Crowding-induced hybridization of single DNA hairpins

It is clear that a crowded environment influences the structure, dynamics, and interactions of biological molecules, but the complexity of this phenomenon demands the development of new experimental and theoretical approaches. Here we use two complementary single-molecule FRET techniques to show that the kinetics of DNA base pairing and unpairing, which are fundamental to both the biological role of DNA and its technological applications, are strongly modulated by a crowded environment. We directly observed single DNA hairpins, which are excellent model systems for studying hybridization, either freely diffusing in solution or immobilized on a surface under crowding conditions. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concentrations of crowder [10% (w/w) polyethylene glycol (PEG)]. These experiments serve both to unambiguously highlight the impact of a crowded environment on a fundamental biological process, DNA base pairing, and to illustrate the benefits of single-molecule approaches to probing the structure and dynamics of complex biomolecular systems

Conformational Heterogeneity in a Fully Complementary DNA Three-Way Junction with a GC-Rich Branchpoint.

DNA three-way junctions (3WJs) are branched structures that serve as important biological intermediates and as components in DNA nanostructures. We recently derived the global structure of a fully complementary 3WJ and found that it contained unpaired bases at the branchpoint, which is consistent with previous observations of branch flexibility and branchpoint reactivity. By combining high-resolution single-molecule Förster resonance energy transfer, molecular modeling, time-resolved ensemble fluorescence spectroscopy, and the first (19)F nuclear magnetic resonance observations of fully complementary 3WJs, we now show that the 3WJ structure can adopt multiple distinct conformations depending upon the sequence at the branchpoint. A 3WJ with a GC-rich branchpoint adopts an open conformation with unpaired bases at the branch and at least one additional conformation with an increased number of base interactions at the branchpoint. This structural diversity has implications for branch interactions and processing in vivo and for technological applications

DNA hairpin dynamics under molecular crowding conditions

We are interested in the effects of the cellular environment on the behaviour of DNA. We have studied diffusing and immobilised DNA hairpins, which are excellent model systems for studying DNA hybridization, under molecular crowding conditions. We used two complementary single-molecule fluorescence methods to probe the structure and dynamics of the hairpins. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concentrations of crowder [10% w/w polyethylene glycol (PEG)]. We interpret the enhanced base pairing in terms of excluded volume effects and increased ion activity. The demonstration of large changes in the base-pairing thermodynamics and kinetics for a simple DNA molecule under relatively dilute crowding conditions suggests that crowding may be even more important for controlling cellular processes than first thought

Branchpoint Structure of DNA Threeway Junctions

Branched structures of nucleic acids are widely observed in nature as intermediates during DNA repair, recombination and replication processes, as well as being a component of vital structures for protein synthesis. Using highresolution single-molecule Fo╮ster resonance energy transfer (SM-FRET), we showed recently that a DNA three way junction was not fully paired at the branchpoint, in spite of being fully complementary.(1) To investigate whether this expanded branchpoint was a general effect, or due to the specific sequence used, we have also studied a three-way junction with a GC-rich branchpoint. We report the results of SM-FRET and ensemble NMR experiments on this new junction, together with initial investigations of the branchpoint reactivity. 1. Sabir, Tara et al. "Branchpoint Expansion in a Fully Complementary ThreeWay DNA Junction." J. Am. Chem. Soc. (2012) 134: 6280 628