The use of single-molecule FRET for the characterization of Holliday junctions containing human telomeric DNA: a methodological approach for nanoscale distance, mobility, and stability measurements

Single-molecule analysis has emerged as a powerful biophysical technique, allowing the investigation of molecular interactions at unprecedented spatial resolutions. In this paper, we outline a step-by-step procedure for the design of DNA sequences to form Holliday junction structures containing repetitive telomeric sequences and exploring conformational dynamics using Single-Molecule Förster Resonance Energy Transfer (smFRET). We describe in detail the setup and optimization of the single-molecule fluorescence microscope, along with data acquisition and analysis procedures. Furthermore, we address potential challenges and provide troubleshooting strategies to ensure successful data collection and interpretation. The presented methodology serves as a valuable resource for researchers aiming to employ smFRET to study Holliday junctions and other biomolecular systems at the nanoscale, paving the way for further advancements in the field of single-molecule biophysics.