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.