Article Cite This: J. Phys. Chem. B 2018, 122, 4249−4266
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Evaluation of Blue and Far-Red Dye Pairs in Single-Molecule Förster Resonance Energy Transfer Experiments Niels Vandenberk,† Anders Barth,‡ Doortje Borrenberghs,† Johan Hofkens,† and Jelle Hendrix*,†,§
J. Phys. Chem. B 2018.122:4249-4266. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/06/18. For personal use only.
†
Laboratory for Photochemistry and Spectroscopy, Division for Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium ‡ Physical Chemistry, Department of Chemistry, Munich Center for Integrated Protein Science, Nanosystems Initiative Munich and Centre for Nanoscience, Ludwig-Maximilians-Universität München, 80539 Munich, Germany § Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre and Biomedical Research Institute, Hasselt University, Agoralaan C (BIOMED), Diepenbeek, B-3590, Belgium S Supporting Information *
ABSTRACT: Förster resonance energy transfer (FRET) is a powerful tool to probe molecular interactions, activity, analytes, forces, and structure. Single-molecule (sm)FRET additionally allows real-time quantifications of conformation and conformational dynamics. smFRET robustness critically depends on the employed dyes, yet a systematic comparison of different dye pairs is lacking. Here, we evaluated blue (Atto488 and Alexa488) and far-red (Atto647N, Alexa647, StarRed, and Atto655) dyes using confocal smFRET spectroscopy on freely diffusing double-stranded (ds)DNA molecules. Via ensemble analyses (correlation, lifetime, and anisotropy) of singlelabeled dsDNA, we find that Alexa488 and Atto647N are overall the better dyes, although the latter interacts with DNA. Via burstwise analyses of double-labeled dsDNA with interdye distances spanning the complete FRET-sensitive range (3.5−9 nm), we show that none of the dye pairs stands out: distance accuracies were generally 0.8), R0, and the standard deviation of the Gaussian linker dye distribution of 5.6 Å, the static FRET line (including linker dynamics) was 4252
DOI: 10.1021/acs.jpcb.8b00108 J. Phys. Chem. B 2018, 122, 4249−4266
Article
The Journal of Physical Chemistry B
background count rates in donor and acceptor channels after donor excitation, (ii) the donor crosstalk correction parameter (β = 0.01), (iii) the direct acceptor excitation correction parameter (α = 0.01), and (iv) the relative detection efficiency of donor and acceptor (FRET-pair specific). β and α were determined as described previously.16,19 Furthermore, a FRETpair specific R0 was used (Table 1), which was calculated using
simulated. The latter parameter was obtained by a subensemble TCSPC fit of the FRET donor decay to a Gaussian distance distribution model. The used approach generates fluorescenceweighted lifetimes, which for molecules exhibiting multiple lifetimes, will be weighted toward the long-lifetime species. Burstwise steady-state fluorescence anisotropies of the FRET donor (rD) and FRET acceptor (rA) were calculated from the respective fluorescence intensities r=
Table 1. Summary of the Förster Distance of Each FRET Pair
GF − F⊥ GF + F⊥
(15)
where G is the correction factor for different detection efficiencies in the two polarization channels, F∥ is the intensity in time gate BB∥ or RR∥, and F⊥ is the intensity in time gate BB⊥ or RR⊥. Perrin equations were calculated with r0 r= (1 + τ /θ ) (16)
donor
acceptor
R0 (Å)a
R0 (Å)b
Atto488 Atto488 Atto488 Atto488 Alexa488 Alexa488 Alexa488 Alexa488
Atto647N Alexa647 Atto655 StarRed Atto647N Alexa647 Atto655 StarRed
51.7 57.4 50.0
51.2 56.8 46.4 49.4 50.0 55.6 46.5 48.6
49.9 55.5
a
Calculated using published data. bCalculated using the quantum yield determined via FCS.
