A Time-Gated FRET Platformfor Screening SARS-CoV-2−1 Programmed Ribosomal Frameshifting Inhibitors

Abstract To address the emerging need for high-throughput and sensitive methodologies in RNA-targeted drug discovery, this study developed and validated a novel biosensing platform based on time-gated Förster resonance energy transfer (TG-FRET). The highly conserved RNA pseudoknot of SARS-CoV-2 served as our model system and target, a structure that regulates the essential viral –1 programmed ribosomal frameshift (−1 PRF). We first constructed a fluorescent protein (mOrange2-mCherry)-based FRET reporter system to visualize and monitor –1 PRF efficiency in living cells. To enable high-throughput screening, we adapted the system into a TG-FRET assay. This assay was based on a HaloTag-mCherry fusion protein, in which the HaloTag moiety was site-specifically labeled with CoraFluor, a new class of macrotricyclic terbium complexes. This configuration enables homogeneous, time-gated detection directly in cell lysates, effectively eliminating interference from biomatrix autofluorescence. Using this platform, we quantified ivacaftor’s inhibition of −1 PRF (IC50 = 7.6 μM) and ruled out direct inhibition by MTDB. The established TG-FRET platform combines the precision for detecting molecular interactions with the high-throughput capability compatible with microplate operations, providing a powerful and versatile screening tool for drug discovery targeting functional RNA structures.