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

Jack Maurer; Claire S. Albrecht; Patrick Herbert; Dylan Heussman; Anabel Chang; Peter H. von Hippel; Andrew H. Marcus

doi:10.1117/12.3001962

13 March 2024 Studies of DNA breathing in exciton-coupled (iCy3)₂ dimer-labeled DNA constructs by polarization-sweep single-molecule fluorescence (PS-SMF) microscopy

Jack Maurer, Claire S. Albrecht, Patrick Herbert, Dylan Heussman, Anabel Chang, Peter H. von Hippel, Andrew H. Marcus

Author Affiliations +

Proceedings Volume 12863, Quantum Effects and Measurement Techniques in Biology and Biophotonics; 128630C (2024) https://doi.org/10.1117/12.3001962
Event: SPIE BiOS, 2024, San Francisco, California, United States

Abstract

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.

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Jack Maurer, Claire S. Albrecht, Patrick Herbert, Dylan Heussman, Anabel Chang, Peter H. von Hippel, and Andrew H. Marcus "Studies of DNA breathing in exciton-coupled (iCy3)₂ dimer-labeled DNA constructs by polarization-sweep single-molecule fluorescence (PS-SMF) microscopy", Proc. SPIE 12863, Quantum Effects and Measurement Techniques in Biology and Biophotonics, 128630C (13 March 2024); https://doi.org/10.1117/12.3001962

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