Submolecular dissection reveals strong and specific binding of polyamide–pyridostatin conjugates to human telomere interface

To modulate biological functions, G-quadruplexes in genome are often non-specifically targeted by small molecules. Here, specificity is increased by targeting both G-quadruplex and its flanking duplex DNA in a naturally occurring dsDNA–ssDNA telomere interface using polyamide (PA) and pyridostatin (PDS) conjugates (PA-PDS). We innovated a single-molecule assay in which dissociation constant (K(d)) of the conjugate can be separately evaluated from the binding of either PA or PDS. We found K(d) of 0.8 nM for PA-PDS, which is much lower than PDS (K(d) ∼ 450 nM) or PA (K(d) ∼ 35 nM). Functional assays further indicated that the PA-PDS conjugate stopped the replication of a DNA polymerase more efficiently than PA or PDS. Our results not only established a new method to dissect multivalent binding into actions of individual monovalent components, they also demonstrated a strong and specific G-quadruplex targeting strategy by conjugating highly specific duplex-binding molecules with potent quadruplex ligands.