Figure 2.

Synthesis of metabolite-capped RNAs by in vitro T7 transcription. T7 RNA polymerase promoter sequence and the desired transcript sequence (underlined) with adenine at the + 1 transcribing position. The sequence lacks internal adenosines, allowing transcription in the absence of ATP. The terminal methylated cytosines encourage T7 RNA polymerase dissociation from the promoter DNA upon completion of one round of RNA synthesis. Cytosines are excluded from the transcript sequence, and CTP is not added to the reaction to prevent copy-back transcription. The dsDNA promoter is incubated with T7 RNA polymerase and other reagents described in the Methods to generate the desired 39-mer RNA transcript, purified from remaining NTPs and abortive products using Urea-PAGE, gel electroelution, and ethanol precipitation. The purity of these ssRNAs was assessed using LC–MS, shown in the table, with percentages of each RNA species in parenthesis. If no percentage is shown, the purity is 100%. The ssRNAs were annealed to a chemically synthesized, complementary RNA with a 3-nt DNA overhang at the 5′-end to prevent RIG-I binding at the opposite end from the 5′PP-cap end, as well as having a fluorescein moiety on the second DNA position for biochemical assays. Created with Biorender.com.