m7G(5')ppp(5')(2'OMeA)pG

In vitro transcription of mRNA (IVT; 20μL system)^[1]:

This plan is based on literature, please modify it to meet your specific needs.

Buffer: 40mM Tris-HCl, pH=7.9; 10mM MgCl₂; 1mM DTT; 2mM spermidine

Template: 50ng/μL linearized plasmid

1. Add 10U/μL T7 RNA polymerase; 1U/μL RNase inhibitor; 2mM ATP/CTP/UTP; 0.5mM GTP and 3mM m7G(5')ppp(5')(2'OMeA)pG to the buffer and incubate at 37°C for 2 hours.

Note: Adding m7G(5')ppp(5')(2'OMeA)pG to GTP at a 6:1 ratio improves capping efficiency.

2. Add 1U/μL DNase I and continue incubation at 37°C for 30 minutes.

3. Crude mRNA was purified using NucleoSpin RNA Clean-up XS; uncapped mRNA was removed using 5′-polyphosphatase and Xrn1.

4. RP-HPLC removal of dsRNA impurities: A linear gradient of buffer B (0.1M triethylamine acetate, pH=7.0, containing 25% acetonitrile) in buffer A (0.1M triethylamine acetate, pH=7.0) was applied from 5% to 50% for 18min at a flow rate of 0.9mL/min. mRNA was recovered from the collected fraction by isopropanol precipitation.

References:
[1] Sikorski PJ, Warminski M, Kubacka D, et al. The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5' cap modulates protein expression in living cells. Nucleic Acids Res. 2020;48(4):1607-1626.