title: Different fluorophore labeling strategies and designs affect millisecond kinetics of DNA hairpins.
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.
authors.affiliation: B CUBE, Center for Molecular Bioengineering, Technische Universität Dresden, Dresden 01307, Germany. hartmann@bcube-dresden.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Hartmann
authors.affiliation: B CUBE, Center for Molecular Bioengineering, Technische Universität Dresden, Dresden 01307, Germany. krainer@bcube-dresden.de.
authors.forename: Georg
authors.initials: G
authors.lastname: Krainer
authors.affiliation: B CUBE, Center for Molecular Bioengineering, Technische Universität Dresden, Dresden 01307, Germany. schlierf@bcube-dresden.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Schlierf
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D009690:Nucleic Acid Conformation
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA