Fluorescence polarization-based RNA synthesis assay
Influenza virus is a major threat to global public health. Essential to virus propagation is the viral RNA-dependent RNA polymerase (FluPol) which amplifies the viral genome and transcribes mRNAs coding for viral proteins. FluPol, a ~260 kDa multifunctional heterotrimeric complex, is an attractive target for anti-infl uenza drug discovery. Here, we present a novel fluorescence polarization assay that directly reads out RNA synthesis by FluPol.
The assay utilizes fluorescence-polarization changes of FAM-labelled model template RNAs associated with FluPol-catalyzed product RNA formation. The assay is high throughput compatible and can easily be performed in 384 well microplates. It reliably reports on compounds inhibiting RNA synthesis, readily can be miniaturized to 0.2 pmole/reaction of recombinant (active) FluPol and might provide an attractive choice for drug discovery campaigns.