Coding region of LANA, EBNA1, and vIRF3 were inserted to pZMV plasmid that contains a T7 promoter. The capped mRNA was synthesized from corresponding linearized pZMV plasmid using the HiScribe T7 High Yield RNA Synthesis Kit (NEB) and Cleancap reagent (Trilink). After a total incubation for 2 hours at 37°C, the DNA was digested by DNase I treatment and mRNA was purified using RNA Clean & Concentrator-5(Zymo). Polyadenylation was performed by using E.coli Poly(A) Polymerase (NEB).The mRNA was again purified using RNA Clean & Concentrator-5. mRNA concentration was determined on a NanoDrop OneC (Thermo Fisher). mRNA quality was determined by 1% formaldehyde agarose gel. In vitro transcribed mRNA was transfected to HEK-293T cells using TransIT reagent (Mirus).
Cleancap reagent
Manufactured by TriLink
Cleancap is a reagent used for the enzymatic removal of unwanted DNA and RNA from laboratory samples. It functions to purify and clean the sample, preparing it for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (2 mentions)
E coli poly a polymerase, by New England Biolabs (1 mentions)
Nanodrop onec, by Thermo Fisher Scientific (1 mentions)
Rna clean concentrator 5, by Zymo Research (1 mentions)
Transit reagent, by Mirus Bio (1 mentions)
In vitro transcription kit, by Promega (1 mentions)
Agilent 1100, by Agilent Technologies (1 mentions)
N1 methylpseudouridine 5 triphosphate, by TriLink (1 mentions)
Monarch rna purification columns, by New England Biolabs (1 mentions)
5 protocols using cleancap reagent
1
Synthesis and Transfection of Viral mRNA
2
Synthesis and Purification of SARS-CoV-2 Spike mRNA
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All the SARS-CoV-2 spike mRNAs were produced and purified using a proprietary custom process at TriLink BioTechnologies (San Diego, CA, USA). A method for synthesizing mRNA was employed using a single in vitro transcription (IVT) reaction with T7 RNA polymerase and linearized DNA plasmids to create codon-optimized mRNAs (PVX1010, PVX1020, PVX1040, and PVX1050). The reaction utilized TriLink’s CleanCap® reagent, which introduced a modification known as 3’OMe on the m7-Guanosine. Notably, the co-transcriptional capping process achieved an efficiency of over 95% in capping the mRNA. The primary objective of this technique was to enhance translation efficiency within living cells and organisms. The IVT reaction was designed to allow for the complete substitution of regular NTPs with modified Pseudo-UTP instead of UTP. Following the synthesis, the RNA was purified using a spin column-based method, effectively removing unincorporated nucleotides, proteins, and salts. Subsequently, the concentration of the purified mRNA was determined by examining the ultraviolet absorbance at 260 nm, while the molecular weight was determined through gel electrophoresis.
3
Generation and Purification of GFP mRNA
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To construct mRNA generation template, GFP sequence was codon optimized and clone into T7 promoter and polyA encoded plasmid. After template linearization, GFP mRNA was generated through in vitro transcription kit (Promega) and Cleancap reagent (TriLink). The High performance Liquid Chromatography (HPLC, Agilent 1100, Agilent Technologies, Santa Clara, CA, United States) was applied for mRNA purification. All mRNAs were checked via agarose gel electrophoresis and stored frozen at −80°C.
4
In vitro synthesis of prime editor, base editor, and MLH1dn mRNA
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The prime editor, ABE, cytosine base editor and MLH1dn-encoded mRNA were generated using in vitro transcription as previously described 61 (link) . Briefly, the prime editor, base editor or MLH1dn transcripts, containing a 5′ untranslated region, Kozak sequence, prime editor or MLH1dn open reading frame, and 3′ untranslated region were PCR amplified from a template plasmid containing an inactive T7 (dT7) promoter. PCR primers repaired this dT7 promoter and also installed a 119 nt poly(A) tail. The purified PCR double-stranded DNA amplicon was used as an in vitro transcription template using the HiScribe T7 high-yield RNA synthesis kit (NEB). In vitro transcription followed the manufacturer's optional protocol to include CleanCap reagent https://doi.org/10.1038/s41551-024-01233-3 AG (Trilink) and substitute N1-methylpseudouridine-5′-triphosphate (Trilink) for uridine triphosphate. Lithium chloride precipitation was used to purify mRNA from complete in vitro transcription reactions, and mRNA transcripts were reconstituted in nuclease-free water.
5
Optimized mRNA Vaccine Formulation
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mRNAs encoding for HBsAg (NCBI accession number: YP_009173871) were synthesized by T7 polymerase-mediated in vitro transcription (IVT) based on a linearized DNA template (pUC57-GW-Kan) containing codon-optimized HBsAg gene flanked with 5’ and 3’ untranslated regions (UTRs) and a 100 nt poly-A tail. During IVT procedure, mRNAs were modified with N1-Methyl-pseudouridine (Synthgene) and capped using CleanCap Reagent (TriLink). After this, IVT products were purified with Monarch RNA purification columns (NEW ENGLAND BioLabs Inc. MA, USA) and resuspended in a TE buffer at a desired concentration. For mRNA encapsulation into LNP, lipid components were dissolved in ethanol at molar ratios of 50:10:38.5:1.5 (ionizable lipid: DSPC: cholesterol: DMG-PEG2000). The ionizable lipid (YX-02) was designed and has been patented by Firestone Biotechnologies. The lipid cocktail was mixed with mRNAs dissolved in 10 mM citrate buffer (pH4.0) at an N/P ratio of 5.3 :1 and a volume ratio of 3: 1 using a microfluidic-based equipment (INanoTML from Micro&Nano Biologics) at a total flow rate of 12 mL/min. Formulations were diluted with PBS and ultrafiltrated using 50-kDa Amicon ultracentrifugal filters. Vaccine formulation was characterized for particle diameter, polymer dispersity index (PDI) and zeta potentials using NanoBrook Omni ZetaPlus (Brookhaven Instruments).
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