title: Quantification of siRNA Duplexes Bound to Gold Nanoparticle Surfaces.
abstract: 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.
authors.affiliation: Department of Biomedical Engineering, University of Delaware, 161 Colburn Lab, Newark, DE, 19716, USA.
authors.forename: Jilian R
authors.initials: JR
authors.lastname: Melamed
authors.affiliation: Department of Biomedical Engineering, University of Delaware, 161 Colburn Lab, Newark, DE, 19716, USA.
authors.forename: Rachel S
authors.initials: RS
authors.lastname: Riley
authors.affiliation: Department of Biomedical Engineering, University of Delaware, 161 Colburn Lab, Newark, DE, 19716, USA.
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Valcourt
authors.affiliation: Department of Biomedical Engineering, University of Delaware, 161 Colburn Lab, Newark, DE, 19716, USA.
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Billingsley
authors.affiliation: Department of Biomedical Engineering, University of Delaware, 161 Colburn Lab, Newark, DE, 19716, USA.
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Kreuzberger
authors.affiliation: Department of Biomedical Engineering, University of Delaware, 161 Colburn Lab, Newark, DE, 19716, USA. emilyday@udel.edu.|Helen F. Graham Cancer Center and Research Institute, Newark, DE, 19713, USA. emilyday@udel.edu.|Department of Materials Science and Engineering, University of Delaware, Newark, DE, 19716, USA. emilyday@udel.edu.
authors.forename: Emily S
authors.initials: ES
authors.lastname: Day
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D053768:Metal Nanoparticles / Q000737:chemistry; D034741:RNA, Small Interfering / Q000032:analysis* / Q000737:chemistry*; D011933:Reagent Kits, Diagnostic; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; D011933:Reagent Kits, Diagnostic; D006046:Gold
keywords: Conjugation; Gene regulation; Loading; Nanoparticles; Nucleic acids; siRNA