AbstractThe ability to manipulate the RNA interference (RNAi) machinery to specifically silence the expression of target genes could be a powerful therapeutic strategy. Since the discovery that RNAi can be triggered in mammalian cells by short double‐stranded RNAs (small interfering RNA, siRNA), there has been a tremendous push by researchers, from academia to big pharma, to move siRNAs into clinical application. The challenges facing siRNA therapeutics are significant. The inherent properties of siRNAs (polyanionic, vulnerable to nuclease cleavage) make clinical application difficult due to poor cellular uptake and rapid clearance. Side effects of siRNAs have also proven to be a further complication. Fortunately, numerous chemical modification strategies have been identified that allow many of these obstacles to be overcome. This unit will present an overview of (1) the chemical modifications available to the nucleic acid chemist for modifying siRNAs, (2) the application of chemical modifications to address specific therapeutic obstacles, and (3) the factors that must be considered when assessing the activity of modified siRNAs. Curr. Protoc. Nucleic Acid Chem. 39:16.3.1‐16.3.22. © 2009 by John Wiley & Sons, Inc.