Single-Molecule Studies of DNA Self-Diffusion in Entangled Blends of Linear and Circular DNA

Abstract

Using fluorescence microscopy and single-particle tracking we have measured the self-diffusion coefficients for single DNA molecules in varying blends of entangled linear and relaxed circular (ring) DNA. We have determined the dependence of self-diffusion of both species on the fraction of linear vs. ring species in the blend, the overall solution concentration, and the molecular length. The diffusion of relaxed circular DNA (DR) was found to depend strongly on the fraction of linear DNA (ϕL) in the solution with DR undergoing a dramatic decline as ϕL was incrementally increased to ∼ 0.3, followed by a much slower decline as ϕL approached 1. This phenomenon can be attributed in part to the tendency of linear polymers to thread their circular counterparts, prohibiting diffusion via reptation and forcing ring DNA to diffuse via the slow mechanism of constraint release. For linear DNA, a surprising non-monotonic dependence of self-diffusion (DL) on ϕL was observed with DL reaching a minimum at ϕL ∼ 0.6. This behavior was not anticipated by previous theoretical models, but the results are in good agreement with predictions from new simulations using a minimal constraint release model that assumes our exact experimental conditions. Molecular entanglements are essential for observing these dramatic effects on diffusion within DNA blends, as we have found that below the critical threshold for molecular entanglement there exists only a weak correlation between self-diffusion and blend composition.