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Title:Temperature and sequence dependence of DNA flexibility
Author(s):Kayikcioglu, Gurcan Tunc
Advisor(s):Ha, Taekjip
Department / Program:School of Molecular & Cell Biology
Discipline:Biophysics & Computational Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Deoxyribonucleic Acid (DNA)
single molecule
Abstract:DNA fragments shorter than the persistence length display about five orders of magnitude higher flexibility than predictions of the semi-flexible polymer models based on its persistence length. Via single molecule cyclization assays, flexibility of different constructs at various conditions can be quantitatively compared. Briefly, short double stranded DNA oligos (dsDNA) of interest with single stranded overhangs are sparsely immobilized on a slide surface. Linear and circular constructs are distinguished by the extent of fluorescence resonance energy transfer between two fluorophores covalently attached at the two termini. This observation is repeated at regular time intervals after introduction of a high salt buffer that promotes looping. Rate constants can be deduced from the relaxation towards the new equilibrium state in this new condition. We performed single molecule cyclization experiments at different temperatures to investigate the affect of temperature on the looping kinetics. Our results suggest that both looping and unlooping rates increase dramatically as temperature is increased. We then further investigated whether this effect was due to a change of the flexibility of DNA or other temperature dependent factors by measuring the bimolecular linear dimerization rate under similar conditions. Annealing and melting rates were also highly temperature dependent. In that regard, we quantified the j-factors (a.k.a. Jacobson-Stockmayer factors), which measures the effective concentration of one end of a polymer at the location of the opposite end. Our results indicated that j-factors were poorly correlated with temperature. Next, we developed a mathematical approach to characterize a generic experiment designed to selectively enrich flexible molecules out of a dsDNA pool with different j-factors. We used this model to predict the expected outcomes for certain specific cases based on initial properties of libraries. We then tried to experimentally characterize a single molecule enrichment protocol of flexible molecules by their loop formation tendencies that had been proposed in our laboratory. Using a DNA library containing 40 000 constructs with different base sequences of the same length, we enriched rapidly looping molecules by digestion of the unlooped molecules via exonuclease treatment. We first verified that our modus operandi indeed acts as a selective filter favoring more flexible molecules by comparing the looping rates of the remaining surface bound molecules with that of the initial state. Afterwards, we performed such a selection assay consecutively for multiple rounds by using the output of one selection as input for the next round. We performed single molecule cyclization assays on those input, intermediate and output libraries and observed an unexpected depletion of high j-factor species during this process. We also confirmed that there is not a PCR bias towards more or less flexible molecules. Our looping rate measurements hence revealed that our technique involving multiple PCR modifications rendered the library stiffer, which we attribute to undesirable by-products that arise as a result of our protocol.
Issue Date:2015-12-04
Rights Information:Copyright 2015 Gurcan Kayikcioglu
Date Available in IDEALS:2016-03-02
Date Deposited:2015-12

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