Unmodified and modified RNA molecules were transcribed
in vitro using the
MEGAscript T7 kit (Thermo Fisher Scientific). Modified nucleotides triphosphates (
2′O methyl-ATP,
N6 methyl-ATP,
pseudouridine triphosphate) were purchased from TriLink Biotechnologies.
In vitro transcription reactions were assembled according to recommendation from the kit manufacturer and incubated overnight (16 h) at 37°C. Co-transcriptional capping was performed by substituting part of the GTP in the reaction with
cap analog m7G(5′)ppp(5′)G (Thermo Fisher Scientific). After transcription, capped transcripts were treated with 1 μL thermosensitive shrimp alkaline phosphatase (tSAP; Promega) for 15 min at 37°C. All capped and uncapped transcripts were then treated with DNase I for 15 min at 37°C and immediately purified using the
RNeasy Mini Kit (Qiagen). Post-transcriptional polyadenylation was performed on purified transcripts by adding 1 μL
E. coli poly(A) polymerase (5 U/μL; New England Biolabs) and 1 mM ATP and incubating at 37°C for 30 min. Polyadenylation reactions were stopped by purification with the
RNeasy Mini Kit. All transcripts were checked for purity and integrity by UV-vis spectrophotometry and denaturing poly-acrylamide gel electrophoresis (PAGE).
Valentini P., Pierattini B., Zacco E., Mangoni D., Espinoza S., Webster N.A., Andrews B., Carninci P., Tartaglia G.G., Pandolfini L, & Gustincich S. (2022). Towards SINEUP-based therapeutics: Design of an in vitro synthesized SINEUP RNA. Molecular Therapy. Nucleic Acids, 27, 1092-1102.