where r is the single-molecule steady-state anisotropy, r0 = 0.4 is the fundamental anisotropy, τ is the fluorescence lifetime, and θ is the rotational correlation time. Multidimensional histogramthe measurements result in intensity, lifetime, and anisotropy information for both donor and acceptor dyes. This information was combined into a single multidimensional histogram, including a FRET efficiency versus stoichiometry histogram (E−S), two FRET efficiency (eq 10) versus donor or acceptor lifetime histograms (E−τD and E−τA, respectively), and two donor and acceptor steady-state anisotropy versus lifetime histograms (rD−τD and rA−τA, respectively). For the E−τD plot, the static FRET line (eq 14) is additionally plotted, whereas a Perrin equation (eq 16) is plotted onto the r−τ plots. Photon Distribution Analysis. Static PDA was carried out to obtain the absolute interdye distance distribution from the observed shot-noise limited FRET efficiency histograms.23 Practically, for each FRET dataset, raw bursts were binned in time bins of 1 ms and a histogram was constructed and analyzed. Data were plotted in an E versus S plot to deselect bins with complex acceptor photophysics, and only bins with at least 20 and maximally 250 photons (to reduce calculation time) were used for PDA analysis. A three-state model for a Gaussian distance distribution23 was fitted to the experimental EPR histogram using a reduced-χ2-guided simplex search algorithm. The histograms of the nine different D/A spacing datasets of one FRET pair were analyzed simultaneously. The mean and width of all Gaussian distributed substates were fitted for the nine datasets. Next, the standard deviation σ of the distance distributions was globally optimized at a fraction F of the corresponding distance to increase fitting robustness, which has been shown before to be reasonable for FRET experiments with a blinking FRET acceptor.24,26 In summary, for a comparative analysis, only a few parameters needed to be optimized (R1, R2, R3, F, A1, A2, and A3). R is the FRET-averaged donor−acceptor distance ⟨RDA⟩E,26 and An is the fraction of the specific distance between D and A. A probability density function (PDF) was calculated per state using the R and σ parameters obtained from PDA that describe the underlying Gaussian distributed states. The summed PDF was scaled to a total area of unity, with each state’s PDF area scaled to the corresponding fraction of molecules. Criteria for a good fit were a low (100 kHz brightness at the laser powers typically used for smFRET (50 μW), with Atto647N the brightest (Figure 1E, right). Interestingly, the brightness of Atto655-DNA was much lower than that of the free dye (Figure 1E, right). The relative brightness (εbright, corrected for the presence of dark states, eq 6) between all dyes (except Atto655) (Figure S8C−F) was compared well to what we predicted for the used microscope (Table S1). However, the population of dark states for Alexa647 and StarRed did render these dyes on average quite dimmer than Atto647N (Figure 1E, right). Next, we quantified the fluorescence lifetimes and anisotropies of the acceptor dyes attached to DNA. Atto647N and StarRed showed similar fluorescence decays (Figure 1F) that could be well-described with a monoexponential model, resulting in a fluorescence lifetime of around 4 ns, slightly larger than the corresponding manufacturer’s values. Alexa647 exhibited a 1.1 ns lifetime, comparable to the manufacturer’s value, which did not change when attached to DNA (Figure 1F). The fluorescence decay for free Atto655, on the other hand, could only be described well with a biexponential model (85% of 1.82 ns and 15% of 0.21 ns). When attached to DNA, the fluorescence lifetime of Atto655 was reduced considerably, resulting in an increased fraction of the short lifetime component (30% of 2.27 ns and 70% of 0.34 ns in PBS). [In Figure 1F, the species-averaged lifetime is displayed and the intensity-averaged lifetime (eq 8) was 1.77 ns]. This is likely caused by quenching through oxazine-guanine photoinduced electron transfer (PET), as has been observed before.53,57,58 In line with this, the observed fluorescence lifetime of Atto655 depended on the labeling position, whereby the attachment site surrounded by the most guanine residues exhibited the most pronounced quenching (Figure