Submillisecond Conformational Transitions of Short Single-Stranded DNA Lattices by Photon Correlation Single-Molecule...

Thermally driven conformational fluctuations (or "breathing") of DNA play important roles in the function and regulation of the "macromolecular machinery of genome expression." Fluctuations in double-stranded (ds) DNA are involved in the transient exposure of pathways to protein binding sites within the DNA framework, leading to the binding of regulatory proteins to single-stranded (ss) DNA templates. These interactions often require that the ssDNA sequences, as well as the proteins involved, assume transient conformations critical for successful binding. Here, we use microsecond-resolved single-molecule Förster resonance energy transfer (smFRET) experiments to investigate the backbone fluctuations of short [oligo(dT)] templates within DNA constructs that also serve as models for ss-dsDNA junctions. Such junctions, together with the attached ssDNA sequences, are involved in interactions with the ssDNA binding (ssb) proteins that control and integrate the functions of DNA replication complexes. We analyze these data using a chemical network model based on multiorder time-correlation functions and probability distribution functions that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT) tails of ss-dsDNA constructs interconvert, on submillisecond time scales, between three macrostates with distinctly different end-to-end distances. These are (i) a "compact" macrostate that represents the dominant species at equilibrium; (ii) a "partially extended" macrostate that exists as minority species; and (iii) a "highly extended" macrostate that is present in trace amounts. We propose a model for ssDNA secondary structure that advances our understanding of how spontaneously formed nucleic acid conformations may facilitate the activities of ssDNA-associating proteins.