Development of a His-Tag-mediated pull-down and quantification assay for G-quadruplex containing DNA sequences†

In this study, we developed a simple pull-down assay using peptide nucleic acids (PNAs) equipped with a His-Tag and a G-quadruplex (G4) ligand for the selective recognition and quantification of G4-forming DNA sequences. Efficient and specific target recovery was achieved using optimized buffer conditions and magnetic Ni–NTA beads, while quantification was realized by employing the enzyme-like properties of the G4/hemin complex. The assay was validated through HPLC analysis and adapted for a 96-well plate format. The results show that higher recovery can be achieved using His-Tag with Ni–NTA magnetic beads as compared to the more common biotin–streptavidin purification. The inclusion of the G4-ligand as an additional selectivity handle was shown to be beneficial for both recovery and selectivity.