Single Molecule Investigations of Protein and Small Molecule Interactions with G-Quadruplexes and their Impact on Telomere Structure

Abstract

G-quadruplex (GQ) structures form in guanine-rich segments of the genome and are particularly more prominent at telomeres and promoters. Telomeric GQ structures (GQs) have emerged as potential targets for small molecule drugs that aim to inhibit telomerase activity in cancer cells, while GQs in promoters are considered to play a role in transcription and translation level gene expression regulation. Due to their high thermal stability, GQs require activity of DNA binding proteins or helicases to be unfolded. Stabilizing small molecules are typically characterized based on their impact on thermal stability of GQs. As a physiologically more relevant approach, we determined the efficacy of several prominent small molecules based on their impact of helicase activity. We measured binding kinetics of individual small molecules and their impact on dynamics and steady state Bloom helicase (BLM) mediated GQ unfolding activities. We also studied Replication Protein A (RPA), GQ, and small molecule interactions while the GQ is maintained under tension by a looped DNA. Finally, we present FRET-PAINT measurements where we studied the accessibility of long telomeric repeats that form multiple tandem GQs and observed length dependent compaction in telomere structure.