Hepatitis C is the leading cause of liver based illnesses and diseases according to both the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO). The hepatitis C Virus (HCV) RNA-dependent RNA polymerase NS5B has been a drug target for many years with ribavirin being the cornerstone for the treatment of Hepatitis C for four decades. In almost all of the research to date, researchers have primarily used radioactively-labeled nucleotides to analyze the products of the NS5B polymer reaction. This is problematic since dedicated space is required to work with open radioactive material and this generates hazardous waste. A more convenient method of detection that still offers relatively-high sensitivity for measuring steady-state kinetics is fluorescence. An ideal fluorescence chromophore is required to have a minimal impact on the affinity of the enzyme for it’s substrate(s). This was accomplished by substituting adenosine-5'-triphosphate (ATP) with the analog 2-amino-adenosine-5'-triphosphate. This nucleotide analog’s fluorescence is altered when incorporated into double-stranded RNA. An assay was evaluated using two different RNA templates, one with a stem-loop secondary structure and one without, measuring NS5B RNA polymerase’s ability to incorporate 2-amino-adenosine-5'-triphosphate in to each RNA sequence. Findings regarding non-competitive assays, and competitive assays evaluating the incorporation of 2-amino-adenosine-5'-triphosphate in the presence of ATP, are reported.

Development of a fluorescence assay for the evaluation of nucleotide-analog incorporation during the elongation stage of RNA synthesis by hepatitis C virus NS5B RNA polymerase

253rd National Meeting (2017)

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