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A single molecule visualization of DNA diffusion and partitioning in model porous materials

Dmytro Nykypanchuk, University of Massachusetts Amherst


We developed an experimental approach that enables molecule visualizations of macromolecular diffusion and partitioning within well-defined pores. By colloidal templating, two-dimensional arrays of open, submicron cavities interconnected by small holes were created in dense polyacrylamide gels. Cavity size of the arrays varied from 600 to 1400 nm, with the corresponding holes about 4–5 times smaller. DNA molecules of sizes from 2.69 to 48.5 kbp were inserted into the cavity arrays and monitored by fluorescent microscopy. In video sequences, individual chain positions identified as the chains diffused under Brownian motion over a period of seconds to tens of minutes. For larger chains, dynamic configurations were resolved during the motion. Over full range of molecular and pore sizes, we found that chain dynamics could be understood through the entropic barriers transport mechanism. At high confinement (large molecules in small cavities), this mechanism produces unexpected trends, for example, independence of diffusion coefficient on molecular size or faster diffusion of molecules in smaller pores. These trends reflect segmental excluded volume. Complicated dynamics akin to motion of an inchworm characterized the largest DNA chains, those with radius of gyration larger than the cavity radius. Diffusion of linear and circular DNA molecules was compared for different molecular sizes, and the resulting differences in diffusion coefficient explained by differences in diffusion mechanism; linear molecules translocate through holes by forming loops, while linear chains predominantly translocate by threading one chain end. A similar colloidal templating approach was also employed to create isolated cavity pair interconnected by a small hole. When templated by bidisperse colloid, the two cavities have unequal diameters. A DNA chain trapped inside such pair partitions unevenly, preferring the larger cavity, which afford greater configurational freedom. This sort of partitioning underlies many separation technologies but had not been visualized previously. The partition coefficient between cavities was measured visually for many combinations of cavity and molecular sizes, trends in this coefficient were then compared to existing theories for polymer partitioning. Good agreement over a two orders-of-magnitude variation of partition coefficient was obtained when effect of excluded volume on confinement free energy was introduced in a mean-field manner.

Subject Area


Recommended Citation

Nykypanchuk, Dmytro, "A single molecule visualization of DNA diffusion and partitioning in model porous materials" (2005). Doctoral Dissertations Available from Proquest. AAI3163693.