| DNA cyclization is potentially the most powerful approach for systematic quantitation of sequence-dependent DNA bending and flexibility. For decades its application had been limited by the labor required to prepare DNA constructs and measure their cyclization rates, and by the time consumed by data interpretation using Monte Carlo simulation. It was not uncommon for the analysis of a single DNA sequence to take more than a year. This thesis reports two advances: a high throughput approach for preparing DNA constructs and measuring their cyclization rates and a completely new theory for cyclization modeling based on statistical mechanics. Using these tools, it has been possible to quantitate the curvature and flexibility of many sequences in a single week. The apparent flexibility of a variety of DNA homopolymers and dinucleotide and trinucleotide repeats has been measured at different temperatures and analyzed based on a model of isotropic bending flexibility, revealing much smaller sequence-dependent bending and flexibility than previously thought. However, our data suggests that there might be highly asymmetric flexibility favoring the bending towards the minor groove at AT-rich region. An enhanced torsional flexibility of two kinds of nicked DNA has also been discovered. An in vitro selection for the sequence motifs for DNA curvature and flexibility is presented. |
