Abstract
Small molecules can inhibit cellular processes such as replication and transcription by binding to the promoter regions that are prone to form G-quadruplexes. However, since G-quadruplexes exist throughout the human genome, the G-quadruplex binders suffer from specificity issues. To tackle this problem, a G-quadruplex binder (Pyridostatin, or PDS) is conjugated with a ligand (Polyamide, or PA) that can specifically recognize DNA sequences flanking the G-quadruplex forming region. The binding mechanism of this hybrid ligand to the hTERT promoter region (hTERT 5-12) is then elucidated using optical tweezers. During mechanical unfolding processes, different intermediate structures of hTERT 5-12 in presence of PDS, PA, or PA-PDS conjugate are observed. These intermediate structures are consistent with two folding patterns of G-quadruplexes in the hTERT 5-12 fragment. While the duplex DNA binder PA facilitates the folding of a hairpin-G-quadruplex structure, the PDS assists the formation of two tandem G-quadruplexes. Both replication stop assay in vitro and dual luciferase assay in vivo established the effectiveness of the PA-PDS conjugate for hTERT 5-12 targeting. We expect such a ligand dependent folding dynamics will provide guidelines to the development of drugs that not only target hTERT expressions, but also other oncogenes via interactions with specific G-quadruplex structures formed in their promotor regions.