Using single molecule polarization-sweep spectroscopy to monitor site-specific fluctuations of internal (iCy3)2 dimer-labeled DNA constructs at ss-ds DNA junctions.

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. In this work, we monitored spectroscopic signals from single-molecules of DNA fork constructs labeled with two cyanine optical probes [(iCy3)2], which were rigidly inserted into the sugar-phosphate backbones at opposite positions of complementary single-strands. We thus studied the local conformational fluctuations of the DNA sugar-phosphate backbones at specific (iCy3)2 dimer-labeled positions relative to a single-stranded (ss) double-stranded (ds) DNA junction. These experiments are based on a newly-developed single-molecule spectroscopic method that uses a linearly polarized continuous wave laser in which the polarization direction is continuously rotated at radio frequencies. The emitted fluorescence from the single-molecule sample contains information about conformational changes of the exciton-coupled (iCy3)2 dimer probe, which monitor conformational fluctuations of the vicinal DNA sugar-phosphate backbones. Our results indicate that the local conformation of the DNA sugar-phosphate backbones at positions near ss-dsDNA junctions adopts four topologically-relevant macrostates. The probability that a particular macrostate is occupied depends on backbone position. We apply a kinetic network approach to interpret our observations of DNA backbone fluctuations at and near ss-dsDNA junctions, and we propose conformational assignments based on exciton-coupling models for the macrostates that we observe. This polarization-sweep single-molecule fluorescence method and analysis can be applied to study protein binding and macromolecular function at and near ss-dsDNA junctions, thus providing site-specific information about the free energy landscapes at various functionally relevant positions within such macromolecular complexes.