Sub-millisecond conformational transitions of short single-stranded DNA lattices by photon correlation single-molecule FRET

Abstract

Thermally-driven conformational fluctuations (or 'breathing') of DNA plays 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 functional and 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 F&oumlrster Resonance Energy Transfer (smFRET) experiments to investigate the backbone fluctuations of short (ss) oligo- oligo(dT)n templates within DNA constructs that can also serve as models for ss-dsDNA junctions. Such junctions, as well as the attached ssDNA sequences, are involved in the binding of ssDNA binding (ssb) proteins that control and integrate the mechanisms of DNA replication complexes. We have used these data to determine multi-order time-correlation functions (TCFs) and probability distribution functions (PDFs) that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT)n tails of ss-dsDNA constructs inter-convert, on sub-millisecond 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 a 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.

Excited-State Lifetimes of DNA-Templated Cyanine Dimer, Trimer, and Tetramer Aggregates: The Role of Exciton Delocalization, Dye Separation, and DNA Heterogeneity

“…Additionally, in recent work by Marcus, von Hippel, and co-workers in which they performed single molecule studies of Cy3−Cy5 pairs attached to single-strand-duplex DNA junctions, they demonstrated the tendency for singlestranded DNA segments to adopt configurations that could be grouped into three sets, or macrostates, which they described as compact, partially extended, and fully extended. 66 The restricted and extended bubble configurations we propose to explain the additional aggregate and monomer subpopulations observed here may be consistent with this interpretation of discrete, thermally accessible, transient dye−dye configurations.…”

Tuning Optical Absorption and Emission Using Strongly Coupled Dimers in Programmable DNA Scaffolds

“…87,88 Beyond strand displacement reactions, the dependence of the photophysics on the complementary central crossover strand has the potential to enable studies of DNA hybridization and other properties of nucleic acids. 89 Finally, the DNA scaffold means that the construct is biocompatible and easily functionalized for specific conjugation to desired targets.…”