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

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.