Antarctic phosphatase kit

The synthetic mRNAs (eGFP, CD39) were prepared as previously described.4 (link),16 (link) The vectors with coding sequences were amplified with a HotStar HiFidelity Polymerase Kit (Qiagen, Germany) using two primers, TTG GAC CCT CGT ACA GAA GCT AAT ACG as the forward primer (Ella Biotech, Germany) and T₁₅₀ CTT CCT ACT CAG GCT TTA TTC AAA GAC CA as the reverse primer (Ella Biotech, Germany). DNA sequences were purified (Qiaquick PCR purification Kit, Qiagen, Germany), and in vitro transcription (IVT) was performed using a MEGAscript® T7 Kit (Ambion, Scotland), according to the manufacturer's instructions. The mRNA was modified using the 3'-0-Me-m⁷G(5') ppp(5')G RNA cap structure analog (New England Biolabs, Germany), pseudouridine-5'-triphosphate (TriLink Biotech, U. S. A.), and 5-methylcytidine-5'-triphosphate (TriLink Biotech, U. S. A.). Additionally, an RNase inhibitor (Themo Scientific, U. S. A.) was added. The IVT was incubated for 4 h at 37 °C.
Afterwards the mRNA was cleaned up with an RNeasy kit (Qiagen, Germany), and eluted in 40 µl nuclease-free water. Next the dephosphorylation of mRNA was implemented with an Antarctic Phosphatase Kit (New England Biolabs, Germany), cleaned up and then eluted in the same amount of nuclease-free water. The concentration of mRNA was confirmed photometrically and the purity of mRNA with 1% agarose gel.

The synthesis of modified mRNA via in vitro transcription (IVT) was described earlier.53 (link), 54 (link) Briefly, the plasmid DNA sequences of EGFP and AAT (Eurofins Medigenomix) were multiplied by using HotStar HiFidelity Polymerase Kit (QIAGEN), as well as a forward primer (5′-TTG GAC CCT CGT ACA GAA GCTA ATA CG-3′; Ella Biotech) and a reverse primer (poly T-tail of 120 thymidines [T₁₂₀] 5′-CTT CCT ACT CAG GCT TTA TTC AAA GAC CA-3′; Ella Biotech). After subsequent purification (QIAquick PCR purification kit; QIAGEN) and gel electrophoresis, the DNA was used as a template for IVT using the MEGAscript T7 kit (Ambion). The following mRNA modifications were implemented: 3′-0-Me-m⁷G(5′)ppp(5′)G RNA cap structure analog (ARCA; New England Biolabs), pseudouridine-5′-triphosphate (TriLink Biotech), and 5-methylcytidine-5′-triphosphate (TriLink Biotech). For the fluorescent labeling of the mRNA, cy3-cytidine-triphosphate (PerkinElmer) was used. For RNase inhibition, an RNase inhibitor (Thermo Scientific) was added. The reaction mix was incubated for 4 hr at 37°C. Afterward, the mRNA was purified with RNeasy kit (QIAGEN), dephosphorylated using the Antarctic Phosphatase kit (New England Biolabs), purified again, and controlled with 1% agarose gel.

The RNAs were dephosphorylated with the Antarctic Phosphatase kit (according to the manufacturer, New England Biolabs) and the resulting RNAs (10 pmol) were phosphorylated with [γ³²P]ATP (2 μL; 6 000 Ci (222 TBq)/mmol in 50 mM Tricine (pH 7.6), PerkinElmer) using the T4 Polynucleotide Kinase (PNK) kit (according to the manufacturer, Promega). Then, the radioactive RNAs were purified through electrophoresis using denaturing (8 M urea) 5% polyacrylamide gels (19:1).