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Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers
The HMO Research Network Center for Education and Research in Therapeutics
Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA
Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA
Department of Radiation Oncology, University of Iowa, Iowa City, IA 52242, USA
Molecular & Cellular Biology Program, University of Iowa, Iowa City, IA 52242, USA
These authors contributed equally to this work.
* Author to whom correspondence should be addressed.
Received: 11 January 2013; in revised form: 9 February 2013 / Accepted: 1 March 2013 / Published: 14 March 2013
Abstract: Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method (“QUSIM”) and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.
Keywords: RNA aptamers; targeted delivery; siRNA delivery; cell-SELEX; cell-internalizing aptamers
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MDPI and ACS Style
Hernandez, L.I.; Flenker, K.S.; Hernandez, F.J.; Klingelhutz, A.J.; McNamara, J.O.; Giangrande, P.H. Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers. Pharmaceuticals 2013, 6, 295-319.
Hernandez LI, Flenker KS, Hernandez FJ, Klingelhutz AJ, McNamara JO, Giangrande PH. Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers. Pharmaceuticals. 2013; 6(3):295-319.
Hernandez, Luiza I.; Flenker, Katie S.; Hernandez, Frank J.; Klingelhutz, Aloysius J.; McNamara, James O.; Giangrande, Paloma H. 2013. "Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers." Pharmaceuticals 6, no. 3: 295-319.