Competitive non-SELEX for the selective and rapid enrichment of DNA aptamers and its use in electrochemical...

The SELEX (Systematic Evolution of Ligands by EXponential enrichment) method has been used successfully since 1990, but work is still required to obtain highly specific aptamers. Here, we present a novel approach called 'Competitive non-SELEX' (and termed as 'SELCOS' (Systematic Evolution of Ligands by COmpetitive Selection)) for readily obtaining aptamers that can discriminate between highly similar targets. This approach is based on the theoretical background presented here, in which under the co-presence of two similar targets, a specific binding type can be enriched more than a nonspecifically binding one during repetitive steps of partitioning with no PCR amplification between them. This principle was experimentally confirmed by the selection experiment for influenza virus subtype-specific DNA aptamers. Namely, the selection products (pools of DNA aptamers) obtained by SELCOS were subjected to a DEPSOR-mode electrochemical sensor, enabling the method to select subtype-specific aptamer pools. From the clonal analysis of these pools, only a few rounds of in vitro selection were sufficient to achieve the surprisingly rapid enrichment of a small number of aptamers with high selectivity, which could be attributed to the SELCOS principle and the given selection pressure program. The subtype-specific aptamers obtained in this manner had a high affinity (e.g., K_D = 82 pM for H1N1; 88 pM for H3N2) and negligible cross-reactivity. By making the H1N1-specific DNA aptamer a sensor unit of the DEPSOR electrochemical detector, an influenza virus subtype-specific and portable detector was readily constructed, indicating how close it is to the field application goal.