Fast and convenient hit validation for DNA-encoded libraries

Focal molography measures DNA-tagged hits from DNA-encoded library (DEL) screens without labels, using the DNA tag for immobilization. This application note, made with Philochem AG, shows the workflow with three known ligands of human carbonic anhydrase IX (CAIX).

The Challenge: Moving From DEL Screen to Kinetic Data

A DEL screen can test millions to billions of DNA-tagged compounds against a target. Each enriched hit still needs affinity and kinetic data before it can move forward. This second selection round should be simple and should use little protein.

The Approach: Three Oligo-Tagged Ligands and CAIX

Acetazolamide (AAZ), para-sulfamoylbenzoic acid (SABA) and meta-sulfamoylbenzoic acid (mSABA) were conjugated to 20-base-pair DNA fragments. Each ligand went to its own molograms on a 3-plex sensor chip by DNA-directed immobilization (DDI), in three sequential injections. Single-cycle kinetics in PBS-T used CAIX from 0.36 nM to 7.1 µM with a final dissociation of 20 minutes. The data were fitted with a Langmuir 1:1 model.

Key Results for AAZ, SABA and mSABA Binding to CAIX

• AAZ: K_D of 8.2 ± 1.8 × 10⁻¹⁰ M, with k_on of 5.7 × 10⁴ M⁻¹s⁻¹ and k_off of 4.8 × 10⁻⁵ s⁻¹.
• SABA: K_D of 3.2 ± 0.4 × 10⁻⁷ M, with k_on of 2.0 × 10⁴ M⁻¹s⁻¹ and k_off of 6.5 × 10⁻³ s⁻¹.
• mSABA: K_D of 3.2 ± 0.3 × 10⁻⁶ M, with k_on of 5.5 × 10³ M⁻¹s⁻¹ and k_off of 1.7 × 10⁻² s⁻¹.
• Clear separation: the three compounds form three separate clusters in the iso-affinity plot.
• Stable capture: no significant detachment of the captured compounds was seen after DDI.

Why It Matters for DEL Hit Validation

The same workflow takes up to 54 uniquely oligo-tagged hits in a single injection on a sensor chip with 54 molograms. The complementary sequence can be added by chemical synthesis or enzymatic ligation. One protein titration then gives kinetic data for all hits on the sensor chip.