Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment

Agata Levay, Randall Brenneman, Jan Hoinka, David Sant, Marco Cardone, Giorgio Trinchieri, Teresa M. Przytycka, Alexey Berezhnoy

Research output: Contribution to journalArticle

34 Scopus citations

Abstract

Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying crossspecies aptamers that can bind human receptors and cross-react with their murine orthologs.

Original languageEnglish (US)
Article numbere82
JournalNucleic acids research
Volume43
Issue number12
DOIs
StatePublished - 2015

ASJC Scopus subject areas

  • Genetics

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