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Molecules 2014, 19(9), 13735-13754;

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

B CUBE, Center for Molecular Bioengineering, Technische Universität Dresden, Dresden 01307, Germany
Molecular Biophysics, University of Kaiserslautern, Kaiserslautern 67663, Germany
These authors contributed equally to this work.
Author to whom correspondence should be addressed.
Received: 18 July 2014 / Revised: 21 August 2014 / Accepted: 26 August 2014 / Published: 3 September 2014
(This article belongs to the Special Issue Single Molecule Techniques)
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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 ∆G0 = −3.6 kJ/mol to ∆G0 = −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. View Full-Text
Keywords: DNA hairpin; labeling strategy; single-molecule FRET; hairpin design DNA hairpin; labeling strategy; single-molecule FRET; hairpin design

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

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