Biomedical Nanotechnology

Jilian R. Melamed, Rachel S. Riley, Danielle M. Valcourt, Margaret M. Billingsley, Nicole L. Kreuzberger, Emily S. Day

Sarah Hurst Petrosko, Emily S. Day

RNA interference (RNAi)-based gene regulation has recently emerged as a promising strategy to silence genes that drive disease progression. RNAi is typically mediated by small interfering ribonucleic acids (siRNAs), which, upon delivery into the cell cytoplasm, trigger degradation of complementary messenger RNA molecules to halt production of their encoded proteins. While RNAi has enormous clinical potential, its in vivo utility has been hindered because siRNAs are rapidly degraded by nucleases, cannot passively enter cells, and are quickly cleared from the bloodstream. To overcome these delivery barriers, siRNAs can be conjugated to nanoparticles (NPs), which increase their stability and circulation time to enable in vivo gene regulation. Here, we present methods to conjugate siRNA duplexes to NPs with gold surfaces. Further, we describe how to quantify the resultant amount of siRNA sense and antisense strands loaded onto the NPs using a fluorescence-based assay. This method focuses on the attachment of siRNAs to 13 nm gold NPs, but it is adaptable to other types of nucleic acids and nanoparticles as discussed throughout the protocol.