In vitro transcription of m7G- or NAD-capped RNAs

The short m⁷GpppA-RNA and NAD-RNA molecules were synthesized in vitro by T7 polymerase following the published method (Zhang et al. 2016). The double-stranded DNA templates were modified as follows. Our DNA template was formed by annealing the two single-stranded DNA oligonucleotides (5′-GATCACTA ATACGACTCACTATTACTGAAGCGGGC-3′ and 5′ GCCCGC TTCAGTAATAGTGAGTCGTATTAGTGATC-3′, the transcription start site is underlined). The transcription reactions were performed in 1 mL buffer containing 1 μM DNA template, 4 mM NAD or 4 mM m⁷GpppA, 4 mM rCTP, 4 mM rUTP, 50 mM Tris-HCl, pH 8.0, 20 mM MgCl₂, 2 mM spermidine, 0.01% Triton X-100, 40 mM DTT and 0.1 mg/mL T7 RNA polymerase at 37°C for 4 h. The short NAD-RNA was isolated using a 1-mL Mono Q column (GE Healthcare, Chicago, Illinois, USA). Nuclease P1 was used to verify the short length of the NAD-RNA.

The DNA template for the 38-nt NAD-RNA was made by annealing the following two single-stranded DNA oligonucleotides (5′-CAGTAATACGACTCACTAT TAGGCCTCTCGCTCTGCTGGGTGTGCGCTTGCTTGGCTT-3′ and 5′-AAGCCAAGCAAGCGCACACCCAGCAGAGCGAGA GGCCTAATAGTGAGTCGTATTACTG-3′, the transcription start site is underlined). The synthetic NAD-RNA was isolated using a 15% denaturing Polyacrylamide Gel (Urea-PAGE) containing 8 M urea. NAD-RNA was extracted from the gel following the previously described method (Winz et al. 2017).

NAD- or m⁷G-capped luciferase mRNA with a poly (A₆₀) tail was generated following the protocol described previously (Jiao et al. 2017), with minor modifications. In brief, the pMD19-Luciferase plasmid was generated by adding the 5′ leader ϕ2.5A-CA2, which contained the ϕ2.5 A T7 promoter (Coleman et al. 2004) lacking adenosines, except the first transcribed nucleotide, to the firefly luciferase open reading frame. ϕ2.5A-CA2-Luciferase-A₆₀ was amplified by PCR using the 3′ primer containing 60 thymidines at its 5′ end and was used as DNA template for in vitro transcription. The transcription reactions were first incubated at 37°C for 15 min by mixing the DNA template, NAD or m⁷GpppA cap analog, T7 RNA polymerase (Promega) and rCTP, rUTP and rGTP (without rATP) and then incubated with 200 μg/mL heparin to inhibit transcription reinitiation and rATP to the mixture for another 30 min incubation. MicroSpin G-50 Columns (GE Healthcare) used to isolate thein vitro transcribed NAD or m⁷G-capped RNAs.