HAPIscreen, a method for high-throughput aptamer identification

Eric Dausse,Carmelo Di Primo,Saïd Taouji,Laetitia Evadé,Eric Chevet,Jean-Jacques Toulmé

doi:10.1186/1477-3155-9-25

Abstract

BackgroundAptamers are oligonucleotides displaying specific binding properties for a predetermined target. They are selected from libraries of randomly synthesized candidates through an in vitro selection process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment) alternating selection and amplification steps. SELEX is followed by cloning and sequencing of the enriched pool of oligonucleotides to enable comparison of the selected sequences. The most represented candidates are then synthesized and their binding properties are individually evaluated thus leading to the identification of aptamers. These post-selection steps are time consuming and introduce a bias to the expense of poorly amplified binders that might be of high affinity and are consequently underrepresented. A method that would circumvent these limitations would be highly valuable.ResultsWe describe a novel homogeneous solution-based method for screening large populations of oligonucleotide candidates generated from SELEX. This approach, based on the AlphaScreen® technology, is carried out on the exclusive basis of the binding properties of the selected candidates without the needs of performing a priori sequencing. It therefore enables the functional identification of high affinity aptamers. We validated the HAPIscreen (High throughput APtamer Identification screen) methodology using aptamers targeted to RNA hairpins, previously identified in our laboratory. We then screened pools of candidates issued from SELEX rounds in a 384 well microplate format and identify new RNA aptamers to pre-microRNAs.ConclusionsHAPIscreen, an Alphascreen®-based methodology for the identification of aptamers is faster and less biased than current procedures based on sequence comparison of selected oligonucleotides and sampling binding properties of few individuals. Moreover this methodology allows for screening larger number of candidates. Used here for selecting anti-premiR aptamers, HAPIscreen can be adapted to any type of tagged target and is fully amenable to automation.

Highlights

Aptamers are oligonucleotides displaying specific binding properties for a predetermined target
We validated the HAPIscreen (High throughput APtamer Identification screen) methodology using aptamers targeted to RNA hairpins, previously identified in our laboratory
HAPIscreen, an Alphascreen®-based methodology for the identification of aptamers is faster and less biased than current procedures based on sequence comparison of selected oligonucleotides and sampling binding properties of few individuals

Summary

Introduction

Aptamers are oligonucleotides displaying specific binding properties for a predetermined target They are selected from libraries of randomly synthesized candidates through an in vitro selection process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment) alternating selection and amplification steps. Aptamers are DNA, RNA or chemically-modified oligonucleotides selected from random pools of candidates containing up to 1015 different sequences on the basis of their ability to bind to other molecules [1,2,3] or to catalyze predetermined reactions [4,5]. The current approaches require sequencing of the selected clones at the end of the in vitro selection This is followed by a limiting step relying on sequence comparison and individual evaluation of few candidates for the identification of aptamers (Figure 1). This methodology is slow, incompatible with automation and is strongly biased since efficiently amplified poor affinity binders may mask low copy/high affinity aptamer candidates

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