Critical evaluation of photocrosslinking parameters for the implementation of efficient DNA-encoded chemical library selections.

The growing importance of DNA-encoded chemical libraries (DECLs) as tools for the discovery of protein binders has sparked an interest for the development of efficient screening methodologies, capable of discriminating between high- and medium-affinity ligands. Here, we present a systematic investigation of selection methodologies, featuring a library dis-played on single-stranded DNA, which could be hybridized to a complementary oligonucleotide carrying a diazirine photore-active group. Model experiments, performed using ligands of different affinity to carbonic anhydrase IX, revealed a recovery of preferential binders up to 10%, which was mainly limited by the highly-reactive nature of carbene intermediates generated during the photocrosslinking process. Ligands featuring acetazolamide or p-phenylsulfonamide exhibited a higher recovery compared to their counterparts based on 3-sulfamoyl benzoic acid, which had a lower affinity towards the target. A systemat-ic evaluation of experimental parameters revealed conditions that were ideally suited for library screening, which were used for the screening of a combinatorial DECL library, featuring 669240 combinations of two sets of building blocks. Compared to conventional affinity capture procedures on protein immobilized on solid supports, photocrosslinking provided a better discrimination of low-affinity CAIX ligands over the background signal and therefore can be used as a tandem methodology with the affinity capture procedures.