Scriptcap 2 o methyltransferase enzyme

Reporter mRNA for was transcribed with a SP6-Scribe Standard RNA IVT Kit (CellScript). Polyadenylated BTF3_Fluc mRNA was transcribed from pSP36T-5′UTR_BTF3-FLuc-A50 linearized with HpaI. mRNAs were capped using the ScriptCap m⁷G Capping System and the ScriptCap 2′-O-methyltransferase enzyme (CellScript) according to the manufacturer's manual.

Rabbit beta-globin mRNA was transcribed by T7 RNA polymerase from pET28a-BetaGlob-bGlob linearized with EcoRI. Firefly luciferase mRNA with 5′ UTR from rabbit beta-globin mRNA was transcribed by SP6 RNA polymerase from pSP36TBetaGlobFLucA50 linearized with SmaI. The transcription was performed using a SP6-Scribe Standard RNA IVT Kit (CellScript, WI, USA). For co-transcriptional rabbit beta-globin mRNA capping, the GTP concentration in the reaction mixture was reduced to 0.2 mM, and the m⁷G(5′)ppp(5′)G RNA cap structure analog (NEB, Ipswich, MA, USA) was added to 3.8 mM. The capped mRNA transcript for in vitro translation was obtained using a ScriptCap™ m⁷G Capping System and ScriptCap 2′-O-Methyltransferase Enzyme (CellScript, Madison, WI, USA) according to the manufacturers’ recommendations.
To obtain m⁶A-containing mRNA for experiments on the formation of 48S complexes, ATP was completely replaced by m⁶ATP at the same concentration (4 mM) in the transcriptional mixture. For translation in a cell-free system, mRNA containing 50% m⁶A was used. To obtain such mRNA, a mixture of 2 mM ATP and 2 mM m⁶ATP was used for in vitro transcription. The quality of the obtained mRNAs was checked by electrophoresis in 6% polyacrylamide gel (PAGE) containing 7 M urea.

3′-end biotinylation was performed as described previously26 (link). Briefly, a 50 μg RNA sample was oxidized in 100 μl of fresh solution of 5 mM NaIO₄ in 75 mM NaOAc buffer (pH 4.5). The reaction was performed in the dark at 4 °C for 45 min. After ethanol precipitation, the RNA was dissolved in 100 μl of fresh solution of 10 mM biotin hydrazide (Calbiochem, USA) in dimethyl sulfoxide. Then the samples were incubated for 4 h at 37 °C, and each of them was supplemented with 100 μl of 200 mM NaBH₄ and 200 μl of 1 M Tris-HCl (pH 8.0) with subsequent incubation in the dark at 4 °C for 30 min. Then the RNA was ethanol-precipitated and dissolved in the required volume of H₂O.
After 3′-biotinylation RNA fragments were capped using a ScriptCap™ m⁷G Capping System and ScriptCap 2′-O-Methyltransferase Enzyme (CellScript) according to the suppliers’ recommendations.