M7gpppa rna cap structure analog

NAD, FAD and m⁷G cap containing RNAs were synthesized by in vitro transcription from a synthetic double stranded DNA template Φ2.5-NAD/FAD-40 containing the T7 Φ2.5 promoter and a single adenosine within the transcript contained at the transcription start site (Supplementary Table S2). For m⁷G-capped RNA, m⁷GpppA RNA Cap Structure Analog (New England Biolabs) was included in the transcription reaction. In vitro transcription was carried out at 37°C overnight, using HiScribe™ T7 High yield RNA Synthesis kit (New England Biolabs). To generate ³²P uniformly labeled RNA, the transcription reactions were performed in the presence of [α-³²P] GTP.

RNAs containing NAD and m⁷G cap structures were synthesized by in vitro transcription from a synthetic double stranded DNA template ɸ2.5-NAD-40 containing the T7 ɸ2.5 promoter and a single adenosine within the transcript contained at the transcription start site (Supplementary Table 1)^{10 (link)}. For m⁷G-capped RNA, m⁷GpppA RNA Cap Structure Analog (New England Biolabs) was included in the transcription reaction. In vitro transcription was carried out at 37 °C overnight, using HiScribe^TM T7 High yield RNA Synthesis kit (New England Biolabs).
To generate ³²P-labeled NAD-capped RNA, transcription was carried out in the presence of ³²P-NAD (PerkinElmer) instead of ATP using ɸ2.5-AG-30 (Supplementary Table 1)^{10 (link)}. To generate ³²P uniformly labeled RNA, the transcription reactions were performed in the presence of [α-³²P] GTP.