A Dual-Halogen Labeling Strategy to Detect and Quantify Double-Stranded RNAs Using NanoSIMS: A Proof-of-Concept Study.
JSON
Vicentini Q, Kurczy M, Estupiñán HY, Hekman D, Becquart C, Haag L, Andersson S, El-Andaloussi S, Dahlén A
ACS Omega 11 (33) 49739-49745 [2026-08-25; online 2026-08-14]
Oligonucleotide drugs, such as small interfering RNAs (siRNAs) and small activating RNAs (saRNAs), are rapidly advancing in the clinic. 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.
PubMed 42662369
DOI 10.1021/acsomega.6c04146
Crossref 10.1021/acsomega.6c04146