Vaccinia capping enzyme

All constructs were amplified with a T7 containing forward primer and a T₅₀ tailed reverse primer to prepare templates for in vitro transcription. In vitro transcription and m7G-capping reactions were carried out with T7 RNA Polymerase (NEB) and Vaccinia Capping enzyme (NEB) respectively according to the manufacturer’s protocol. ApppG (NEB) was added to transcription reaction mixture in a proportion of 10:1 to GTP for preparation of A-capped RNAs. m7G-cap and A-capped RNAs were purified with RNA purification kit (Macherey-Nagel).

Three micrograms of E. coli RNA was incubated in 50 μl 1× VCE buffer (NEB) supplemented with 0.1 mM S-adenosyl methionine, and 0.5 mM DTB-GTP and 50 units of Vaccinia Capping Enzyme (NEB), for 30 min at 37 °C. The RNA was purified on a Zymo Research Clean and Concentrator-5 column for 200 nucleotide and greater RNA per manufacturer’s instructions with a total of 4 washes with RNA wash buffer. The RNA was eluted in 100 μl of 1 mM Tris pH 7.5, 0.1 mM EDTA (low TE).

In vitro transcription reactions were performed using PCR products generated with primers encoding a flanking T7 RNA polymerase promoter and a poly-A tail. Reactions were set up, as previously described (66 (link)), with 20 mM Tris-HCl pH 7.5, 35 mM MgCl₂, 2 mM spermidine, 10 mM DTT, 1 u/ml pyrophosphatase (Sigma), 7.5 mM of each NTP, 0.2 u/ml RiboLock RNase Inhibitor (ThermoFisher), 0.1 mg/ml T7 RNA polymerase and 40 ng/μl PCR-generated DNA. After 3 h incubation at 37 °C, 0.1 u/μl DNase I (Promega) was added to the reactions, which were incubated at 37 °C for 30 min to remove the template DNA. RNA was precipitated for 2–3 h at −20 °C after adding 0.5x volume of 7.5 M LiCl/50 mM EDTA, and the resulting pellet was washed with cold 70% ethanol and dissolved with RNase-free water. The mRNA was further purified by using a Zymo RNA Clean and Concentrator (Zymo Research) before use in in vitro translation reactions.
DNA templates were amplified from a plasmid containing the corresponding 5’ UTR and the NanoLuc Luciferase coding sequence. Primers used for this amplification added a 30T sequence at the 3′ end to form a poly(A) tail after transcription. The HBB 5’ UTR containing mRNA was then capped using Vaccinia Capping enzyme (New England Biolabs) and 2′O-methylated using Vaccinia 2′O Methyltransferase (New England Biolabs). The IRES-containing mRNAs were uncapped and polyadenylated.

RNAs were in vitro transcribed and purified as described previously (Lahr et al., 2015 (link)). For studying the un-capped RNAs, transcribed RNAs were treated with alkaline phosphatase (Roche Life Sciences (Indianapolis, IN), cat. no. M183A) and 5’ end-radiolabeled with [γ-³²P]-ATP. To generate capped RNAs, the 5’ triphosphate required for the capping reaction was regenerated in TOP RNAs by incubation of 50 nM RNA with T4 PNK in PNK buffer A (ThermoFisher Scientific cat. no. EK0032) with 3 mM ATP for 20 min at 37°C followed by addition of 5 units of nucleoside monophosphate kinase (Roche Life Sciences, cat. no. 10107948001). RNA was purified by phenol-chloroform extraction, MicroSpin G-25 desalting columns (GE Healthcare Life Sciences (Marlborough, MA), cat. no. 27-5325-01), and ethanol precipitation. RNAs with a 5’ triphosphate were subsequently capped and radiolabeled using vaccinia capping enzyme (NEB (Ipswich, MA), cat. no. M2080S) and [α-³²P]-GTP or GTP according to the manufacturer’s protocol.

mRNA was synthesized in vitro by T7 RNAP-mediated transcription at 37 °C using 100% substituted N1-methylpseudouridine-triphosphate and a linearized DNA template, which incorporated the 5′ and 3′ untranslated regions and a polyadenosine tail. Reactions with WT and mutant enzymes were treated similarly. NTPs were included at equimolar concentrations. After transcription, the Cap 1 structure was added to the 5′ end using Vaccinia capping enzyme (New England Biolabs) and Vaccinia 2′O-methyltransferase (New England Biolabs). The mRNA was purified by oligo-dT affinity purification. For mRNAs described as ‘with RP’, ion-paired reversed-phase (RP) chromatography was subsequently used for purification. All mRNAs were buffer exchanged by tangential flow filtration into sodium citrate, pH 6.5, and sterile filtered. The mRNA was kept frozen at –20 °C until further use.