Orientational and Dynamical Heterogeneity of Rhodamine 6G Terminally Attached to a DNA Helix Revealed by NMR and Single-Molecule Fluorescence Spectroscopy

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• 作者：Heike Neubauer ; Natalia Gaiko ; Sylvia Berger ; J&ouml ; rg Schaffer ; Christian Eggeling ; Jennifer Tuma ; Laurent Verdier ; Claus A. M. Seidel ; Christian Griesinger ; Andreas Volkmer
• 刊名：Journal of the American Chemical Society
• 出版年：2007
• 出版时间：October 24, 2007
• 年：2007
• 卷：129
• 期：42
• 页码：12746 - 12755
• 全文大小：402K
• 年卷期：v.129,no.42(October 24, 2007)
• ISSN：1520-5126

文摘

The comparison of Förster resonance energy transfer (FRET) efficiencies between twofluorophores covalently attached to a single protein or DNA molecule is an elegant approach for deducinginformation about their structural and dynamical heterogeneity. For a more detailed structural interpretationof single-molecule FRET assays, information about the positions as well as the dynamics of the dye labelsattached 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 isinvestigated by both single-molecule multiparameter fluorescence detection (MFD) experiments and NMRspectroscopy. Rhodamine 6G is commonly employed in nucleic acid research as a FRET dye. MFDexperiments directly reveal the equilibrium of the dye bound to DNA between three heterogeneousenvironments, which are characterized by distinct fluorescence lifetime and intensity distributions as a resultof 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- tomilliseconds and species-specific triplet relaxation times. Two-dimensional NMR spectroscopy corroboratesthis information by the determination of the detailed geometric structures of the dye-nucleobase complexand their assignment to each population observed in the single-molecule fluorescence experiments. Fromboth methods, a consistent and detailed molecular description of the structural and dynamical heterogeneityis obtained.