The Influence of Single-Stranded or Double-Stranded DNA Tags on Ligand Binding Affinity in DNA-Encoded Libraries.

DNA-encoded libraries have become widely used in drug discovery, and several different setups to link chemical compounds to DNA have been employed in the field, including single-stranded and double-stranded DNA tags as well as a variety of linker chemistries. In our previous study, we observed distinct differences in binding affinities between ligands coupled either to single-stranded or double-stranded DNA; however, the molecular basis for these differences remained unclear. Here, we present a native ion mobility mass spectrometry approach that incorporates gas- and solution-phase activation techniques to systematically investigate these differences, specifically the impact of DNA tags on binding performance in protein-ligand interactions. The results of this study reveal that the binding affinity of DNA-coupled compounds is strongly affected by the DNA setup used. The differences in binding affinities of On-DNA protein-ligand complexes correlated with their differences in thermal stability in solution, with a synergistic binding effect observed exclusively for ligands coupled to single-stranded DNA. This trend is consistent across various protein-ligand systems, which differ in size, binding sites, and ligand structures. Here, we highlight how mass spectrometry can be used to elucidate the impact of different DNA tags on binding dynamics, which is essential for screening and hit validation in DNA-encoded library technology.