Evaluation of Blue and Far-Red Dye Pairs in Single-Molecule FRET

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Evaluation of Blue and Far-Red Dye Pairs in Single-Molecule FRET Experiments Niels Vandenberk, Anders Barth, Doortje Borrenberghs, Johan Hofkens, and Jelle Hendrix J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b00108 • Publication Date (Web): 15 Mar 2018 Downloaded from http://pubs.acs.org on March 15, 2018

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The Journal of Physical Chemistry

Evaluation of Blue and Far-Red Dye Pairs in Single-Molecule FRET Experiments

1 2 3 4

Niels Vandenberk1, Anders Barth2, Doortje Borrenberghs1, Johan Hofkens1 and Jelle

5

Hendrix1,3,*

6 7

1

8

Photonics, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven,

9

Belgium.

Laboratory for Photochemistry and Spectroscopy, Division for Molecular Imaging and

10

2

11

Nanosystems Initiative Munich and Centre for Nanoscience, Ludwig-Maximilians-Universität

12

München, 80539 Munich, Germany

13

3

14

Institute, Hasselt University, Agoralaan C (BIOMED), B-3590, Diepenbeek, Belgium

Physical Chemistry, Department of Chemistry, Munich Center for Integrated Protein Science,

Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre and Biomedical Research

15 16

*Address correspondence to

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Prof. Jelle Hendrix

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Agoralaan C (BIOMED), B-3590 Diepenbeek

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[email protected]

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+32 (0) 11 269213

21

1 ACS Paragon Plus Environment

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Abstract

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Förster resonance energy transfer (FRET) is a powerful tool to probe molecular interactions,

24

activity, analytes, forces and structure. Single-molecule (sm) FRET additionally allows real-

25

time quantifications of conformation and conformational dynamics. SmFRET robustness

26

critically depends on the employed dyes, yet a systematic comparison of different dye pairs is

27

lacking. Here, we evaluated blue (Atto488 and Alexa488) and far-red (Atto647N, Alexa647,

28

StarRed and Atto655) dyes using confocal smFRET spectroscopy on freely diffusing dsDNA

29

molecules. Via ensemble analyses (correlation, lifetime and anisotropy) of single-labeled

30

dsDNA, we find that Alexa488 and Atto647N are overall the better dyes, although the latter

31

interacts with DNA. Via burstwise analyses of double-labeled dsDNA with inter-dye distances

32

spanning the complete FRET-sensitive range (3.5-9 nm), we show that none of the dye pairs

33

stands out: distance accuracies were generally 0.8), R0 and the

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standard deviation of the Gaussian linker dye distribution of 5.6 Å, the static FRET line

291

(including linker dynamics) was simulated. The latter parameter was obtained by a sub-

292

ensemble TCSPC fit of the FRET donor decay to a Gaussian distance distribution model. The

293

used approach generates fluorescence-weighted lifetimes, which, for molecules exhibiting

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multiple lifetimes, will be weighted towards the long-lifetime species.

295

Burstwise steady-state fluorescence anisotropies of the FRET donor (rD) and FRET acceptor

296

(rA) were calculated from the respective fluorescence intensities: 𝐺𝐺𝐹𝐹 −𝐹𝐹

𝑟𝑟 = 𝐺𝐺𝐹𝐹ǁ +𝐹𝐹⊥ , ǁ

Eq. 15

⊥

297

where G is the correction factor for the different detection efficiencies in the two polarization

298

channels, 𝐹𝐹ǁ the intensity in time gate BBǁǁ or RRǁǁ and 𝐹𝐹⊥ the intensity in time gate BB⊥ or RR⊥.

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Perrin equations were calculated with: 𝑟𝑟

𝑟𝑟 = (1+𝜏𝜏0⁄𝜃𝜃 ) ,

Eq. 16

300

where r is the single molecule steady state anisotropy, r0 = 0.4 the fundamental anisotropy, τ

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the fluorescence lifetime and θ the rotational correlation time.

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Multi-dimensional histogram - The measurements result in intensity, lifetime and anisotropy

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information for both donor and acceptor dyes. This information was combined into a single

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multi-dimensional histogram, including a FRET efficiency versus stoichiometry histogram (E-

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S), two FRET efficiency (Eq. 10) versus donor or acceptor lifetime histograms (E-τD and E-τA,

306

respectively) and two donor and acceptor steady-state anisotropy versus lifetime histograms

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(rD-τD and rA-τA, respectively). For the E-τD plot, the static FRET line (Eq. 14) is additionally

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plotted, while a Perrin equation (Eq. 16) is plotted onto the r-τ plots.

