title: Characterisation of aptamer-target interactions by branched selection and high-throughput sequencing of SELEX pools.
abstract: Nucleic acid aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX) has shown great promise for use in the development of research tools, therapeutics and diagnostics. Typically, aptamers are identified from libraries containing up to 10(16) different RNA or DNA sequences by 5-10 rounds of affinity selection towards a target of interest. Such library screenings can result in complex pools of many target-binding aptamers. New high-throughput sequencing techniques may potentially revolutionise aptamer selection by allowing quantitative assessment of the dynamic changes in the pool composition during the SELEX process and by facilitating large-scale post-SELEX characterisation. In the present study, we demonstrate how high-throughput sequencing of SELEX pools, before and after a single round of branched selection for binding to different target variants, can provide detailed information about aptamer binding sites, preferences for specific target conformations, and functional effects of the aptamers. The procedure was applied on a diverse pool of 2'-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin plasminogen activator inhibitor-1 (PAI-1) through five rounds of standard selection. The results demonstrate that it is possible to perform large-scale detailed characterisation of aptamer sequences directly in the complex pools obtained from library selection methods, thus without the need to produce individual aptamers.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark Danish-Chinese Centre for Proteases and Cancer, Aarhus University, 8000 Aarhus C, Denmark dmd@mbg.au.dk.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Dupont
authors.affiliation: iNANO Interdisciplinary Nanoscience Center, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Niels
authors.initials: N
authors.lastname: Larsen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark Danish-Chinese Centre for Proteases and Cancer, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark Danish-Chinese Centre for Proteases and Cancer, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark iNANO Interdisciplinary Nanoscience Center, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Jørgen
authors.initials: J
authors.lastname: Kjems
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001665:Binding Sites; D059014:High-Throughput Nucleotide Sequencing; D008024:Ligands; D009154:Mutation; D009690:Nucleic Acid Conformation; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D052156:SELEX Aptamer Technique; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D017395:Plasminogen Activator Inhibitor 1; D012313:RNA