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Molecules 2014, 19(9), 13735-13754; https://doi.org/10.3390/molecules190913735

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

1
B CUBE, Center for Molecular Bioengineering, Technische Universität Dresden, Dresden 01307, Germany
2
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)
Full-Text   |   PDF [1621 KB, uploaded 3 September 2014]   |  

Abstract

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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