Strand-Resolved Mapping of siRNA Trafficking in Cells by Nanoscale Secondary Ion Mass Spectrometry

Abstract Small interfering RNAs (siRNAs) are double-stranded nucleic acid therapeutics (NATs) that silence target mRNAs and have emerged as a useful therapeutic modality for treating human disease. However, their intracellular trafficking, strand separation, and organelle-specific distribution remain poorly understood. Here, we introduce a nanoscale secondary ion mass spectrometry (NanoSIMS)-based imaging platform for high-resolution, strand-resolved visualization of chemically labeled siRNA duplexes in mammalian cells. By incorporating nuclease-stable 2′-O-methyl–modified halogenated nucleotides into single-stranded overhangs, we achieved orthogonal labeling of antisense and sense strands with bromine and iodine, respectively, or in reverse, enabling simultaneous mapping of each strand at organelle-level resolution. NanoSIMS imaging revealed that endolysosomes serve as major accumulation sites for both strands, with heterogeneous signal intensities and partial strand dissociation in the cytoplasm. In addition, siRNA localization was observed in nuclear bodies and filopodia, suggesting complex trafficking beyond canonical RNA-induced silencing complex (RISC) pathways. Comparative analysis of antisense oligonucleotides (ASOs) exhibited a markedly different subcellular distribution from siRNAs, showing significantly greater accumulation in the nucleus when delivered under the same conditions. These findings establish the utility of NanoSIMS analyses and imaging for strand-specific profiling of RNA therapeutics, providing insights for guiding the design and intracellular delivery of NATs.