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Developing rapid, sensitive, and specific in situ virus\u2010detection technologies is crucial for tracking viral genome release, replication in host cells, and intercellular transmission, thus holding great promise in understanding viral infection mechanisms, enabling early diagnosis, screening antiviral drugs, and formulating prevention and treatment strategies. In this study, we present a multifunctional tetrahedral DNA nanostructure (TDN)\u2010based system designed to monitor viral RNA levels in host cells in situ. The system employs a TDN\u2010based hyperbranched catalytic hairpin assembly (CHA) reaction to achieve rapid and powerful signal amplification, generating specific fluorescence resonance energy transfer (FRET) signals in response to target viral RNA. Using Japanese encephalitis virus (JEV) as a model, we developed the <jats:bold>THA@JEV<\/jats:bold> system responding to JEV\u2010RNA. This system demonstrated rapid and sensitive detection of JEV\u2010RNA within 12 minutes in a cell\u2010free environment, enabling real\u2010time imaging of JEV\u2010RNA in situ within host cells. The integrated TDN structure enhanced the biological stability of the probes and ensured their reliability under various stress conditions and microenvironments. This TDN\u2010based sensor system shows significant potential for elucidating viral infection pathways and advancing related virology research, as well as for screening antiviral drugs.<\/jats:p>","DOI":"10.1002\/chem.202501329","type":"journal-article","created":{"date-parts":[[2025,5,23]],"date-time":"2025-05-23T08:59:42Z","timestamp":1747990782000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["DNA Tetrahedron\u2010Enhanced Catalytic Hairpin Assembly for in Situ Monitoring of Viral RNA in Host Cells"],"prefix":"10.1002","volume":"31","author":[{"given":"Sha","family":"Lu","sequence":"first","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences College of Chemistry, Nankai University  Tianjin 300071 P. R. 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