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.
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Acknowledgement
The authors acknowledge support from the University of Delaware Research Foundation, the W.M. Keck Foundation, Grant IRG14-251-07-IRG from the American Cancer Society, and an Institutional Development Award (IDeA) from the National Institutes of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under grant number U54-GM104941. J.R.M. received support from a National Defense Science and Engineering Graduate Fellowship from the Department of Defense.
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Melamed, J.R., Riley, R.S., Valcourt, D.M., Billingsley, M.M., Kreuzberger, N.L., Day, E.S. (2017). Quantification of siRNA Duplexes Bound to Gold Nanoparticle Surfaces. In: Petrosko, S., Day, E. (eds) Biomedical Nanotechnology. Methods in Molecular Biology, vol 1570. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6840-4_1
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DOI: https://doi.org/10.1007/978-1-4939-6840-4_1
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