T7 RNA Polymerase NTP Discrimination Assay

Studies on NTP discrimination by T7 RNA polymerase were conducted using the MEGAscript T7 transcription kit or the SP6 RNA polymerase using the HiScribe SP6 RNA synthesis kit with some changes to the manufacturer's protocol as described. The NTP concentrations were 2 mM for ATP, GTP and CTP, while the UTP and ΨTP or m¹ΨTP were 1 mM each to achieve a total concentration of U and its derivative of 2 mM. To ensure the UTP and ΨTP or m¹ΨTP were at a 1:1 ratio, the stock solution concentrations were determined using UV-vis spectroscopy with established extinction coefficients (UTP: λ_{262 nm} = 10 000 l mol⁻¹cm⁻¹; ΨTP: λ_{262 nm} = 7550 l mol⁻¹cm⁻¹; m¹ΨTP: λ_{271 nm} = 8870 l mol⁻¹cm⁻¹). The IVT reactions were allowed to progress for 2 h at 37°C before termination by the addition of DNase I following the manufacturer's protocol. Further studies on T7 RNA polymerase NTP selection were conducted with N1-ethylpseudouridine triphosphate (e¹ΨTP: λ_{271 nm} = 7800 l mol⁻¹cm⁻¹) or N1-propylpseudouridine triphosphate (p¹ΨTP: λ_{271 nm} = 8900 l mol⁻¹cm⁻¹; Trilink Biotechnologies) similarly to those described above. Replicate experiments were conducted to obtain errors.