RNAs containing NAD and m7G cap structures were synthesized by in vitro transcription from synthetic double-stranded DNA template ɸ2.5-NAD-40, ɸ2.5-NAD-75 containing the T7 ɸ2.5 promoter, and a single adenosine within the transcript positioned at the transcription start site (Supplementary Table 3 ). For m7G-capped RNA, m7G-(5′) PPP (5′)-A RNA Cap Structure Analog (New England Biolabs) was included in the transcription reaction, whereas for NAD-RNA, NAD was used instead of ATP. In vitro transcription was carried out at 37 °C overnight, using HiScribeTM T7 High yield RNA Synthesis kit (New England Biolabs (NEB)). Following in vitro transcription, RNA was purified using Monarch®RNA Cleanup Columns (NEB) as per the manufacturer’s instructions.
M7g 5 ppp 5 a rna cap structure analog
Manufactured by New England Biolabs
The M7G(5′)ppp(5′)A RNA cap structure analog is a synthetic compound that mimics the natural cap structure found at the 5' end of eukaryotic mRNA. It is used in various research applications to study mRNA processing, translation, and stability.
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3 protocols using m7g 5 ppp 5 a rna cap structure analog
1
Synthesis of NAD and m7G-capped RNAs
2
Generation of Mutant Dengue Virus
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DV2(KIKP) was generated based on an infectious clone of DV2 16681, pD2/IC-30P, kindly provided by C. Huang (Center for Disease Control and Prevention) and described previously30 (link), 31 (link). PCR was used to generate mutant pD2/IC-30P harboring R64K and E66K mutations in the NS3 gene. The WT and mutant infectious clone plasmids were linearized by XbaI digestion and in vitro transcribed using the T7 promoter (RiboMAX Large Scale RNA Production System, Promega) with the addition of a m7G(5′)ppp(5′)A RNA cap structure analog (New England Biolabs). The in vitro transcribed RNA was purified using Micro Bio-Spin columns (Bio Rad) and transfected into Vero cells using Lipofectamine 2000. Viral supernatants were harvested and used to propagate the WT and mutant virus in Vero cells. Vero cells were further used to titer the recombinant viruses by serial dilution of the supernatant and staining with anti-prM antibody, followed by FACS analysis45 (link).
3
Generation of Mutant Dengue Virus
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