The method of equilibrium cyclization for the determination of the curvature, flexibility and helical repeats of DNA will be developed and calibrated. This innovative method is based on fluorescence detection of the reversible cyclization of DNA. The initial rate of the irreversible cyclization of DNA has been previously used to examine the bending and flexibility of DNA. The methods used to quantify the kinetics of irreversible cyclization will be used to analyze the equilibrium cyclization results. The DNAs to be studied will be duplexes that have single stranded tails that are terminated in sticky ends of complementary sequences. The DNA will be cyclized when the sticky ends form a duplex. The equilibrium between the open and cyclized forms will be monitored by the fluorescence of 2-aminopurine, 2AP. 2AP will be incorporated into the complementary region. 2AP is highly fluorescent in single stranded DNA and weakly fluorescent in duplex DNA. The equilibrium cyclization will be calibrated as a function of the end to end distance of the DNA. The end to end distance will be varied by changing the length of the duplex region. The equilibrium cyclization will be calibrated for several values of the length of the single stranded and sticky end regions. Equlibrium cyclization will be applied to DNAs containing single and triple mismatches. The equilibrium cyclization results will be obtained as a function of the phasing of the mismatched sites. The results will be analyzed using the approaches developed for kinetic cyclization to determine the flexibility, curvature and helical repeat of the mismatched sites. Equilibrium cyclization will be applied to DNAs containing dA tracts to determine the curvature, helical repeat and flexibility of dA tracts. DNAs with the dA tracts at different phases with respect to the helical repeat will be examined. The results will be analyzed using the approaches developed for kinetic cyclization to determine the flexibility, curvature and helical repeat of the mismatched sites. The effects of magnesium, temperature and netropsin on the curvature, flexibility and helical repeat of dA tracts will be examined.
