Screening of a Combinatorial Library of Triazine-Scaffolded Dipeptide-Mimic Affinity Ligands to Bind Plasmid DNA.

Plasmid DNA (pDNA) purification plays a key role in the development of vaccines and gene therapies. Affinity chromatography stands out as a promising method for plasmid purification, leveraging a range of biological and synthetic ligands to achieve selectivity. This study investigates the potential of a synthetic ligand library consisting of triazine-based bifunctional compounds designed to mimic the side chains of amino acids that are known to bind nucleic acids. A high-throughput screening method was employed to assess the binding ability of 158 ligands within the library to single-stranded, FITC-labeled homo-oligonucleotides (G and T), each comprising 20 nucleotides, under both hydrophilic and hydrophobic conditions. High-affinity ligands were identified for both T and G oligonucleotides. Follow-up microscale chromatographic screening uncovered some false positives from the initial FITC-based screening, narrowing the selection to 22 ligands for further investigation. In the next phase of the study, the binding affinity of these ligands towards double-stranded oligonucleotides (AT and CG) was assessed. Ligand 1/2, a mimic of Ala-Lys or Gly-Lys, and ligand 2/3, a mimic of Lys-Tyr, were chosen as initial candidates for evaluating plasmid DNA purification from an