*5.2. Functional Aspects of MTase*

The functional analysis of MTase was determined using bioluminescence-based assay with GTP as a substrate. The reaction was performed in white-bottomed 96-well plate (Tarsons) in the presence of 20 mM Tris buffer of pH 8.0, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, and 1 µM SAM. SAM acts as a methyl donor, and GTP acts as the methyl acceptor. In principle, MTase transfers a methyl group from SAM to GTP and converts it into SAH and m7GTP. Then MTase-Glo™ reagent (Promega, Madison, WI, USA) changes SAH to ADP, which is turned into ATP by MTase-Glo™ detection solution (Promega, USA). The luminescence was measured on a Tecan Plate reader. The enzymatic activity was determined under various reaction conditions, such as pH (6–12), temperature (17–45 ◦C), time (5–120 min), and various enzyme (0 to 5 µM) and substrate concentrations. The substrate-specificity assay was performed to test its ability to incorporate methyl groups on different nucleoside triphosphates, ATP, CTP, UTP guanine nucleotides (GMP and GTP), and cap analogues (m7GDP, m7GTP, and dGTP) at 0.5 mM. The standard reaction was carried out at 37 ◦C for 120 min in a 20 µL solution that contained 0.675 µM MTase, 0.4 mM GTP, 20 mM Tris buffer, pH 8.0, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, and 1 µM SAM. The reaction was stopped by adding 0.5% TFA (trifluoroacetic acid) (Sigma) and incubated for 10 min at room temperature, followed by incubation with MTase Glo reagent at room temperature for 30 min. Further, MTase detection reagent was added to the reaction mixture, and luminescence was detected using Tecan Plate Reader after 30 min. Similarly, the MTase activity was determined by increasing the concentrations of Mg2+ and EDTA. A standard curve was generated using a serial dilution of SAH ranging from 0 to 1000 nM to correlate luminescence with SAH concentration. The luminescence was plotted on *Y*-axis against the SAH concentration on *X*-axis, and the straight-line equation (Y = 19.71 × X + 227.60) was calculated using linear regression in GraphPad Prism 9.0. The graphs were plotted as a function of the concentration of SAH produced.
