Droplet digital PCR-based single aptamer selection

Aptamers are potent alternatives to antibodies in applications including diagnostics and disease treatment. These synthetic molecules are generated from sequences identified through specific targets within an aptamer pool of random sequences, approximately 10^{^15} in size, via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. Nevertheless, SELEX encompasses repetitive, time/money-consuming and stochastic methodologies. In this study, we introduce a method for the direct acquisition of target aptamers in a single step, rapidly identifying the aptamers of interest. Single molecules of aptamers are first encapsulated into droplets and amplified therein, and the fluorescence in the droplets will be active upon binding between the aptamers with the target with good affinity. Subsequent identification and sorting of these fluorescing droplets enable the immediate acquisition of desired aptamers without the need for synthesizing them based on selected sequences. This digital selection process bypasses traditional sequencing, thereby reducing stochastic events and costs associated with repeated sequencing, as well as mitigating the uncertainties tied to the synthesis of aptamers. Our proof-of-concept findings suggest that this straightforward yet effective strategy can directly yield aptamers, thereby enhancing the exploration of aptamer biology and promoting the development of aptamer-based applications.