Mathematical extraction of binding constants from electrophoretic selection data from DNA-Encoded Libraries

DNA-encoded libraries (DELs) comprise up to billions of small molecules, enabling the discovery of binders to target proteins. Various selection techniques are used to isolate target binders and assess their binding properties. In our study, we focus on electrophoretic separation of DEL members in media uniformly filled with the target protein. Differences in the electrophoretic velocities of bound and unbound DEL members impact their spatial distribution in the medium. This distribution depends on the binding rate constant (kon) and the equilibrium dissociation constant (KD). We derive theoretical models to determine these constants from data acquired with our novel nDexer technique, and with capillary electrophoresis (CE), by analyzing the mean and variance of DEL members' spatial positions in nDexer, and their arrival times at a detector in capillary electrophoresis. Our findings align well with computer simulations and experimental results.