Understanding G-Quadruplex Biology and Stability Using Single-Molecule Techniques

Thelink between the chemical stability of G-quadruplex (qDNA)structures and their roles in eukaryotic genomic maintenance processeshas been an area of interest now for several decades. This Reviewseeks to demonstrate how single-molecule force-based techniques canprovide insight into the mechanical stabilities of a variety of qDNAstructures as well as their ability to interconvert between differentconformations under conditions of stress. Atomic force microscopy(AFM) and magnetic and optical tweezers have been the primary toolsused in these investigations and have been used to examine both freeand ligand-stabilized G-quadruplex structures. These studies haveshown that the degree of stabilization of G-quadruplex structureshas a significant effect on the ability of nuclear machinery to bypassthese roadblocks on DNA strands. This Review will illustrate how variouscellular components including replication protein A (RPA), Bloom syndromeprotein (BLM), and Pif1 helicases are capable of unfolding qDNA. Techniquessuch as single-molecule fluorescence resonance energy transfer (smFRET),often in conjunction with the aforementioned force-based techniques,have proven extremely effective at elucidating the factors underpinningthe mechanisms by which these proteins unwind qDNA structures. Wewill provide insight into how single-molecule tools have facilitatedthe direct visualization of qDNA roadblocks and also showcase resultsobtained from experiments designed to examine the ability of G-quadruplexesto limit the access of specific cellular proteins normally associatedwith telomeres.