Orientational and dynamical heterogeneity of rhodamine 6G terminally attached to a DNA helix revealed by NMR and single-molecule fluorescence spectroscopy
2007 | journal article; research paper. A publication with affiliation to the University of Göttingen.
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Orientational and dynamical heterogeneity of rhodamine 6G terminally attached to a DNA helix revealed by NMR and single-molecule fluorescence spectroscopy
Neubauer, H.; Gaiko, N.; Berger, S.; Schaffer, J.; Eggeling, C. ; Tuma, J. & Verdier, L. et al. (2007)
Journal of the American Chemical Society, 129(42) pp. 12746-12755. DOI: https://doi.org/10.1021/ja0722574
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Details
- Authors
- Neubauer, Heike; Gaiko, Natalia; Berger, Sylvia; Schaffer, Joerg; Eggeling, Christian ; Tuma, Jennifer; Verdier, Laurent; Seidel, Claus A. M.; Griesinger, Christian ; Volkmer, Andreas
- Abstract
- The comparison of Forster resonance energy transfer (FRET) efficiencies between two fluorophores covalently attached to a single protein or DNA molecule is an elegant approach for deducing information about their structural and dynamical heterogeneity. For a more detailed structural interpretation of single-molecule FRET assays, information about the positions as well as the dynamics of the dye labels attached to the biomolecule is important. In this work, Rhodamine 6G (2-[3'-(ethylamino)-6'-(ethylimino)2',7'-dimethyl-6'H-xanthen-9'-yl]-benzoic acid) bound to the 5'-end of a 20 base pair long DNA duplex is investigated by both single-molecule multiparameter fluorescence detection (MFD) experiments and NMR spectroscopy. Rhodamine 6G is commonly employed in nucleic acid research as a FRET dye. MFD experiments directly reveal the equilibrium of the dye bound to DNA between three heterogeneous environments, which are characterized by distinct fluorescence lifetime and intensity distributions as a result of different guanine-dye excited-state electron transfer interactions. Sub-ensemble fluorescence autocorrelation analysis shows the highly dynamic character of the dye-DNA interactions ranging from nano- to milliseconds and species-specific triplet relaxation times. Two-dimensional NMR spectroscopy corroborates this information by the determination of the detailed geometric structures of the dye-nucleobase complex and their assignment to each population observed in the single-molecule fluorescence experiments. From both methods, a consistent and detailed molecular description of the structural and dynamical heterogeneity is obtained.
- Issue Date
- 2007
- Publisher
- Amer Chemical Soc
- Journal
- Journal of the American Chemical Society
- ISSN
- 0002-7863