Studies of stretching and immobilization of DNA molecules and their interactions with proteins at the single-molecule level.

In this dissertation, the degrees of adsorption and stretching of DNA onto surfaces achieved by various stretching methods that use fluid flows are compared and the processes by which proteins find target sequences along these DNA molecules and either carry out cleavage or transcription are directly observed by fluorescence microscopy. DNA molecules either combed or spin-stretched onto hydrophobic surfaces stretch to a greater degree, but fewer are deposited, at pH 8.0 than at lower pH. At pH 8.0, DNA adhesion occurs primarily only at the DNA extremities, and avoids trapped regions of incompletely stretched DNA. A new technique, protein-assisted DNA immobilization (PADI), is developed to immobilize and stretch, but not overstretch, DNA molecules inside a micro/nanochannel at physiological pH. The biological activity of the immobilized DNA molecules is confirmed by digesting the DNA with restriction enzymes in the microchannel. Single molecule transcription, which has stringent requirements on the immobilized DNA with respect to surface interactions and stretched lengths, is also successfully demonstrated on DNA molecules immobilized by PADI. The one dimensional Brownian motion and transcription elongation of T7 RNA polymerase along aligned DNA molecules bound to substrates by molecular combing are directly visualized. Fluorescent antibodies are used to label T7 RNA polymerase and a shear flow is applied to convect proteins orthogonally to the DNA alignment direction, permitting observation and estimation of the protein diffusivity along the DNA at the single-molecule level. The ID diffusion coefficient varies from molecule to molecule over a large range, suggesting that the individual proteins have distinct diffusivities. From the measured dependence of the rate of transcription on concentration of nucleotide triphosphate, the combed DNA molecules capable of interacting with proteins are inferred to be under an average tension of 25 pN.PhDApplied SciencesBiological SciencesBiophysicsChemical engineeringMolecular physicsPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126739/2/3276206.pd