Single-molecule methods have been used to study telomeric protein-DNA interaction. However, preparing single molecule constructs with the telomeric repetitive motifs remains a challenging task. In this protocol, we outline the steps for expressing and purifying TRF2 protein, preparing telomeric DNA, setting up single-molecule mechanical assays, and analyzing the resulting data.
Single-molecule tools are powerful techniques for exploring telomeric protein-DNA interactions. Single-molecule mechanical methods, such as magnetic tweezers, optical tweezers, and AFM have been used to investigate TRF2-dependent DNA distortion, reveal TRF2-mediated columnar stacking of human telomeric chromatin, and observe the presence of telomerase catalysis among other applications. We investigated telomeric DNA-protein interactions using single molecular magnetic tweezers, enabling precise measurements of changes in extension and the durations of protein-DNA interactions under applied forces.
From these measurements, we derived the dissociation kinetics of the telomeric DNA-protein complexes. Furthermore, the formation of loops within these complexes was revealed. Numerous structures of telomeric-binding proteins in complex with DNA have been resolved using cryo-EM, X-ray crystallography, and NMR.
These structural insights have advanced our understanding of telomeric protein-DNA interactions. To further investigate the dynamics of these interactions, we have developed a single-molecule mechanical methods specifically for studying telomeric protein-DNA interactions. Single-molecule tools are powerful techniques for investigating protein-DNA interactions at telomeres.
These methods are especially valuable for topological conformations and analyzing the kinetics of protein-DNA association and dissociation.
