A Dual-HalogenLabeling Strategy to Detect and QuantifyDouble-Stranded RNAs Using NanoSIMS: A Proof-of-Concept Study

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.