S9B). All other dyes showed no change in fluorescence lifetime or position-dependent quenching when bound to DNA (Figure S9B). We then analyzed the time-resolved anisotropy decays of the labeled DNA molecules (Figure 2E). The rotational correlation time of the probes attached to DNA approximately scaled with their size, being highest for Atto655 (∼3 ns) and around 2−2.5 ns for other dyes (Figure 2F,G). The anisotropy decayed completely only for molecules labeled with Alexa647 and StarRed (Figure 2E). On the other hand, Atto647N and Atto655 did exhibit residual anisotropies (rinf), indicating long-lived interactions with the DNA molecule (Figure 2H). For Atto647N, rinf values where in the range of 0−0.03 and depended slightly on the labeling position (Figure 2H), whereas for Atto655, large rinf values up
to 0.07 were observed that changed significantly with the position on the DNA. This suggests that Atto655 and, to a lesser extent, Atto647N, tend to stick to the DNA molecule. The absence of electrostatic repulsion of these two dyes by the negatively charged DNA (Table S1) is in line with these observations. Summarized, we spectroscopically evaluated four far-red dyes covalently attached to DNA molecules under conditions similar to those used for burstwise smFRET experiments. Atto647N possessed the highest brightness and suffered the least from microsecond blinking, although a partial sticking to the labeled molecule of interest was observed. In the context of DNA, Alexa647 and StarRed each exhibited significant microsecond blinking, whereas Atto655 additionally suffered from quenched fluorescence and sticking to the DNA. A clear positive effect of Trolox (aged or fresh) was not obvious in the performed experiments for any of the tested dyes, although Trolox did seem to provide a slight photoprotection for all dyes except Atto655. All Pairs Exhibit Heterogeneously Distributed FRET Histograms. To test how the dyes behave as a donor and an acceptor in FRET experiments, we carried out a comparative analysis of all DNA(D9A22) (11 bp distance) molecules using multiparameter single-molecule spectroscopy. For this, we diluted the samples to concentrations that allowed singlemolecule observation (typically 10 pM) and recorded 1−2 h time traces per sample. After identifying single-molecule bursts in the recordings, we calculated a number of burst-averaged parameters for every single-molecule event: the intensity-based corrected (absolute) FRET efficiency E (eq 10), the corrected stoichiometry parameter S (eq 11), the fluorescence lifetime of the FRET donor, τD, and acceptor, τA, and the steady-state anisotropy of the FRET donor, rD, and acceptor, rA. We filtered the data by selecting all bursts that contained fluorescence from both FRET donor and acceptor and discarded bursts containing photobleaching events (Figure S3). To be able to visually detect possible correlations between parameters, we displayed the resulting data in a multidimensional histogram (Alexa488 acceptor data in Figure 3, Atto488 acceptor data in Figure S10). The reader is referred to the Materials and Methods section for more details on the multidimensional histogram. Because the subensemble dataset of each FRET pair supposedly consisted of 100% identical molecules, uniform and centrosymmetrical distributions were expected in each of the plots in the multidimensional histogram. Quite surprisingly, this was not observed for any of the samples. For those containing Atto647N as an acceptor, two distinct subpopulations were obvious in the multiparameter plots that each laid on the static FRET line in the E−τD space (Figures 3A and S10A), similarly to what has been observed before by Kalinin et al.29 Interestingly, while the fluorescence lifetime of Atto647N (τA) was similar, we detected a higher burst-averaged anisotropy (rA) for the low-FRET species (Figure S11). This suggests that the high-FRET species is given by DNA molecules with a more freely rotating acceptor moiety, whereas in the low-FRET species, the dye is interacting with the DNA molecule. The lowFRET species was slightly more populated, indicating that the equilibrium is in favor of the DNA bound state. Moreover, as two distinct species were observed, the overall dwell time in each state must be longer than the burst duration (∼1 ms). The presence of aged Trolox in the measurement buffer approximately doubled (from 21 to 43%) the percentage of (clearly identifiable) double-labeled molecules (i.e., the ratio of the D/A bursts to the total number of detected bursts, rescaled to 100%), 4256
