Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

33-POS Board 17 SINGLE-MOLECULE FRET DETECTS INTERMEDIATES AND FAST DYNAMICS OF DNA HOLLIDAY JUNCTIONS Alessandro Valeri 1 ; Suren Felekyan 1 ; Stanislaw Kalinin 1 ; Markus Richert 1 ; Stefan Marawske 1 ; Enno Schweinberger 1 ; Oleg Opanasyuk 1 ; Ivan Rech 2 ; Angelo Gulinatti 2 ; Ralf Kuehnemuth 1 ; Claus A Seidel 1 ; 1 Heinrich-Heine-Universität, Lehrstuhl für Molekulare Physikalische Chemie, Düsseldorf, Germany 2 Politecnico di Milano, Dipartimento di Elettronica, Informazione e Bioingegneria, Milano, Italy Holliday junctions, i.e. DNA four-way junctions, play essential roles in DNA replication, repair, and recombination. In addition they are key building blocks in DNA nanotechnology. Previous studies have shown that in the presence of Mg2+ ions Holliday junctions switch between two stacking conformations with rates strongly dependent on the ion concentration. The molecular mechanism for this is unclear. We performed FRET studies using confocal single-molecule detection (SMD) with diffusing molecules or evanescent excitation of immobilized single molecules as well as ensemble fluorescence lifetime studies. We used the full dynamic time range of fluorescence from ns to s to capture the complex Mg-dependent kinetics. The minimal model that describes the observed kinetics consists of four states, with only two distinct FRET levels. All methods and analysis techniques consistently reveal continuous interconversion also in absence of Mg2+ whereas addition of Mg2+ “locks” the junctions in the respective states. Our results shape a new view on structural properties of four-way DNA junctions, identifying a dynamic equilibrium instead of an accumulation of a single open structure at low Mg2+. DNA four-way junctions do not adopt static conformations but the exchange of Mg2+ ions determines their effective dynamic and structural features in their distinct environments.

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