Local fluctuations of the sugar-phosphate backbones and bases of DNA (a form of DNA ‘breathing’) play a central role in the assembly of protein-DNA complexes. We present a single-molecule fluorescence method to sensitively measure the local conformational fluctuations of exciton-coupled cyanine [(iCy3)2] dimer-labeled DNA fork constructs in which the dimer probes are placed at varying positions relative to the DNA fork junction. These systems exhibit spectroscopic signals that are sensitive to the local conformations adopted by the sugar-phosphate backbones and bases immediately surrounding the dimer probe label positions. The (iCy3)2 dimer has one symmetric (+) and one anti-symmetric (–) exciton with respective transition dipole moments oriented perpendicular to one another. We excite single molecule samples using a continuous-wave, linearly polarized laser with its polarization direction rotated at a frequency of 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and anti-symmetric excitons of the (iCy3)2 dimer probe. Phase-sensitive detection of the signal at the photon-counting level provides information about the distribution of local conformations and conformational dynamics. We analyze our data using a kinetic network model, which we use to parametrize the free energy surface of the system. In addition to observing DNA breathing at and near ss-dsDNA junctions, the approach can be used to study the effects of proteins that bind and function at these sites.
Base stacking is fundamentally important to the stability of double-stranded DNA. However, few experiments can directly probe the local conformations and conformational fluctuations of the DNA bases. Here we report a new spectroscopic approach to study the local conformations of DNA bases using the UV-absorbing fluorescent guanine analogue, 6-methyl isoxanthopterin (6-MI), which can be used as a site-specific probe to label DNA. In these experiments, we apply a two-photon excitation (2PE) approach to two-dimensional fluorescence spectroscopy (2DFS), which is a fluorescence-detected nonlinear Fourier transform spectroscopy. In 2DFS, a repeating sequence of four collinear laser pulses (with center wavelength ~ 675 nm and relative phases swept at radio frequencies) is used to excite the lowest energy electronic-vibrational (vibronic) transitions of 6-MI (with center wavelength ~ 340 nm). The ensuing low flux fluorescence is phase-synchronously detected at the level of individual photons and as a function of inter-pulse delay. The 2PE transition pathways that give rise to electronically excited state populations include optical coherences between electronic ground and excited states and non-resonant (one-photon-excited) virtual states. Our results indicate that 2PE-2DFS experiments can provide information about the electronic-vibrational spectrum of the 6-MI monomer, in addition to the conformation-dependent exciton coupling between adjacent 6-MI monomers within a (6-MI)2 dimer. In principle, this approach can be used to determine the local base-stacking conformations of (6-MI)2 dimer-substituted DNA constructs.
Picosecond fluorescence depolarization measurements of electronic excitation transfer (EET) are used to probe the structures and dynamics of polymer blends. Fluorescent chromophores are covalently incorporated into one of the polymers in the blend, and the rate of fluorescence anisotropy decay is measured using time correlated single photon counting. Analysis of the fluorescence anisotropy decays yields the time-dependence of the EET. EET is a very sensitive probe of interchromophore distance; therefore it can be used to examine the single chain structure and the spatial distribution of chains in the blend. At temperatures above the phase separation temperature, macroscopic phase separation eventually occurs. However, prior to macroscopic phase separation, the structure and dynamics of the onset of phase separation is not understood. Using EET, we are able to observe the aggregation of as few as two polymer chains at the onset of phase separation. These small aggregates are referred to an nanodomains. By lowering the sample temperature below the blend's glass transition temperature, the nanodomains can be trapped in the solid material. The number of chains in the nanodomains and the growth of the nanodomains are determined by analyzing the EET data with a detailed theory that relates the spatial distribution of chromophores to the EET observables. Varying the rate of heating allows us to study the kinetics of formation of these nanoscopic heterogeneities, and to observe the phase separation process at the molecular level, from its onset through macroscopic phase separation.
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