A general double library SELEX strategy for aptamer selection using unmodified nonimmobilized targets.

Aptamer discovery for unmodified nonimmobilized targets has been constantly presenting itself as a significant challenge to the research community. We demonstrate here a novel double library (DL) SELEX strategy and its usefulness and generality toward discovering both ssDNA- and RNA-based aptamers with nanomolar binding affinities toward unmodified targets of both small (e.g., doxycycline) and large (e.g., VEGF165) sizes. The same selection strategy further allows for concurrent selection of an aptamer pair, recognizing discrete epitopes on the same protein, from the same selection cycles for the sandwich aptamer pair-based biosensor development (e.g., one aptamer for the recognition and the other for the signal transduction). These results establish the DL-SELEX method developed here as a valuable and highly accessible selection strategy for aptamer discovery, especially when chemical modifications of target molecules are not preferred or simply impossible.