Focal molography enables high-throughput, label-free kinetic validation of DNA-encoded library (DEL) hits, measuring 24 compounds with subnanomolar accuracy.

The Challenge

DNA-encoded libraries (DELs) let medicinal chemists screen millions to billions of compounds against a target in a single experiment. The bottleneck has shifted downstream. Established hit-validation methods such as surface plasmon resonance (SPR), fluorescence polarization (FP), and isothermal titration calorimetry (ITC) characterize only a fraction of the enriched compounds. Many potentially relevant binders therefore remain uncharacterized.

The Approach

The authors used focal molography on the MACS® Matchmaker to measure binding kinetics of 24 DEL-derived hits. The targets were carbonic anhydrase IX (CAIX) and carbonic anhydrase II (CAII). Each hit was conjugated to a unique 20-mer DNA code and immobilized in parallel via DNA-directed immobilization (DDI) onto spatially encoded molograms. That enabled 18-plex single-cycle kinetic measurement on a single regenerable chip.

Key Results

• Multiplexed throughput: 24 DEL hits plus an SABA reference were characterized on an 18-plex chip, run as two pools and merged at the analysis stage. The 32-plex format supports up to 500 duplicate kinetic measurements per day.
• Subnanomolar accuracy: Four top hits reached K_D values of 0.3 to 0.8 nM against CAIX. These were compounds 4, 8, 12, and 16, all 2R,4R stereoisomers.
• Selectivity profiling: No binding to CAII was detectable for any of the 24 compounds, placing selectivity for the most potent hits above 10,000-fold.
• Cross-platform consistency: Kinetic rank order matched SPR and FP across three reference sulfonamides (AAZ, SABA, mSABA).
• Crude compatibility: Ligation crudes were measured directly without HPLC purification, removing a time-consuming workflow step.