A novel selection approach for DNA-encoded libraries using terminal deoxynucleotidyl transferase

DNA-encoded chemical libraries (DECLs) are an essential tool for hit finding in drug discovery. They are routinely selected against immobilized proteins of interest (POIs) by affinity enrichment. However, this method selects mainly for binders with slow k_off rate constants and leaves an incomplete picture of potential ligands for a target.
Terminal deoxynucleotidyl transferase (TdT) is an unusual DNA polymerase that extends template-independent the 3’-end of DNA with random nucleotides. Herein we establish a strategy for the expression of POI-TdT fusions and demonstrate that induced proximity to TdT by a target binding event of a DECL member selectively extends its DNA. The added nucleotides serve as a stable record of the binding event and are used as a handle for binder separation. We then synthesized a 2 × 10^5-membered DECL and selected it against carbonic anhydrase II as a benchmarking system. Ligands from double-digit nanomolar to double-digit micromolar were identified by TdT extension with excellent data clarity, whereas only the tightest binding ligands were identified by classical affinity enrichment.