Studies of DNA breathing in exciton-coupled (iCy3) 2 dimer-labeled DNA constructs by polarization-sweep single-molecule fluorescence (PS-SMF) microscopy.

Autor: Maurer J; Center for Optical, Molecular and Quantum Science, Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon 97403.; Institute of Molecular Biology, Department of Chemistry and Biochemistry, and University of Oregon, Eugene, Oregon 97403., Albrecht CS; Center for Optical, Molecular and Quantum Science, Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon 97403.; Institute of Molecular Biology, Department of Chemistry and Biochemistry, and University of Oregon, Eugene, Oregon 97403., Herbert P; Center for Optical, Molecular and Quantum Science, Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon 97403.; Institute of Molecular Biology, Department of Chemistry and Biochemistry, and University of Oregon, Eugene, Oregon 97403., Heussman D; Center for Optical, Molecular and Quantum Science, Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon 97403.; Institute of Molecular Biology, Department of Chemistry and Biochemistry, and University of Oregon, Eugene, Oregon 97403., Chang A; Institute of Molecular Biology, Department of Chemistry and Biochemistry, and University of Oregon, Eugene, Oregon 97403., von Hippel PH; Institute of Molecular Biology, Department of Chemistry and Biochemistry, and University of Oregon, Eugene, Oregon 97403., Marcus AH; Center for Optical, Molecular and Quantum Science, Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon 97403.; Institute of Molecular Biology, Department of Chemistry and Biochemistry, and University of Oregon, Eugene, Oregon 97403.
Jazyk: angličtina
Zdroj: Proceedings of SPIE--the International Society for Optical Engineering [Proc SPIE Int Soc Opt Eng] 2024 Jan-Feb; Vol. 12863. Date of Electronic Publication: 2024 Mar 13.
DOI: 10.1117/12.3001962
Abstrakt: 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.
Databáze: MEDLINE
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