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Limited tools exist that are capable of monitoring nucleic acid conformations, fluctuations, and distributions in free solution environments. Single molecule free solution hydrodynamic separation enables the unique ability to quantitatively analyze nucleic acid biophysics in free solution. Single molecule fluorescent burst data and separation chromatograms can give layered insight into global DNA conformation, binding interactions, and molecular distributions. First, we show that global conformation of individual DNA molecules can be directly visualized by examining single molecule fluorescent burst shapes and that DNA exists in a dynamic equilibrium of fluctuating conformations as it is driven by Poiseuille flow through micron-sized channels. We then show that this dynamic equilibrium of DNA conformations is reflected as shifts in hydrodynamic mobility that can be perturbed using salt and ionic strength to affect packing density. Next, we demonstrate that these shifts in hydrodynamic mobility can be used to investigate hybridization thermodynamics and binding interactions. We distinguish and classify multiple interactions within a single sample, and demonstrate quantification amidst large concentration differences for the detection of rare species. Finally, we demonstrate that these differences can resolve perfect complement, 2 bp mismatched, and 3 bp mismatched sequences. Such a system can be used to garner diverse information about DNA conformation and structure, and potentially be extended to other molecules and mixed-species interactions, such as between nucleic acids and proteins or synthetic polymers.

Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution