Multiplexed siRNA Immunoassay Unveils Spatial and Quantitative Dimensions of siRNA Function, Abundance, and Localization In Vitro and In Vivo.

This article focuses on the development and characterization of a monoclonal antibody, designated PNPDB3, that detects chemically modified small interfering RNAs (siRNAs) across diverse sequences and conjugation methods used in therapeutic applications. By integrating this anti-siRNA antibody into multiplex immunoassays, the authors created robust platforms for simultaneous quantification of siRNA uptake, intracellular trafficking, and gene silencing activity both in vitro—using high-content imaging—and in vivo—using immunohistochemistry on tissue microarrays. The multiplex siRNA immunoassay enables spatial, cellular, and subcellular resolution of siRNA biodistribution and activity, providing advantages over conventional bioanalytical methods that lack spatial context. This approach was demonstrated in mouse liver and rat lung tissues treated with various siRNA conjugates, highlighting differences in cellular targeting and knockdown efficiency, and offering a valuable tool for optimizing siRNA therapeutic delivery and safety profiles.