title: Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment.
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 cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.
authors.affiliation: Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
authors.forename: Agata
authors.initials: A
authors.lastname: Levay
authors.affiliation: Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
authors.forename: Randall
authors.initials: R
authors.lastname: Brenneman
authors.affiliation: National Center of Biotechnology Information (NCBI), National Library of Medicine (NLM), National Institutes of Health (NIH), Bethesda, MD 20894, USA.
authors.forename: Jan
authors.initials: J
authors.lastname: Hoinka
authors.affiliation: Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
authors.forename: David
authors.initials: D
authors.lastname: Sant
authors.affiliation: Cancer Inflammation Program, Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health (NIH), Frederick, MD, USA.
authors.forename: Marco
authors.initials: M
authors.lastname: Cardone
authors.affiliation: Cancer Inflammation Program, Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health (NIH), Frederick, MD, USA.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Trinchieri
authors.affiliation: National Center of Biotechnology Information (NCBI), National Library of Medicine (NLM), National Institutes of Health (NIH), Bethesda, MD 20894, USA.
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Przytycka
authors.affiliation: Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA ABerezhnoy@med.miami.edu.
authors.forename: Alexey
authors.initials: A
authors.lastname: Berezhnoy
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000378:metabolism*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008024:Ligands; D051379:Mice; D009154:Mutation; D016133:Polymerase Chain Reaction; D053701:Receptors, Interleukin-10 / Q000378:metabolism; D052156:SELEX Aptamer Technique / Q000379:methods*; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D053701:Receptors, Interleukin-10; C505714:TNFRSF9 protein, human; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9