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Here, the capability is demonstrated to detect target pairs that are in close proximity by exploiting base\u2010stacking interactions between two DNA strands, each labelling one target. The DNA probes hybridize transiently with the each other only when both target molecules are proximal, thus creating a hybridization site for fluorophore\u2010conjugated DNA strand, called imager. In this design, hybridization and co\u2010axial base\u2010stacking act synergistically to enable imager binding, with stacking interactions providing essential stabilization that allows for transient hybridization. This synergy generates the stochastic binding events required for DNA\u2010PAINT imaging, which it call Stack\u2010Proximity\u2010PAINT (Stack\u2010pPAINT). To gain mechanistic insights into hybridization of the probe, atomistic equilibrium and steered molecular dynamics (MD) simulations are performed. The simulations reveal that DNA base\u2010stacking and fluorophore stacking together stabilize the imager. Programmable DNA nanostructures are utilized to benchmark the applicability of Stack\u2010pPAINT. As a cellular proof of concept, microtubular structures are visualized using Stack\u2010pPAINT with antibodies targeting both alpha\u2010 and beta\u2010tubulin molecules. This probe technology offers promising applications in cell biology research aimed at elucidating spatial interactomes at high resolution within cells.<\/jats:p>","DOI":"10.1002\/smll.202507139","type":"journal-article","created":{"date-parts":[[2025,11,22]],"date-time":"2025-11-22T08:19:38Z","timestamp":1763799578000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Proximity\u2010Based Super\u2010Resolution Imaging Enabled by DNA Base\u2010Stacking Interactions"],"prefix":"10.1002","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3985-3014","authenticated-orcid":false,"given":"Abhinav","family":"Banerjee","sequence":"first","affiliation":[{"name":"Department of Biochemistry Indian Institute of Science  Malleswaram Bangalore 560012 India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8113-4067","authenticated-orcid":false,"given":"Saanya","family":"Yadav","sequence":"additional","affiliation":[{"name":"Department of Biotechnology Indian Institute of Technology Hyderabad  Kandi Sangareddy Telangana 502284 India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-3820-8143","authenticated-orcid":false,"given":"Vedanth Shree","family":"Vidwath","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Indian Institute of Science  Malleswaram Bangalore 560012 India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9451-8331","authenticated-orcid":false,"given":"Simanta","family":"Kalita","sequence":"additional","affiliation":[{"name":"New Chemistry Unit and Chemistry and Physics of Materials Unit Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR)  Bangalore 560064 India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7216-0490","authenticated-orcid":false,"given":"Sarit S.","family":"Agasti","sequence":"additional","affiliation":[{"name":"New Chemistry Unit and Chemistry and Physics of Materials Unit Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR)  Bangalore 560064 India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0769-524X","authenticated-orcid":false,"given":"Himanshu","family":"Joshi","sequence":"additional","affiliation":[{"name":"Department of Biotechnology Indian Institute of Technology Hyderabad  Kandi Sangareddy Telangana 502284 India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8176-3322","authenticated-orcid":false,"given":"Mahipal","family":"Ganji","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Indian Institute of Science  Malleswaram Bangalore 560012 India"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,11,22]]},"reference":[{"key":"e_1_2_8_1_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2024.108118"},{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt896"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth947"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/nl103427w"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202009031"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.9b03418"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41565-023-01485-1"},{"key":"e_1_2_8_8_1","unstructured":"D. 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