Direct visualization of RNA virus and evaluation of drug effect in living cells by catalytically transformable DNA...

In situ visualization of viral RNA in living cells is critical for understanding infection dynamics and evaluating antiviral responses, yet offline methods such as polymerase chain reaction are incompatible with real-time analysis. Here, a CAtalytically Transformable DNA Cube (CAT-Cube) is presented as a structurally stable and autonomously cell-permeable DNA nanostructure for real-time viral RNA imaging in living cells. By spatially confining catalytic hairpins within a rigid scaffold, CAT-Cube increases the local reaction concentration and enhances sensitivity toward viral RNA while releasing the RNA after transformation rather than consuming it. The probe produces bright, photostable signals that support continuous tracking of transcription- and replication-competent severe acute respiratory syndrome coronavirus 2 virus-like particles at a multiplicity of infection as low as 0.1. This system also enables evaluation of antiviral agents, including angiotensin-converting enzyme 2 (ACE2) inhibitors, DNA aptamers, and the natural compound cepharanthine, and reveals mechanisms involving ACE2 and spike binding. CAT-Cube therefore provides a generalizable approach for real-time viral RNA imaging and dynamic assessment of antiviral effects in living cells.