Fluorescent Aptamers Go Viral: Tracking the Coseparation of a SARS-CoV-2 RNA Fragment with the Nucleocapsid Protein...

Fluorescent aptamers (FLAPs) are encodable RNA tags that light up upon binding to their cognate fluorogenic dyes, thereby facilitating RNA imaging. While single-molecule imaging remains challenging, RNA assembly into biocondensates can be tracked by using FLAP-based systems. This has been demonstrated for human RNA-protein condensates but not for viral ones, which have recently emerged as promising drug targets. We report the first FLAP-based monitoring of coronavirus-specific condensates, which are scaffolded by the nucleocapsid (N) protein and the hairpin-rich fragment of the genomic RNA (gRNA). We tagged the N-protein-binding gRNA hairpin with Mango II FLAP and confirmed its coseparation with N-protein in human cells. The resulting condensates were sensitive to a well-known condensate-disrupting antiviral and a recently proposed antiviral candidate. The FLAP-tagged RNA showed no apparent cross-reactivity with host proteins and provided an improved signal-to-noise ratio in the quantitative condensate analysis. This supports the usability of FLAP-based systems for intracellular drug-screening assays.