DOI: 10.1021/acs.jpcb.8b00108 J. Phys. Chem. B 2018, 122, 4249−4266
Article
The Journal of Physical Chemistry B
Figure 3. Multiparameter analysis of different double-labeled DNA(D9A22) molecules with Alexa488 as a FRET donor. The FRET acceptors were (A) Atto647N, (B) Alexa647, (C) Atto655, or (D) StarRed. The respective molecules are illustrated on top of the data, with the FRET donor accessible volume in cyan, and the FRET acceptor accessible volume cloud in pink. Measurements were performed in PBS containing aged Trolox using 100 μW of 483 nm and 50 μW of 635 nm excitation, except for Atto655, which was measured in PBS using 75 μW of 483 nm and 150 μW of 635 nm excitation. The 2D contour plots display a 2D histogram of molecule counts (red = high counts, blue = low counts). The 1D bar charts are projections of the 2D histograms on the respective axes. The pink line in the E−τD plot is the static FRET line calculated with eq 14. The pink lines (with explicitly indicated rotational correlation times θ) in the r−τ plots are Perrin equations calculated with eq 16. The corresponding data with Atto488 as a donor are shown in Figure S10. A r−τ comparison for the two FRET states observed for Atto647N is in Figure S11. The effect of Trolox on the data is shown in Figure S12. The effect of the labeling position on the Atto655 data is shown in Figure S14.
whereas the percentage of D-only (from 24 to 14%) and A-only (from 21 to 11%) molecules decreased. The total number of bursts per second (at the same overall dilution) increased by about a factor of two in the presence of aged Trolox. Omitting aged Trolox from the buffer additionally increased the overall FRET population width (Figure S12A, left and middle) and increased the measured D/A distance slightly (Figure S12A, right). The effect of Trolox was not related to the known distance-dependent FRET histogram broadening29 but more likely to the agent reducing photobleaching and, blinking, as also concluded from the ensemble experiments. Samples with Alexa647 as the FRET acceptor exhibited narrower FRET distributions (Figures 3B and S10B), showing one main population that was slightly stretched toward low FRET efficiencies. Here, the presence of aged Trolox again increased the percentage of double-labeled molecules (from 16 to 24%), decreased the percentage of D-only (from 22 to 14%) and A-only (26 to 21%) molecules, increased the total number of bursts, and had a similar effect on the shape and mean of the FRET histograms (Figure S12B). For pairs with Atto655 as an acceptor, the corrected FRET efficiency was much lower compared to the other acceptor dyes
(Figures 3C and S10C) because of the smaller Förster radius (Table 1). Data also appeared much noisier because of the low acceptor brightness. A variety of long-lived (≫1 ms) emissive (i.e., not dark) states of the Atto655 dye, each exhibiting a different fluorescence lifetime, were obvious in the E−S, E−τA, and rA−τA plots, as have been reported before.57 Contrary to the other acceptor dyes, the addition of aged Trolox seemed to have an adverse effect. The percentage of clearly identifiable D/A bursts decreased (from ∼18 to ∼8%), the percentage of D-only bursts increased (from 6 to 28%), and the percentage of A-only bursts decreased from 33 to 5%. Trolox also increased the overall spread on the data and seemed to give rise to even more different emissive states of the dye (Figure S12C). Finally, the samples with StarRed as an acceptor exhibited a densely populated, well-defined high FRET state, together with a long tail of different minor states extending to almost zero FRET efficiency (Figures 3D and S10D). The burst-averaged lifetime of the StarRed dye in all of these states, however, was invariant, suggesting that the dye switches between bright and dark (rather than quenched) states. Aged Trolox increased the percentage of D/A bursts slightly (from ∼24 to ∼29%), decreased the percentage of D-only bursts (from 22 to 12%) 4257
DOI: 10.1021/acs.jpcb.8b00108 J. Phys. Chem. B 2018, 122, 4249−4266
Article
The Journal of Physical Chemistry B
estimation of distance (
