Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

36-POS Board 18 DEVELOPING A SINGLE-MOLECULE PLATFORM TO INVESTIGATE INFLUENZA REPLICATION DYNAMICS Cornelius Lee 1 ; 1 University of Oxford, Department of Physics, Oxford, United Kingdom Influenza A virus genome replication is mediated by the viral RNA-dependent RNA polymerase and requires the host factor ANP32. Structural studies have provided important insights into the architecture of the influenza replication machinery; however, they provide limited information on the conformational dynamics and transient molecular interactions that occur during replication. In particular, the intrinsically disordered low-complexity acidic region (LCAR) of ANP32, which is important for efficient genome replication, remains unresolved by cryo-electron microscopy. Here, we aim to develop a surface-immobilised platform for investigating influenza genome replication and ANP32 dynamics in real time using single-molecule fluorescence approaches. Viral ribonucleoprotein complexes (vRNPs) were isolated from influenza A/WSN/33 (H1N1) virions and immobilised onto a surface using biotinylated DNA probes targeting the non-structural (NS) genome segment. To validate selective immobilisation, individual vRNPs were visualised by DNA-PAINT using an Oxford Nanoimager. Super-resolution imaging revealed fluorescence localisation patterns consistent with the elongated morphology expected for influenza vRNPs. In contrast, negative controls lacking vRNPs showed only sparse localisation clusters attributed to non-specific surface binding. These results provide proof of principle that isolated influenza vRNPs can be specifically immobilised and visualised at the single-molecule level.

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