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

Luiza I Hernandez; Katie S Flenker; Frank J Hernandez; Aloysius J Klingelhutz; James O McNamara II; Paloma H Giangrande

doi:10.3390/ph6030295

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

Journal article

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

Luiza I Hernandez, Katie S Flenker, Frank J Hernandez, Aloysius J Klingelhutz, James O McNamara II and Paloma H Giangrande

Pharmaceuticals (Basel, Switzerland), Vol.6(3), pp.295-319

03/14/2013

DOI: 10.3390/ph6030295

PMCID: PMC3722562

PMID: 23894227

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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 .

cell-SELEX

siRNA delivery

RNA aptamers

targeted delivery

cell-internalizing aptamers

Details

Title: Subtitle: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers
Creators: Luiza I Hernandez - Molecular & Cellular Biology Program, University of Iowa, Iowa City, IA 52242, USA
Katie S Flenker - Molecular & Cellular Biology Program, University of Iowa, Iowa City, IA 52242, USA
Frank J Hernandez - Molecular & Cellular Biology Program, University of Iowa, Iowa City, IA 52242, USA
Aloysius J Klingelhutz - Molecular & Cellular Biology Program, University of Iowa, Iowa City, IA 52242, USA
James O McNamara II - Molecular & Cellular Biology Program, University of Iowa, Iowa City, IA 52242, USA
Paloma H Giangrande - Molecular & Cellular Biology Program, University of Iowa, Iowa City, IA 52242, USA
Resource Type: Journal article
Publication Details: Pharmaceuticals (Basel, Switzerland), Vol.6(3), pp.295-319
DOI: 10.3390/ph6030295
PMID: 23894227
PMCID: PMC3722562
NLM abbreviation: Pharmaceuticals (Basel)
ISSN: 1424-8247
eISSN: 1424-8247
Publisher: MDPI
Language: English
Date published: 03/14/2013
Academic Unit: Microbiology and Immunology; Radiation Oncology; Internal Medicine
Record Identifier: 9984001213402771

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