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Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

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Author(s): Luiza I. Hernandez | Katie S. Flenker | Frank J. Hernandez | Aloysius J. Klingelhutz | James O. McNamara | Paloma H. Giangrande

Journal: Pharmaceuticals
ISSN 1424-8247

Volume: 6;
Issue: 3;
Start page: 295;
Date: 2013;
Original page

Keywords: RNA aptamers | targeted delivery | siRNA delivery | cell-SELEX | cell-internalizing aptamers

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.
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