesis reactions were performed according to the manufacturer's protocol. For mRNAs with CleanCap®, T7 2× NTP/ARCA was substituted with 8 mM CleanCap® Reagent AG (TriLink Biotechnologies, N-7113) and 10 mM of each NTP. Modified uridines used included pseudouridine-5′-triphosphate (TriLink Biotechnologies, N-1019), N1-methyl-pseudouridine-5′-triphosphate (TriLink Biotechnologies, N-1081) or 5-methoxyuridine-5′-triphosphate (TriLink Biotechnologies, N-1093). mRNAs were subsequently purified by the MegaClear Transcription Clean-up kit (Ambion, AM1908) and quantified on the NanoDrop spectrophotometer.
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