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

Abstract

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.

SELEX-based DNA Aptamer Selection: A Perspective from the Advancement of Separation Techniques

“…The development of a unique separation technique to access functional aptamers is also a challenging subject. 104 Reports about functional aptamers are increasing and address higher affinity and stability through a variety of means, including: DNA nanoscaffold 105 and circular DNA; 106 structure-switching aptamers (typically in Capture-SELEX and PD-SELEX) and affinity rulers for applicability in ELONA using aptamers selected by EX-SELEX; 107 different epitope-binding and isotope-binding aptamers; 109,110 and RNA mimics of green fluorescent protein. [111][112][113] However, the compatibility of separation techniques used to acquire these functional aptamers is unclear, and it could be expected that unique separation modes could be identified to extract unique aptamers in the future.…”

High-efficiency enrichment enables identification of aptamers to circulating Plasmodium falciparum-infected erythrocytes

“…The ideal outcome of a SELEX experiment is a number of high frequency sequences identified which can be rapidly characterized for high affinity binding to the target of interest 44 . As such, a successful experiment must allow for the efficient enrichment of the putative aptamers well above background non-binding sequences 48 . Inefficient enrichment can make SELEX prohibitively resource and labor intensive because more rounds of selection are required.…”

“…There are many emerging strategies to improve the success of SELEX experiments such as performing only one or a few rounds of SELEX with the help of improved partitioning 41,48 . These strategies have been successful where capillary electrophoresis or prior knowledge of the specific target is available and accessible.…”

Aptamer Applications in Emerging Viral Diseases

“…Therefore, aptamers have been identified to bind with affinity to HA antigens from different strains of influenza. For further applications in diagnosis and possibly therapy, aptamers can be modified with biotin [ 52 , 53 ], amine groups [ 54 ], fluorophores [ 55 , 56 , 57 , 58 ], and thiol groups that will be attached to nanostructured materials, such as polymers [ 59 , 60 ], gold nanoparticles [ 61 , 62 , 63 ], metallic semiconductors [ 64 , 65 ], carbon-nanotubes [ 66 , 67 ], nanocomposites [ 68 ], and microspheres [ 69 ].…”