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Publication: Different Fluorophore Labeling Strategies and Designs Affect Millisecond Kinetics of DNA Hairpins

Molecules (published 3 Sep 2014)

Changes in molecular conformations are one of the major driving forces of complex biological processes. Many studies based on single-molecule techniques have shed light on conformational dynamics and contributed to a better understanding of living matter. In particular, single-molecule FRET experiments have revealed unprecedented information at various time scales varying from milliseconds to seconds. The choice and the attachment of fluorophores is a pivotal requirement for single-molecule FRET experiments. One particularly well-studied millisecond conformational change is the opening and closing of DNA hairpin structures. In this study, we addressed the influence of base- and terminal-labeled fluorophores as well as the fluorophore DNA interactions on the extracted kinetic information of the DNA hairpin. Gibbs free energies varied from deltaG0 = -3.6 kJ/mol to deltaG0 = -0.2 kJ/mol for the identical DNA hairpin modifying only the labeling scheme and design of the DNA sample. In general, the base-labeled DNA hairpin is significantly destabilized compared to the terminal-labeled DNA hairpin and fluorophore DNA interactions additionally stabilize the closed state of the DNA hairpin. Careful controls and variations of fluorophore attachment chemistry are essential for a mostly undisturbed measurement of the underlying energy landscape of biomolecules.

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A. Hartmann, G. Krainer, M. Schlierf: Different Fluorophore Labeling Strategies and Designs Affect Millisecond Kinetics of DNA Hairpins. Molecules 19(9), 13735-13754 (2014)

DOI: 10.3390/molecules190913735

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