{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T18:30:26Z","timestamp":1789583426417,"version":"build-2803163510"},"reference-count":56,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2025,11,10]],"date-time":"2025-11-10T00:00:00Z","timestamp":1762732800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Key Research and Development Program of China","award":["2022YFB3205600"],"award-info":[{"award-number":["2022YFB3205600"]}]},{"name":"Guangxi Key Technologies R&D Program","award":["AB241484024"],"award-info":[{"award-number":["AB241484024"]}]},{"DOI":"10.13039\/501100002367","name":"Chinese Academy of Sciences","doi-asserted-by":"crossref","award":["Y52A100K10"],"award-info":[{"award-number":["Y52A100K10"]}],"id":[{"id":"10.13039\/501100002367","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001809","name":"National Nature Science Foundation of China","doi-asserted-by":"crossref","award":["61471336"],"award-info":[{"award-number":["61471336"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>The COVID-19 pandemic caused by the SARS-CoV-2 virus has exposed the urgency of research on rapid and efficient virus detection and strategies to inhibit its replication. Previous studies have mostly focused on traditional immunoassay or optical methods, but they have limitations in terms of sensitivity, timeliness, and in-depth analysis of molecular interaction mechanisms. Solid-state nanopore single-molecule detection methods, which can monitor molecular conditions in real time at the single-molecule level, bring new opportunities to solve this problem. The nucleocapsid protein (N protein) of SARS-CoV-2 was systematically investigated under different conditions, such as external drive voltage, pH, nanopore size, and N protein concentration. The translocation of the N protein through the nanopore was then analyzed. Subsequently, we analyzed the translocation characteristics of the N protein, RNA, and N protein\u2013RNA complexes. With the aid of EMSA experiments, we conclusively confirmed that RNA binds to the N protein. Building on this finding, we further explored small molecules that could affect the nanopore translocation of N protein\u2013RNA complexes, such as gallocatechin gallate (GCG), epigallocatechin gallate (EGCG), and the influenza A viral inhibitor Nucleozin. The results show that GCG can disrupt the liquid-phase condensation of the N protein\u2013RNA complex and inhibit the replication of the N protein. Meanwhile, the structural isomer EGCG of GCG and the small molecule Nucleozin can also block RNA-triggered N protein liquid\u2013liquid phase separation (LLPS). Our results confirmed that GCG, EGCG, and Nucleozin exhibit antagonistic effects on the N protein, with differences in their effective concentrations and the potency of their antagonism. Herein, using solid-state nanopore single-molecule detection technology, we developed an experimental method that can effectively detect RNA-induced changes in N protein properties and the regulatory effects of small molecules on the LLPS of N protein\u2013RNA complexes. These findings not only provide highly valuable insights for in-depth research on the molecular interactions involved in viral replication, but also open up promising new avenues for future responses to similar viral outbreaks, the development of a rapid and effective detection method based on solid-state nanopores and single-molecule detection, and antiviral therapies targeting N protein\u2013RNA interactions.<\/jats:p>","DOI":"10.3390\/s25226870","type":"journal-article","created":{"date-parts":[[2025,11,10]],"date-time":"2025-11-10T15:07:31Z","timestamp":1762787251000},"page":"6870","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Solid-State Nanopore Single-Molecule Analysis of SARS-CoV-2 N Protein: From Interaction Exploration to Small-Molecule Antagonism"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9468-7103","authenticated-orcid":false,"given":"Xiaoqing","family":"Zeng","sequence":"first","affiliation":[{"name":"Chongqing University, Chongqing 400044, China"},{"name":"Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, China"},{"name":"Chongqing College, University of Chinese Academy of Sciences, Chongqing 400714, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shinian","family":"Leng","sequence":"additional","affiliation":[{"name":"Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, China"},{"name":"Chongqing College, University of Chinese Academy of Sciences, Chongqing 400714, China"},{"name":"School of Electronic and Information Engineering, Changchun University of Science and Technology, Changchun 130022, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6704-3262","authenticated-orcid":false,"given":"Wenhao","family":"Ma","sequence":"additional","affiliation":[{"name":"Chongqing University, Chongqing 400044, China"},{"name":"Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, China"},{"name":"Chongqing College, University of Chinese Academy of Sciences, Chongqing 400714, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenxin","family":"Wang","sequence":"additional","affiliation":[{"name":"Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, China"},{"name":"Chongqing College, University of Chinese Academy of Sciences, Chongqing 400714, China"},{"name":"School of Electronic Information, University of Chinese Academy of Sciences, Beijing 101408, 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