Fluorescence-Based Alkaline Phosphatase-Coupled Polymerase Assay (FAPA)

The fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA)^[1] (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

• determination of kinetic replication parameters^[1-3]
• identification of potential antiviral drugs^[4]

under high throughput conditions. To this end, 2'-(2-benzothiazoyl)-6'-hydroxybenzothiazole-(BBT)-modified nucleotides have been proposed as well-suited fluorescent probes, combining excellent enzymatic acceptance with

• a high extinction coefficient (ε = 26,484 M^-1cm^-1)
• a large Stokes' shift (λ_max ex/em = 422/566 nm)
• excellent photostability

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 (BBTpp_i). After stopping the replication reaction at a time point of choice, calf intestinal alkaline phosphatase (CIP) is added to convert BBTpp_i into fluorescent BBT for subsequent readout.

Selected References

[1] Niyomrattanakit et al. (2011) A fluorescence-based alkaline phosphatase-coupled polymerase assay for identification of inhibitors of dengue virus RNA-dependent RNA polymerase. J. Biomol. Screen. 16 (2):201.
[2] Niyomrattanakit et al. (2015) Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening. Antiviral Res. 119:36.
[3] 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.
[4] 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.