Single Molecule Flourescence Methods to Monitor Site-Specific Fluctuations of Cy3 Monomer and Dimer Labeled DNA Constructs within Macromolecular Machines

Abstract

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 that are either rigidly inserted into the sugar-phosphate backbones or attached using flexible linkers, it is possible to study local conformational fluctuations within DNA at specific sites, and the dependence of these fluctuations on base sequence and secondary structure. Here we present two experimental single molecule spectroscopic methods, recently developed in our laboratory, to monitor the local fluctuations of Cy3-labeled DNA constructs. Both methods combine single-molecule total internal reflection fluorescence (TIRF) microscopy with polarized, phase-modulated optical excitation to detect, alternatively, linear and nonlinear optical signals. In Method 1 we use a linearly polarized continuous wave (cw) beam to excite the sample, so that the emitted fluorescence contains information about the rotational motion of a Cy3 monomer labeled DNA construct, or alternatively, the relative conformational changes of a Cy3 dimer inserted into the DNA sugar-phosphate backbones. In Method 2, we use sequences of broadband optical pulses to excite the sample, so that the ensuing fluorescence contains information about the linear absorbance spectrum, and alternatively, the two-dimensional fluorescence spectrum (2DFS). The latter experiments can be used to monitor the details of local conformational changes of a Cy3 dimer labeled DNA construct. Together, the two methods can be used to study biomolecular dynamics with varying levels of time resolution and structural information. For example, Method 1 can be applied to study sub-millisecond equilibrium fluctuations of Cy3 labeled DNA constructs, while Method 2 is well suited to monitor conformational transitions of a Cy3 labeled DNA construct during a biochemical reaction.