The fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA) (Fig. 1) is a highly sensitive method to study the kinetic features of RNA-dependent RNA polymerases (RdRPs). It has found broad application in research on flaviviral replication such as the
Figure 1: Workflow of the BBT-based FAPA assay. Genomic viral RNA ((+)ssRNA) is subjected to RdRP-dependent replication in the presence of BBT-modified nucleotides, thereby releasing non-fluorescent BBT pyrophosphate (BBTppi). After stopping the replication reaction at a time point of choice, calf intestinal alkaline phosphatase (CIP) is added to convert BBTppi into fluorescent BBT for subsequent readout.
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 Tay et al. (2016) The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production. PLoS Pathog. 12 (9):e1005886.
 Yokokawa et al. (2016) Discovery of Potent Non-Nucleoside Inhibitors of Dengue Viral RNA-Dependent RNA Polymerase from a Fragment Hit Using Structure-Based Drug Design. J. Med. Chem. 59 (8):3935.