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 1016 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.
Highlights
Library screening methodologies such as phage display and systematic evolution of ligands by exponential enrichment (SELEX) are useful tools for the identification of novel protein-targeting ligands for pharmacological intervention, drug delivery, molecular imaging, and other diagnostic and prognostic analysis [1,2,3,4]
We demonstrate that High-throughput sequencing (HTS) of SELEX pools, before and after a parallel binding selection to a panel of alanine mutants of plasminogen activator inhibitor-1 (PAI-1), enables individual and global characterisation of binding sites for the aptamers in a pool enriched for target-binding RNA
To investigate how selection for target variants can affect SELEX pool composition, we chose to work with an RNA pool from a previously reported experiment in which 2 ́-F-Y aptamers were selected against the PAI-1 protein [14]
Summary
Library screening methodologies such as phage display and systematic evolution of ligands by exponential enrichment (SELEX) are useful tools for the identification of novel protein-targeting ligands for pharmacological intervention, drug delivery, molecular imaging, and other diagnostic and prognostic analysis [1,2,3,4]. In SELEX experiments, up to 1016 unique nucleic acid sequences are screened for their ability to bind a target of interest. The general frequency of target binding nucleic acid molecules (aptamers) in random libraries is in the order of 10−10–10−14 for most proteins [5,6], and through iterative rounds of affinity selection and amplification of the pool, the relative content of target-binding aptamers is increased to a level where they can be identified by sequencing a limited number of clones. Novel strategies are needed to facilitate the identification of aptamers from rare sequence families with desirable binding properties and functional effects
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