Characterizing small molecule hits from DNA-encoded library screenings by focal molography

Focal molography uses the DNA barcode of a small molecule as its immobilization tag. DNA-encoded library (DEL) hits can therefore be measured directly on the sensor chip. This application note shows the workflow with biotin and desthiobiotin as model compounds.

The Challenge: Characterizing DEL Hits After the Screen

A DEL holds millions to billions of compounds, each tagged with a unique DNA barcode. A screen finds the binders, but it does not give their kinetics. Hits are then usually made by off-DNA synthesis and tested one at a time for affinity and activity.

The Approach: DNA-directed Immobilization and Single-Cycle Kinetics

The sensor chip carried 20-base-pair oligonucleotides. Complementary strands with biotin or desthiobiotin (300 nM, 10 minutes) were captured by DNA-directed immobilization (DDI). Single-cycle kinetics in PBS-T used streptavidin up to 10 nM and monovalent streptavidin up to 1000 nM. The data were fitted with a Langmuir 1:1 model, and the sensor chip could be regenerated with 50 mM NaOH.

Key Results for Biotin and Desthiobiotin Binding

• Biotin and streptavidin: K_D of 4.9 × 10⁻¹⁶ M, in line with the literature value of 10⁻¹⁶ M. No dissociation was visible, and at this affinity the sensorgram may not fit the model perfectly.
• Desthiobiotin and streptavidin: K_D of 2.6 × 10⁻¹¹ M with a detectable dissociation rate, in line with the literature value of 10⁻¹¹ M.
• Biotin and monovalent streptavidin: K_D of 6.8 × 10⁻⁸ M, a much weaker interaction than with tetravalent streptavidin.
• Stable DDI layer: the signal changed by less than 1 pg/mm² during a wash of more than four hours at 250 µL/min.

Why It Matters for DEL Hit Validation

A sensor chip with 54 molograms can take up to 54 uniquely barcoded DEL hits in a single injection. One titration series of the target protein then gives k_on, k_off and K_D for all of them. Off-target binding can be checked by injecting protein mixtures, tissue extracts, cell lysates or sera.