Recording Binding Information Directly into DNA-Encoded Libraries Using Terminal Deoxynucleotidyl Transferase

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase.
• DNA-encoded libraries (DELs) are powerful tools for drug discovery.
• Traditional affinity selection methods have limitations in identifying binders with moderate affinity.

Purpose of the Study:

• To develop a novel method for identifying binders from DELs using TdT.
• To enhance the sensitivity and range of affinity selection in DEL screening.
• To create a stable molecular record of binding events within DELs.

Main Methods:

• Target proteins were fused with TdT.
• Incubation of TdT-fused proteins with DELs in the presence of dATP.
• Poly-adenine (polyA) tail synthesis on DNA binders, with tail length correlating to binding affinity.
• Enrichment of binders using magnetic poly(dT)25 beads.
• Sequencing to identify enriched binders.

Main Results:

• TdT extension successfully generated polyA tails on DNA binders.
• PolyA tail length served as a direct measure of binding affinity.
• Ligands with nanomolar to double-digit micromolar binding affinities were identified.
• This method outperformed classical affinity selection for broader affinity range identification.

Conclusions:

• TdT-mediated polyA tail synthesis is a simple and effective method for DEL binder selection.
• The technique significantly expands the detectable binding affinity range compared to traditional methods.
• This approach offers a robust and versatile tool for accelerating drug discovery using DELs.