Characterizing the bending and twisting mechanics of DNA and its mismatches
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Ryan, Michael Lee
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Abstract
DNA holds enormous power over cellular processes, genetic expression, reproduction, and as a consequence life in general. While many genetic interactions are studied and manipulated, in an ideal world we would understand DNA at a physical level and build up all interactions from first principles. The more we know about the theromechanics of this semi-flexible polymer, the greater control we have over it to correct mistakes, utilize it as a tool, or fortify it from degradation. In the interest of exploring this molecule and its intrinsic properties, we target it with bending and torsional stress. In our bending studies, we design small DNA molecules capable of bending into tight loops. Measuring the rates of this cyclization and de-cyclization process gives insight on how rigid the helical backbone is for matched nucleotides and mismatched pairs. We find that matched nucleotides resist helical deformation to a much greater degree than mismatches. We take this a step further and explore bending anisotropy for all of these pairs and mispairs. It is clear in our experimental results that nucleotide pairs have a preferred bending direction and that anisotropy should be considered more relevant than the current field treats it. We also take a novel approach at twisting DNA with a horizontal variant of magnetic tweezers, which we hope to combine with fluorescence to merge two powerful DNA probing techniques. We confirm the viability of this new tweezing technique by reproducing plectonemic hat curves, which are well studied. A fluorescent study of these molecules confirms our tweezing results. In this twisting study, we also developed a new technique using CRISPR to create extremely long, torsionally constrained molecules that show preliminary success.
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2022-08-31
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Dissertation