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)
Megaclear kit
Manufactured by New England Biolabs
The Megaclear kit is a laboratory equipment designed for the purification of DNA from various sample types. It utilizes a silica-based membrane technology to efficiently capture and elute DNA, providing a convenient and reliable method for DNA extraction and purification.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Antarctic phosphatase, by New England Biolabs (5 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (4 mentions)
Cytidine triphosphate, by Thermo Fisher Scientific (4 mentions)
Megaclear kit, by Thermo Fisher Scientific (4 mentions)
Guanosine triphosphate, by Thermo Fisher Scientific (4 mentions)
3 o me m7g 5 ppp 5 g, by TriLink (3 mentions)
N1 methylpseudouridine 5 triphosphate, by TriLink (2 mentions)
Nanodrop spectrometer, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Geneart gene synthesis, by Thermo Fisher Scientific (1 mentions)
5 protocols using megaclear kit
1
Synthesis and Purification of Modified RNA
2
In Vitro Synthesis of Modified mRNA
3
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.
4
In Vitro Transcription of Modified RNA
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DNA plasmids for RIPK3WT, RIPK3S204>A, RIPK3S232>A or RIPK3DM were purchased from GeneArt Gene Synthesis (Thermo Fisher). In vitro transcription was made using clean tailed (120- poly A tail) DNA PCR products. ModRNAs were transcribed in vitro using a custom ribonucleoside 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), N1-Methylpseu-douridine-50-Triphosphate (7.5 mM, TriLink Biotechnologies). The mRNA was purified using a Megaclear kit (Life Technologies) and was treated with Antarctic Phosphatase (New England Biolabs); then it was purified again using the Megaclear kit. The mRNA was quantitated by Nanodrop (Thermo Scientific), precipitated with ethanol and ammonium acetate, and re-suspended in 10 mM TrisHCl and 1 mM EDTA70 (link).
5
Synthesis of Modified RNA for Protein Expression
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ModRNAs of the selected cardiogenic and vasculogenic growth factors were produced as previously described17 (link). Briefly, mRNA was synthesized using T7 RNA polymerase-mediated IVT from a linearized DNA template containing the target gene’s ORF, flanking 5′ and 3′ UTRs and a poly-A tail. A Cap1 structure was enzymatically added to the 5′ end to produce the final mRNA. Uridine was completely substituted with N1-methylpseudouridine to reduce potential immunostimulatory activity and to improve protein expression relative to unmodified mRNA. ModRNA was then purified using the Ambion MEGAclear kit and treated with Antarctic phosphatase (New England Biolabs) for 30 min at 37 °C to remove residual 5’-phosphates. All modRNAs were re-purified and quantified using Nanodrop (Thermo Scientific). After purification, modRNA was resuspended in MEGAclear buffer at 1 μg/μL concentrations and frozen at −80 °C for future use.
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