Wet-Dry-Wet Experiments Enable Simultaneous Aptamer Isolation and Critical Binding Domain Identification.

The critical binding domain (CBD) is typically identified via tedious affinity screening of truncated and mutated sequences of an aptamer. We report here a wet-dry-wet experiment strategy, which enables the isolation of rapamycin-binding aptamers and the identification of the CBD at the same time without the need for affinity testing of numerous rationally designed sequences. In the first wet-experimental module, the pre-enriched library was obtained via 15 rounds of Capture-SELEX. In the dry-experimental module, the two key binding architectures, a three-stem-three-loop (3S3L) motif and a 3S3L-A motif (containing an additional adenosine in the second loop of 3S3L), were identified by comprehensively analyzing the high-throughput sequencing data using K-mer assembly, unsupervised learning (RBM), and mFold structure simulation. The structure-confined mixed secondary library designed based on the two structures exhibited high affinity, validating the importance of structure. In the second wet-experimental module, enriched library 2R6 was obtained after six rounds of second Capture-SELEX. A series of aptamers with nanomolar dissociation constants and high specificity were obtained, along with the identification of 11-nt CBD via analyzing the high-throughput sequencing result of 2R6. The decreased or completely lost binding affinity of the mutated sequences of seed aptamer 1R15-2 confirmed the CDB. A strand-displacement fluorescence sensor was constructed and capable of the detection of rapamycin spiked in 10% human serum with a nanomolar limit of detection. This study provides an efficient method for simultaneous aptamer isolation and CBD identification and can be applied to other targets.