Unlocking chemical diversity in aptamers with DNA orthogonal barcodes

Aptamers are a versatile alternative to antibodies as they are smaller, easier to synthesize and less immunogenic. However, while antibodies are composed of 20 chemically diverse amino acids and are established therapeutics, aptamers are composed of only 4 similar nucleobases, thereby limiting their therapeutic potential. Aptamer chemical modifications are restricted to maintain compatibility with enzymatic selection. Here we introduce aptamer-like encoded oligomers (alenomers), highly chemically modified aptamers that are read and sequenced using a DNA code branching from and corresponding to the target-binding oligomer. We build ~300,000-member DNA-encoded libraries using an automated DNA synthesizer and split-and-pool methods, and screen them for protein binding via next-generation sequencing. In contrast to aptamers, alenomers are not restricted by the need for conservative enzyme-compatible modifications. They can thus explore an almost limitless chemical space, enabling the discovery of highly stable, high-affinity protein-binding aptamers, while offering structural insights into their interactions with target molecules.