Intermolecular static and dynamic fluorescence quenching constants
of eight coumarin derivatives by nucleobase
derivatives have been determined in aqueous media. One common
sequence of the quenching efficiency has
been found for the nucleobases. The feasibility of a photoinduced
electron transfer reaction for the nucleobase-specific quenching of fluorescent dyes is investigated by the
calculation of the standard free energy changes
with the Rehm−Weller equation. A complete set of one-electron
redox potential data for the nucleobases
are determined electrochemically in aprotic solvents for the first
time, which are compared with values obtained
by various other methods. Depending on the redox properties of the
fluorescent dyes, the sequences of the
quenching efficiencies can be rationalized by the orders of
electrochemical oxidation potentials (vs NHE) of
nucleosides (dG (+1.47 V) < dA < dC ≈ dT < U (≥ +2.39 V)) and
reduction potentials (dG (< −2.76 V)
< dA < dC < dT < U (−2.07 V)). The correlation between the
intermolecular dynamic quenching constants
and the standard free energy of photoinduced electron transfer
according to the classical Marcus equation
indicates that photoinduced electron transfer is the rate-limiting
step. However, an additional, water-specific
gain of free energy between −0.5 and −0.9 eV shows that additional
effects, like a coupled proton transfer
and a hydrophobic effect, have to be considered, too. Furthermore,
the capability of the nucleobases to form
ground state complexes with fluorescent dyes is influenced by their
redox potentials. The relevance of these
observations to current efforts for DNA sequencing with a detection by
laser-induced fluorescence and their
application to other dyes are discussed.
The photostability of fluorescent dyes is of crucial importance for the statistical accuracy of single-molecule detection (SMD) and for the image quality of scanning confocal microscopy. Concurrent results for the photostability were obtained by two different experimental techniques. First, the photostabilities of several coumarin and rhodamine derivatives in aqueous solution were obtained by monitoring the steady-state fluorescence decay in a quartz cell. Furthermore, an epi-illuminated microscope, continuous wave (CW) excitation at 514.5 nm, and fluorescence correlation spectroscopy (FCS) with a newly developed theory were used to study the photobleaching characteristics of rhodamines under conditions used for SMD. Depending on the rhodamine structure, the probability of photobleaching, p(b), is in the order of 10(-)(6)-10(-)(7) for irradiances below 10(3) W/cm(2). However, a considerable increase of p(b) for irradiances above this level was observed which can only be described by photobleaching reactions from higher excited states (two-step photolysis). In view of these observations, the probability of photobleaching, p(b), as well as a closed expression of its dependence on the CW excitation irradiance considering a five-level molecular electronic state model with the possibility of photobleaching from higher excited electronic states, is derived. From this model, optimal conditions for SMD with respect to the number of emitted fluorescence photons and to the signal-to-background ratio are discussed, taking into account both saturation and photobleaching. The additional photobleaching due to two-step photolysis limits the applicable irradiance.
We present a comprehensive toolkit for Förster resonance energy transfer (FRET)-restrained modeling of biomolecules and their complexes for quantitative applications in structural biology. A dramatic improvement in the precision of FRET-derived structures is achieved by explicitly considering spatial distributions of dye positions, which greatly reduces uncertainties due to flexible dye linkers. The precision and confidence levels of the models are calculated by rigorous error estimation. The accuracy of this approach is demonstrated by docking a DNA primer-template to HIV-1 reverse transcriptase. The derived model agrees with the known X-ray structure with an r.m.s. deviation of 0.5 Å. Furthermore, we introduce FRET-guided 'screening' of a large structural ensemble created by molecular dynamics simulations. We used this hybrid approach to determine the formerly unknown configuration of the flexible single-strand template overhang.
Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.
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