Pseudouridine triphosphate

Synthetic modified RNA (modRNA) was generated as described [29 (link)]. Briefly, RNA was synthesized with the MEGAscript T7 kit (Ambion, Austin, TX). A custom ribonucleoside blend was used, comprising 6 mM 5’ cap analog (New England Biolabs), 7.5 mM adenosine triphosphate and 1.5 mM guanosine triphosphate (USB, Cleveland, OH), 7.5 mM 5-methylcytidine triphosphate and 7.5 mM pseudo-uridine triphosphate (TriLink Biotechnologies, San Diego, CA). Transfections of modRNA and multiple siRNA targeted against Sirt1 and Satb2 (both from Santa Cruz) were carried out with RNAiMAX (Invitrogen), as per the manufacturer’s instructions.

In vitro transcription was performed as previously described^{29 (link),30 (link)}. Briefly, plasmids except for that containing B18R gene was linearized with Spe-I, purified and used as template for in vitro transcription using T7 High Yield RNA Synthesis Kit (Cat #E2040, NEB) in the presence of anti-reverse cap analogue (Trilink) according to manufacturer’s protocol with a capping efficiency of ~80% (based on 4:1 ratio of ARCA cap to GTP). Pseudouridine (Ψ)-modified mRNA was synthesized by the same method with uridine triphosphate completely replaced by pseudouridine triphosphate (Trilink). Plasmid containing B18R gene was linearized with Sac-I, purified and transcribed. The transcript was then subjective to enzymatic poly A tail reaction with E.coli poly (A) polymerase (Cat #M0276, NEB). Successful poly A tailling was confirmed with gel electrophoresis. All synthesized mRNA was purified with RNeasy kit (Qiagen), quantified by spectrophotometry and analyzed by agarose gel electrophoresis to confirm the synthesis of full-length mRNA.

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).

Messenger RNAs were generated as previously described [19 (link)], using linearized plasmids (pTEV-CPD-PL-A101 and pTEVeGFP-A101) encoding codon-optimized Potorous CPD-photolyase (CPD-PL Ψ-mRNA) and enhanced green fluorescent protein (eGFP Ψ-mRNA). The CPD-photolyase gene from Potorous tridactylus (rat kangaroo) was synthesized by Entelechon (Bad Abbach, Germany). The Megascript T7 RNA polymerase kit (Ambion, Austin, TX) was used for transcription, and UTP was replaced with pseudouridine triphosphate (TriLink, San Diego, CA) [21 (link)]. To remove the template DNA Turbo DNase (Ambion) was added to the reaction mix. Pseudouridine-modified mRNAs were HPLC-purified as described [36 (link)] and provided with cap1 generated by using the m7G capping enzyme and 2′-O-methyltransferase according to the manufacturer (CellScript, Madison, WI). The mRNAs were transcribed to contain 101 nt-long 3’ poly(A) tail. Small aliquots of RNA samples were stored in siliconized tubes at -20°C.