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

Abstract

Abstract:The comparison of Fö rster 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.

Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules

“…Cyanine dyes were found to stack frequently on the end of the DNA helix [62][63][64] and also rhodamines, terminally attached to a DNA duplex, show a dynamic equilibrium between various structural states. [61,65] Vaiana et al were among the first to perform MD studies on fluorophore/quencher pairs that were known to show efficient PET-quenching upon molecular contact. [12] They developed molecular force field parameters for the oxazine dye MR121 and the rhodamine dye R6G.…”

Silicon Photon-Counting Avalanche Diodes for Single-Molecule Fluorescence Spectroscopy

“…Although a rich and fascinating topic, its discussion would distract us from our main topic. We refer the interested reader to a recent introductory discussion in the context of detector development [14] as well as specialized and review articles [29][30][31][32][33][34][35][36][37][38][39][40][41]. We will briefly return to smFRET in Section VII, when discussing experimental results with SPAD arrays.…”

Fluorescent Probes and Delivery Methods for Single‐Molecule Experiments

“…This observation fits nicely to recent studies on the conformations of rhodamine 6G terminally attached to double-stranded DNA. [57] Additionally, we found that the rates of fluorescence fluctuations of donor emission observed after acceptor bleaching depend on the donor-acceptor distance. The duration of the probe in the fluorescent state increased with donor-acceptor separations and was at its longest on constructs where no acceptor was present.…”

“…The observed fluctuations are then explained by structural fluctuations (bubble formation) in the DNA. [57] Our current interpretation is that a cationic guanosine radical is produced after ET occurs between ATTO 520 and the guanosine residue (DG 0 = 0.07 eV). This radical cation is then reduced by Cy5 through the p-stack of the DNA (DG 0 = À0.71 eV).…”