The effect of Cytoskeletal crowding on the Mobility and Conformational Dynamics of circular and linear DNA

In order to carry out processes such as gene transcription and cell replication, DNA must diffuse through a highly crowded cellular environment. Previous studies aimed at understanding intracellular DNA transport have mainly focused on the effect of small mobile crowders. However, the cytoskeleton, composed of filamentous proteins such as semiflexible actin and rigid microtubules, has been identified as a key factor suppressing viral transfection and gene therapy. Here, we investigate the effect that cytoskeletal proteins have on the transport properties of linear and circular DNA. Specifically, we use fluorescence microscopy and custom single-molecule tracking algorithms to measure center-of-mass transport and time-varying conformational changes of single DNA molecules diffusing in in vitro composite networks of actin and microtubules. We determine the role that DNA topology as well as cytoskeletal filament rigidity (actin vs microtubules) has on DNA transport and conformational states. We specifically quantify DNA diffusion coefficients, degrees of anomalous diffusion, and conformational changes within protein networks of varying concentrations and polymerization states.