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Although Subak substrates offer advantages such as ratiometric readouts and low manufacturing costs over traditional FRET substrates, the mechanism governing AgNC color switching remains unclear. Here, using a site-specific cleavage strategy, we identify color-switching hotspots and demonstrate that AgNC transformation can be controlled by the cleavage positions within the nucleic acid host. Our data support a cleavage-driven reorganization of the AgNC coordination environment, converting a non-emissive precursor into a red-emitting cluster, rather than direct enzyme-cluster interactions. Leveraging this insight, we engineer rSubak, an RNA-incorporated Subak that displays 95 nm red shift (530 to 625 nm) upon RNase cleavage. In amplification-free CRISPR\/Cas13 assays for SARS-CoV-2, influenza A (A\/H5N1), and measles viruses (MV) detection, rSubak achieved a limit of detection of 0.3 pM, superior to that of the commercial RNaseAlert (\u223c250 pM). Collectively, our results establish Subak as a generalizable, non-FRET platform for sensitive ratiometric reporting the activities of diverse nucleases.<\/jats:p>","DOI":"10.64898\/2026.03.11.710959","type":"posted-content","created":{"date-parts":[[2026,3,14]],"date-time":"2026-03-14T01:35:18Z","timestamp":1773452118000},"source":"Crossref","is-referenced-by-count":2,"title":["Mechanistic insights into the color transformation of a non-FRET substrate for RNase activity detection"],"prefix":"10.64898","author":[{"given":"Sohyun","family":"Kim","sequence":"first","affiliation":[{"name":"Department of Biomedical Engineering, The University of Texas at Austin, Texas, 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Soonwoo","family":"Hong","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, The University of Texas at Austin, Texas, 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jada N.","family":"Walker","sequence":"additional","affiliation":[{"name":"Department of Chemistry, The University of Texas at Austin, Texas, 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yujie","family":"He","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, The University of Texas at Austin, Texas, 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anh-Thu","family":"Nguyen","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, The University of Texas at Austin, Texas, 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei-Ru","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, The University of Texas at Austin, Texas, 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saeed","family":"Seifi","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, The University of Texas at Austin, Texas, 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuan-I","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, The University of Texas at Austin, Texas, 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu-An","family":"Kuo","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, The University of Texas at Austin, Texas, 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer S.","family":"Brodbelt","sequence":"additional","affiliation":[{"name":"Department of Chemistry, The University of Texas at Austin, Texas, 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hsin-Chih","family":"Yeh","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, The University of Texas at Austin, Texas, 78712, USA"},{"name":"Texas Materials Institute, The University of Texas at Austin, Texas, 78712, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026031703200762000_2026.03.11.710959v1.1","doi-asserted-by":"crossref","first-page":"810","DOI":"10.1038\/s41565-024-01612-6","article-title":"A non-FRET DNA reporter that changes fluorescence colour upon nuclease digestion","volume":"19","year":"2024","journal-title":"Nature nanotechnology"},{"key":"2026031703200762000_2026.03.11.710959v1.2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.20.11241"},{"key":"2026031703200762000_2026.03.11.710959v1.3","doi-asserted-by":"publisher","DOI":"10.1126\/science.aat4422"},{"key":"2026031703200762000_2026.03.11.710959v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/84397"},{"key":"2026031703200762000_2026.03.11.710959v1.5","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1038\/s41592-021-01341-x","article-title":"Engineered HaloTag variants for fluorescence lifetime multiplexing","volume":"19","year":"2022","journal-title":"Nature methods"},{"key":"2026031703200762000_2026.03.11.710959v1.6","doi-asserted-by":"crossref","first-page":"718","DOI":"10.1038\/s41589-021-00775-w","article-title":"The HaloTag as a general scaffold for far-red tunable chemigenetic indicators","volume":"17","year":"2021","journal-title":"Nature chemical biology"},{"key":"2026031703200762000_2026.03.11.710959v1.7","doi-asserted-by":"publisher","DOI":"10.1126\/science.aav6416"},{"key":"2026031703200762000_2026.03.11.710959v1.8","doi-asserted-by":"crossref","first-page":"1560","DOI":"10.1021\/acs.biochem.9b00048","article-title":"Spectral tuning by a single nucleotide controls the fluorescence properties of a fluorogenic aptamer","volume":"58","year":"2019","journal-title":"Biochemistry"},{"key":"2026031703200762000_2026.03.11.710959v1.9","doi-asserted-by":"crossref","unstructured":"Kuo, Y.-A. , Chen, Y.-I. , Wang, Y. , Korkmaz, Z. , Yonas, S. , He, Y. , Nguyen, T. 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