Flow-cell based technology for massively parallel characterization of base-modified DNA aptamers

Aptamers incorporating chemically modified bases can achieve superior affinity and specificity compared to natural aptamers, but their characterization remains a labor-intensive, low-throughput task. Here we describe the ‘non-natural aptamer array’ (N2A2) system, in which a minimally modified Illumina MiSeq instrument is used for the high-throughput generation and characterization of large libraries (∼106) of base-modified DNA aptamer candidates on the basis of both target affinity and specificity. We first demonstrate the capability to screen multiple different base modifications to identify the optimal chemistry for high-affinity target binding. We next use N2A2 to generate aptamers that can maintain excellent specificity even in complex samples, with equally strong target affinity in both buffer and diluted human serum. Given that N2A2 requires only minor mechanical modifications to the MiSeq, we believe N2A2 offers a broadly accessible tool for generating high-quality affinity reagents for diverse applications.