Resolving the dynamics of atypical DNA structures using smFRET

B-form DNA is a structural icon in biology. Noncanonical DNA conformations are increasingly recognized as essential drivers of genomic phenomena. These atypical structures, including hairpins, G-quadruplexes, and multistranded junctions, are often transient and highly dynamic, making them difficult to characterize using traditional methods. Single-molecule fluorescence resonance energy transfer (smFRET) has emerged as an indispensable tool for resolving these rapid conformational changes and elucidating their role in human disease. Here, we discuss the sub-millisecond folding dynamics of single-stranded DNA, the mechanisms of protein-induced DNA compaction, and the pathogenic strand-slippage of tandem repeats associated with neurodegenerative diseases. Finally, we highlight the challenges of translating in vitro findings to live cells and the potential for fluorophore labels to perturb the delicate energy landscape.