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Conventional fluorescence microarrays provide high throughput but lack molecular resolution, whereas single\u2010molecule sensors such as nanopores offer exquisite precision but limited scalability. Here, we present a DNA carrier\u2010based nanoarray that integrates programmable nucleic acid hybridization with solid\u2010state nanopore readout for direct, multiplexed RNA detection. Each modular DNA carrier is pre\u2010assembled with spatially defined probe sites that sequence\u2010specifically capture RNA targets of varying lengths and conformations, in which poly(dT) sequences both structurally facilitate target binding and enhance signal strength without the need for fluorescent or protein labeling. Upon nanopore translocation, hybridized carrier\u2010target complexes generate binary ionic current signatures, allowing single\u2010molecule identification of target occupancy at each sensing site. Using a ternary coordinate encoding system, we constructed a nanoarray comprising 27 carriers with 81 addressable sensing sites, enabling simultaneous detection of multiple bacterial and viral RNA targets within a single assay directly from total RNA extracts without target\u2010specific isolation, amplification, or enrichment. By combining the multiplexing capability of traditional microarrays with the single\u2010molecule precision of nanopore sensing, this carrier\u2010based nanoarray establishes a scalable, programmable, and universally adaptable framework for high\u2010throughput molecular diagnostics in complex biological backgrounds.<\/jats:p>","DOI":"10.1002\/advs.75319","type":"journal-article","created":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T09:50:05Z","timestamp":1777369805000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["DNA Nanoarray for Multiplexed RNA Detection With Single\u2010Molecule Readout"],"prefix":"10.1002","volume":"13","author":[{"given":"Yunxuan","family":"Li","sequence":"first","affiliation":[{"name":"Cavendish Laboratory University of Cambridge  Cambridge UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yesheng","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Biomedical Engineering Faculty of Medicine Dalian University of Technology  Dalian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunmiao","family":"Yu","sequence":"additional","affiliation":[{"name":"School of Biomedical Engineering Faculty of Medicine Dalian University of Technology  Dalian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Siyu","family":"Hou","sequence":"additional","affiliation":[{"name":"Cavendish Laboratory University of Cambridge  Cambridge UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elli","family":"Mylona","sequence":"additional","affiliation":[{"name":"Cambridge Institute of Therapeutic Immunology &amp; Infectious Disease (CITIID) Jeffery Cheah Biomedical Centre Cambridge Biomedical Campus University of Cambridge  Cambridge UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Plamena","family":"Naydenova","sequence":"additional","affiliation":[{"name":"Cambridge Institute of Therapeutic Immunology &amp; Infectious Disease (CITIID) Jeffery Cheah Biomedical Centre Cambridge Biomedical Campus University of Cambridge  Cambridge UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingyuan","family":"Zhao","sequence":"additional","affiliation":[{"name":"Central Hospital of Dalian University of Technology (Dalian Municipal Central Hospital)  Dalian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong","family":"Yuan","sequence":"additional","affiliation":[{"name":"Central Hospital of Dalian University of Technology (Dalian Municipal Central Hospital)  Dalian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingsong","family":"Duan","sequence":"additional","affiliation":[{"name":"School of Biomedical Engineering Faculty of Medicine Dalian University of Technology  Dalian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hendrik F. 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