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).
3 o me m7g 5 ppp 5 g
Manufactured by TriLink
Sourced in United States
3'-O-Me-m7G(5')ppp(5')G is a chemically modified guanine nucleotide used as a cap analog in in vitro transcription reactions. It serves as a structural analog of the 5' cap found on eukaryotic mRNA, facilitating efficient translation of the synthesized RNA.
Automatically generated - may contain errors
Lab products found in correlation
Megaclear kit, by Thermo Fisher Scientific (5 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (5 mentions)
Cytidine triphosphate, by Thermo Fisher Scientific (5 mentions)
N1 methylpseudouridine 5 triphosphate, by TriLink (4 mentions)
Guanosine triphosphate, by Thermo Fisher Scientific (4 mentions)
Antarctic phosphatase, by New England Biolabs (4 mentions)
Nanodrop spectrometer, by Thermo Fisher Scientific (3 mentions)
Megaclear kit, by New England Biolabs (3 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Antarctic phosphatase, by Merck Group (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Geneart, by Thermo Fisher Scientific (1 mentions)
5 protocols using 3 o me m7g 5 ppp 5 g
1
Synthetic mRNA Production and Purification
2
Synthesis and Purification of Modified RNA
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ModRNAs were synthesized in house. All DNA plasmid templates used for modRNA production were purchased from GeneArt and Invitrogen and were verified by sequencing. PCR-tailed DNA plasmids (120 poly(A) tail) were used for in vitro transcription to generate modRNAs (see Table S1 for complete list of open reading frame sequences used for modRNA synthesis in this study) with a customized ribonucleotide 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 1-mψU (7.5 mM, TriLink Biotechnologies), as previously described.37 (link), 38 (link), 39 (link), 40 (link) We purified the mRNA with the Megaclear kit (Life Technologies) and treated it with Antarctic phosphatase (New England Biolabs) for 1 hr, before repurification with the Megaclear kit. The mRNA was precipitated by incubation overnight with ammonium acetate and ethanol at 4°C and resuspended in 10 mM TrisHCl, 1 mM EDTA. We quantified the mRNA with a Nanodrop spectrometer (Thermo Scientific) and checked its quality with a bioanalyzer (Agilent Technologies). A more detailed description of the protocol for modRNA synthesis is provided elsewhere.39 (link)
3
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.
4
In Vitro Synthesis of Modified mRNA
5
In vitro Transcription of ModRNA
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ModRNAs were transcribed in vitro from plasmid templates (see Table S1 (Supporting Information) for a complete list of open reading frame sequences used to make the modRNA for this study) using a customized ribonucleotide blend of anti‐reverse cap analog; 3 ´‐O‐Me‐m7G (5')ppp(5')G (6 × 10−3m , TriLink Biotechnologies); guanosine triphosphate (1.5 × 10−3m , Life Technology); adenosine triphosphate (7.5 × 10−3m , Life Technology); cytidine triphosphate (7.5 × 10−3m , Life Technology); and N1‐Methylpseudouridine‐5'‐Triphosphate (7.5 × 10−3m , TriLink Biotechnologies) as described previously in the recent protocol paper.[52 ] The mRNA was purified using the Megaclear kit (Life Technology) and treated with antarctic phosphatase (New England Biolabs), followed by re‐purification using the Megaclear kit. The mRNA was quantitated by Nanodrop (Thermo Scientific), precipitated with ethanol and ammonium acetate, and resuspended in 10 × 10−3m TrisHCl, 1 × 10−3m EDTA.
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