Versatile Analysis of DNA–Biomolecule Interactions in Solution by Hydrodynamic Separation and Single Molecule Detection

DNA can interact with a wide array of molecules with a range of binding affinities, stoichiometry, and size-scales. We present a sensitive, quantitative, and versatile platform for sensing and evaluating these diverse DNA–biomolecule interactions and DNA conformational changes in free solution. Single molecule free solution hydrodynamic separation utilizes differences in hydrodynamic mobility to separate bound DNA–biomolecule complexes from unbound DNA and determine the associated size change that results from binding. Single molecule detection enables highly quantitative analysis of the fraction of DNA in the bound and unbound state to characterize binding behavior including affinity, stoichiometry, and cooperativity. A stacked injection scheme increases throughput to enable practical analysis of DNA–biomolecule interactions using only picoliters of sample per measurement. To demonstrate analysis of DNA–protein interactions on a local scale, we investigate binding of the E. coli single stranded binding protein to two DNA oligos both individually and in direct competition. We show that stoichiometry and cooperativity is a function of DNA length and verify these differences in binding characteristics through direct competition. To demonstrate analysis of DNA–small molecule interactions and global conformational changes, we also assess DNA condensation with the polyamine spermidine. We use hydrodynamic mobility to evaluate the size of spermidine-condensed DNA and single molecule burst analysis to evaluate DNA packing within the condensed globules relative to free-coiled DNA. This platform thus presents a versatile tool capable of quantitative and sensitive evaluation of diverse biomolecular interactions, complex properties, and binding characteristics.