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Photon distribution analysis

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Static photon distribution analysis (PDA) was carried out to obtain the absolute inter-dye

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distance distribution from the observed shot-noise limited FRET efficiency histograms.23

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Practically, for each FRET dataset, raw bursts were binned in time bins of 1 ms and a histogram

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was constructed and analyzed. Data was plotted in a E vs. S plot to deselect bins with complex

314

acceptor photophysics and only bins with at least 20 and maximally 250 photons (to reduce

315

calculation time) were used for PDA analysis. A three-state model for a Gaussian distance

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distribution23 was fitted to the experimental EPR histogram using a reduced-χ2-guided simplex 12 ACS Paragon Plus Environment

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search algorithm. The histograms of the nine different D/A spacing datasets of one FRET-pair

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were analyzed simultaneously. The mean and width of all Gaussian distributed sub-states were

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fitted for the nine datasets. Next, the standard deviation σ of the distance distributions was

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globally optimized at a fraction F of the corresponding distance to increase fitting robustness,

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which has been shown before to be reasonable for FRET experiments with a blinking FRET

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acceptor.24,26 In summary, for a comparative analysis, only a few parameters needed to be

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optimized (R1, R2, R3, F, A1, A2 and A3). R is the FRET-averaged donor-acceptor distance

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E

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function (PDF) was calculated per state using the R and σ parameters obtained from PDA

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analysis, that describe the underlying Gaussian distributed states. The summed PDF was scaled

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to a total area of unity, with each state’s PDF area scaled to the corresponding fraction of

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molecules. Criteria for a good fit were a low (< 3) reduced χ2 value, as well as a weighted

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residuals plot free of trends. During PDA analysis the following parameters were taken into

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account: (i) the average background count rates in donor and acceptor channels after donor

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excitation, (ii) the donor crosstalk correction parameter (β = 0.01), (iii) the direct acceptor

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excitation correction parameter (α = 0.01), and (iv) the relative detection efficiency of donor

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and acceptor (FRET-pair specific). β and α were determined as described previously.16,19

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Furthermore, a FRET-pair specific R0 was used (Table 1), that was calculated using the

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measured dye spectra (Table S1, Figure S6), an orientation factor κ2 = 2/3, the FCS-based50

336

measured quantum yield Φ = 0.72 for Atto488, Φ = 0.77 for Alexa488 (Figure S5), a refractive

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index n = 1.33, and the literature acceptor extinction coefficient (ε) Table S1). The direct

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acceptor excitation parameter varied between 0.01 and 0.02. The used gamma factor is

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determined as described in M&M, which is dye pair specific.

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Accessible volume simulation

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The geometry of the different dyes, including the linker length (measured from the C5 of

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Thymidine to the geometrical center of the dye), linker width and 3D radius was determined

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with the Molecular Operating Environment (MOE, chemical computing group, Montreal,

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Canada) after stretching the molecule maximally with force fields available in the software and

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maximum energy tolerance.26 The different parameters are summarized in Table S3. The FPS

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tool26 was used to simulate the accessible volume per dye in the context of the actual dsDNA,

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using standard settings (i.e. search nodes = 3, clash tolerance = 1.0 Å). This information,

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together with R0 (Table 1), was used to estimate the simulated FRET averaged D/A distance,

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, and An the fraction of the specific distance between D and A. Probability density



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〈RDA〉E.

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http://milou.science.uu.nl/services/3DDART/ (Figure S1C).

The

employed

DNA

structure

was

calculated

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using

3D-DART;

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Results

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We chose to compare eight different 488/633-nm excitable FRET pairs: the two donor (D)

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probes Atto488 and Alexa488, in combination with either of the four FRET acceptor (A) probes

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Atto647N, Alexa647, StarRed and Atto655. Per FRET pair, we generated nine D/A-labeled

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dsDNA molecules, each with different D/A distances (8-27 base pairs), covering the 35-90-Å

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range over which the FRET method is sensitive. Per dye, we additionally generated three single-

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labeled dsDNA molecules, each carrying the dye at one possible attachment site. In the

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Supporting Information, the specific dye characteristics (Table S1), a list of all dsDNA

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molecules (Table S2), the structure of all thymidine-dye complexes (Figure S1A), and the

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sequence of all DNA strands (Figure S1B) can be found. Before carrying out actual smFRET

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experiments, we examined the structure of the different single-labeled dsDNA molecules, as

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well as their photophysics in PBS buffer in absence or presence of 1 mM aged (containing the

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redox pair Trolox/Trolox-quinone) or fresh (containing only the reductant Trolox) Trolox (the

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reader is referred to the Materials and Methods section for more details on the buffer

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preparation).

