Orientational and dynamical heterogeneity of rhodamine 6G terminally attached to a DNA helix revealed by NMR and single-molecule fluorescence spectroscopy.
The comparison of Förster 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.
Neubauer
Heike
H
Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, 37077, Göttingen, Germany.
Gaiko
Natalia
N
Berger
Sylvia
S
Schaffer
Jörg
J
Eggeling
Christian
C
Tuma
Jennifer
J
Verdier
Laurent
L
Seidel
Claus A M
CA
Griesinger
Christian
C
Volkmer
Andreas
A
D004247:DNA / Q000737:chemistry*; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008968:Molecular Conformation; D036103:Nanotechnology; D009690:Nucleic Acid Conformation; D017785:Photons; D017433:Protein Structure, Secondary; D012235:Rhodamines / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013237:Stereoisomerism; D013997:Time Factors
D005456:Fluorescent Dyes; D012235:Rhodamines; C026188:rhodamine 6G; D004247:DNA