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Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection
Journal article   Open access   Peer reviewed

Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection

William H Thiel, Thomas Bair, Andrew S Peek, Xiuying Liu, Justin Dassie, Katie R Stockdale, Mark A Behlke, Francis J Miller Jr and Paloma H Giangrande
PloS one, Vol.7(9), pp.e43836-e43836
2012
DOI: 10.1371/journal.pone.0043836
PMCID: PMC3433472
PMID: 22962591
url
https://doi.org/10.1371/journal.pone.0043836View
Published (Version of record) Open Access

Abstract

The broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX) with high-throughput sequencing (HTS) and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers. We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs). Several rounds of positive (VSMCs) and negative (endothelial cells; ECs) selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs. We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.
 Computational Biology Drug Delivery Systems Gene Library Muscle, Smooth, Vascular - metabolism Endothelial Cells - metabolism Humans Aptamers, Nucleotide - chemistry Muscle, Smooth, Vascular - cytology RNA - chemistry RNA - genetics Aptamers, Nucleotide - genetics Biological Transport Endothelial Cells - cytology SELEX Aptamer Technique Aptamers, Nucleotide - isolation & purification High-Throughput Nucleotide Sequencing Myocytes, Smooth Muscle - cytology Myocytes, Smooth Muscle - metabolism

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