RNA methylation is a ubiquitous post-transcriptional modification found in diverse RNA classes and is a critical regulator of gene expression. In this study, we used Zika virus RNA methyltransferase (MTase) to develop a highly sensitive microplate assay that uses a biotinylated RNA substrate and radiolabeled AdoMet coenzyme. The assay is fast, highly reproducible, exhibits linear progress-curve kinetics under multiple turnover conditions, has high sensitivity in competitive inhibition assays, and significantly lower background levels compared with the currently used method. Using our newly developed microplate assay, we observed no significant difference in the catalytic constants of the full-length nonstructural protein 5 enzyme and the truncated MTase domain. These data suggest that, unlike the Zika virus RNA-dependent RNA polymerase activity, the MTase activity is unaffected by RNA-dependent RNA polymerase-MTase interdomain interaction. Given its quantitative nature and accuracy, this method can be used to characterize various RNA MTases, and, therefore, significantly contribute to the field of epitranscriptomics and drug development against infectious diseases.
Mensah, Isaiah K.; Norvil, Allison B.; He, Ming; Lendy, Emma; Hjortland, Nicole; Tan, Hern; Pomerantz, Richard T.; Mesecar, Andrew; and Gowher, Humaira, "Development of a Sensitive Microplate Assay for Characterizing RNA Methyltransferase Activity: Implications for Epitranscriptomics and Drug Development" (2023). Department of Biochemistry and Molecular Biology Faculty Papers. Paper 245.
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