Presenter: Anabel Chang − Biochemistry
Faculty Mentor(s): Andrew Marcus, Jack Maurer
(In-Person) Poster Presentation
Local fluctuations of the sugar-phosphate backbones of DNA (a form of DNA ‘breathing’) play key roles in protein-DNA assembly and enzymatic function. By monitoring spectroscopic signals from single-molecules of DNA constructs labeled with optical probes rigidly inserted within the sugar- phosphate backbones at opposite positions within complementary single-strands, it is possible to study local conformational fluctuations within DNA at specific sites. Here we present an experimental single-molecule spectroscopic method, to monitor the local fluctuations of Cy3-labeled DNA constructs at varying positions near a single-stranded (ss)-double-stranded (ds)DNA junction. The method combines single-molecule total internal reflection fluorescence (TIRF) microscopy with polarized, phase-modulated optical excitation to detect linear optical signals. We use a linearly polarized continuous wave (cw) laser beam to excite the single-molecule sample, so that the emitted fluorescence contains information about the relative conformational changes of an exciton-coupled Cy3 dimer probe that labels the DNA sugar-phosphate backbones. Our results indicate that the local conformation of DNA at positions near ss-dsDNA junctions adopts four topologically-relevant macrostates. We apply a kinetic network model approach to interpret our observations of DNA breathing fluctuations at and near the ss-dsDNA junction.