One on-DNA ligand, three species variants of its target and five unrelated proteins, profiled on one focal molography chip layout. The same runs show which DNA-tag format keeps the ligand able to bind.
The Challenge
Fibroblast activation protein (FAP) is a target for radioligands, CAR-T cells and small-molecule drug conjugates. Its catalytic pocket lies about 30 Å deep, so FAP ligands are sensitive to linkers and attachment points. When a hit from a DNA-encoded library (DEL) is validated on its DNA tag, two questions decide whether the numbers hold: does it bind the species variants used in animal studies, and does the tag interfere with binding?
The Approach
The clinical-stage FAP ligand oncoFAP was coupled to DNA through a C6 or a PEG4-C6 linker, each single- and double-stranded at the surface end. The constructs were immobilized on individual molecular holograms (molograms) by DNA-directed immobilization (DDI). Human, mouse and canine FAP and five control proteins were titrated over the same layout by single-cycle kinetics.
Key Results
- Species profile: the single-stranded constructs bind human, mouse and canine FAP with KD values from 700 pM to 10 nM.
- Clean selectivity: no response above the 0.5 pg/mm² noise level on five control proteins, at concentrations up to 100 µM for BSA.
- A DNA-tag design rule: double-stranded constructs do not bind at all, while linker length changes the rates only about two-fold.
- Self-referencing readout: the control traces stay flat without any reference subtraction.
Why It Matters
For a lead program, the species profile translates directly into the choice of animal model, and the selectivity panel shows that the signal comes from the target. The duplex rule applies to any DEL follow-up on a deep binding pocket.
Data from Favalli N et al., Journal of Medicinal Chemistry (2026), in collaboration with Philochem AG. https://doi.org/10.1021/acs.jmedchem.6c01097
