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However, a complete picture of their intracellular uptake, trafficking, and fate remains elusive. Additionally, the double-stranded nature of the compounds makes it challenging to track both the guide and passenger strands individually. To address this, we present a proof-of-concept study of nanoscale secondary ion mass spectrometry (NanoSIMS) in combination with a dual-labeling strategy using 5-bromo- and 5-iodo-modified uracil to detect, discriminate, and quantify both strands without relying on bulky fluorescent tags. By evaluating GalNAc-conjugated siRNAs and saRNAs in HepG2 cells, we demonstrate that NanoSIMS can effectively codetect guide and passenger strands within the endolysosomal compartments. For the siRNA design, imaging revealed, as expected, a robust correlation between the strands. Conversely, we highlight the use of NanoSIMS as a potential aid in the optimization of guide strand stability by exploring different chemical designs for the saRNA. This work highlights the potential of NanoSIMS to quantitatively monitor individual double-stranded RNA strands and their subcellular localization in vitro, offering a valuable tool to generate critical trafficking and quantitative data for oligonucleotide therapeutics.<\/jats:p>","DOI":"10.1021\/acsomega.6c04146","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T21:01:14Z","timestamp":1786741274000},"page":"49739-49745","source":"Crossref","is-referenced-by-count":0,"title":["A Dual-Halogen\nLabeling Strategy to Detect and Quantify\nDouble-Stranded RNAs Using NanoSIMS: A Proof-of-Concept Study"],"prefix":"10.1021","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-7659-6164","authenticated-orcid":true,"given":"Quentin","family":"Vicentini","sequence":"first","affiliation":[{"name":"AstraZeneca , , ,","place":["Mo\u0308lndal, Sweden, 431 83"]},{"name":"Karolinska Institutet , , , ,","place":["Huddinge, Stockholm, Sweden, 141 52"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6579-9691","authenticated-orcid":true,"given":"Michael","family":"Kurczy","sequence":"additional","affiliation":[{"name":"AstraZeneca , , ,","place":["Mo\u0308lndal, Sweden, 431 83"]},{"name":"University of Gothenburg , , ,","place":["Gothenburg, Sweden, 413 90"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"H. Yesid","family":"Estupin\u0303a\u0301n","sequence":"additional","affiliation":[{"name":"Karolinska Institutet , , , ,","place":["Huddinge, Stockholm, Sweden, 141 52"]},{"name":"Universidad Industrial de Santander , , ,","place":["Bucaramanga, Colombia, 680002"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dennis","family":"Hekman","sequence":"additional","affiliation":[{"name":"AstraZeneca , , ,","place":["Mo\u0308lndal, Sweden, 431 83"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ce\u0301cile","family":"Becquart","sequence":"additional","affiliation":[{"name":"AstraZeneca , , ,","place":["Mo\u0308lndal, Sweden, 431 83"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lars","family":"Haag","sequence":"additional","affiliation":[{"name":"Karolinska Institutet , , , ,","place":["Huddinge, Stockholm, Sweden, 141 52"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shalini","family":"Andersson","sequence":"additional","affiliation":[{"name":"AstraZeneca , , ,","place":["Mo\u0308lndal, Sweden, 431 83"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samir","family":"El-Andaloussi","sequence":"additional","affiliation":[{"name":"Karolinska Institutet , , , ,","place":["Huddinge, Stockholm, Sweden, 141 52"]},{"name":"Karolinska University Hospital , , ,","place":["Stockholm, Sweden, 141 86"]},{"name":"Karolinska ATMP Center, Karolinska Institutet , , ,","place":["Huddinge, Stockholm, Sweden, 14152"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2641-7918","authenticated-orcid":true,"given":"Anders","family":"Dahle\u0301n","sequence":"additional","affiliation":[{"name":"AstraZeneca , , ,","place":["Mo\u0308lndal, Sweden, 431 83"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,14]]},"reference":[{"issue":"29","key":"2026083119123259400_cit1","doi-asserted-by":"publisher","first-page":"10549","DOI":"10.1021\/acs.analchem.2c02111","article-title":"Intracellular\nAbsolute Quantification of Oligonucleotide Therapeutics by NanoSIMS","volume":"94","author":"Becquart","year":"2022","journal-title":"Anal. 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