On-DNA Hit Validation from DNA-Encoded Library Screenings by Focal Molography

Focal molography measures DNA-encoded library (DEL) hits while they stay on their DNA tag, with up to 64 hits on one sensor chip. This poster, made with Philochem AG, was presented at the 13th International Symposium on DNA-Encoded Chemical Libraries in Boston, USA (August 14 to 15, 2025).

The Challenge: Kinetics After the DEL Screen

Hit characterization remains a bottleneck after a DEL screen, in particular for kinetic and affinity data. Researchers need k_on, k_off and K_D for many hits, ideally with little protein and a simple workflow.

The Approach: From DNA Tag to Sensor Chip

An adapter oligo complementary to the sensor spots is added by on-DNA resynthesis or by splint ligation to an existing DNA tag. The MACS Sampler then injects up to 64 hits as one mixture, and DNA-directed immobilization (DDI) loads the 64 molograms in 10 minutes. Single-cycle kinetics follow, guided by the software, in about 60 minutes.

Key Results for Three CAIX Ligands

• Model system: three hits against human carbonic anhydrase IX (CAIX): acetazolamide (AAZ), sulfamoylbenzoic acid (SABA) and meta-sulfamoylbenzoic acid (mSABA).
• Immobilization: location-specific DDI with no cross-immobilization during sequential injection.
• Affinity clusters: a Langmuir 1:1 fit gave three well-separated clusters in the iso-affinity plot.

Why It Matters for DEL Campaigns

One protein concentration series gives kinetic data for all hits on the sensor chip, with low protein consumption. Off-target binding can be checked in tissue extracts and cell lysates. This supports the secondary screening phase of a DEL campaign.