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Alexa488 slightly outperforms Atto488

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First, we compared the two FRET donor dyes Atto488 and Alexa488 attached to DNA (Figure

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1A-D). Although both dyes share the same basic sulforhodamine skeleton (Figure S1A), the

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linker between the Alexa488 dye and the reactive group is directly attached to the dye’s

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carboxyphenyl moiety, while for Atto488, the linker is longer, and attached via an amide bond

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to the acid function of the carboxyphenyl moiety. Atto488 is also 1 net charge less negative

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than Alexa488 (Table S1). When attached to dsDNA, the accessible volume for both dyes is of

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comparable size due to their high structural similarity (Figure 1A and Table S3).

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We carried out FCS experiments to compare the brightness and blinking behavior of both dyes

376

freely diffusing in solution and bound to DNA (D11 construct) (Figure 1B). The amplitude of

377

the correlation function reports on the concentration of molecules, which allows quantifying

378

the brightness of the fluorophores (Eq. 5). Additionally, the time dependence of the correlation

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function allows investigating the diffusion time of molecules (τ ≈ 0.1-10 ms), as well as other

380

processes that give rise to intensity fluctuations, such as photoblinking due to the population of

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dark triplet states (τ ≈ 1-100 µs). The correlation functions for both dyes revealed a similar

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diffusion time Figure 1B, left). However, at the excitation intensities used for smFRET

383

experiments (100 µW, see Eq. 1 for the conversion to power density), both dyes show a blinking 15 ACS Paragon Plus Environment


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term with a relaxation time of ~10 µs. To quantitatively analyze the FCS curves, we fitted the

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data with a model including a single diffusion and blinking term (Eq. 3). The blinking was more

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pronounced for Alexa488, which showed a higher population of the dark state (Fblinking = 0.2

387

vs. 0.15) (Figure S7A-B). The blinking behavior was also observed for the DNA-attached dyes

388

and is in line with previous reports.51 The apparent average molecular brightness εavg (Eq. 5)

389

was found to be about ~20% higher for Alexa488 as compared to Atto488 (Figure 1B, right).

390

This increased brightness was not due to a difference in absorption (Table 1) or optical

391

saturation of the dyes at the employed excitation intensities (Figure S8A-B). Based on the

392

slightly blue-shifted spectrum and higher expected quantum yield (0.92 vs. 0.8) of Alexa488,

393

the brightness of the latter was indeed expected to be slightly (13%) higher under the

394

experimental conditions (Figure S6 and Table S1). For both dyes, aged Trolox increased the

395

relaxation time and amplitude of the blinking process and thus stabilized the triplet state (Figure

396

S7A-B). Both in presence and absence of Trolox, higher excitation intensities decreased the

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relaxation time of the blinking process, while increasing the blinking fraction (Figure S7A-B).

398

In the presence of Trolox, the apparent diffusion time of both dyes increased (data not shown).

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This observation hints at a photostabilizing effect of Trolox by reducing bleaching or rescuing

400

the fluorophores from long-lived dark states. In general, there was no significant difference in

401

blinking terms between aged and fresh Trolox (Figure S7A-B).

402

Next, we analyzed the fluorescence lifetime of the dyes by ensemble time-correlated single

403

photon counting. The fluorescence decays could be fitted well to a single-exponential model

404

(Figure 1C, left) and free dye lifetimes were, within experimental error, the same as reported

405

by the manufacturers (Figure 1C, right). Dye lifetimes did not vary with their attachment

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position on the DNA (Figure S9A), but the DNA attached dye lifetimes were about 10% lower

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in PBS and 6% lower in PBS/Trolox (Figure 1C, right). To obtain insights into the mobility of

408

the DNA attached dyes, we calculated (Eq. 9) and analyzed the time-resolved anisotropy

409

decays. For both dyes, the anisotropy decayed completely within 8 ns (Figure 2A). The

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rotational correlation time was around 1.3 ns when attached to DNA, compared with 0.3 ns for

411

the free dye Figure 2B). These values confirm an overall free mobility of the dye when attached

412

to DNA, which is an important control for quantitative FRET measurements to address the

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effect of dipole orientation on the transfer process (commonly known as the “κ2 problem”).

414

Again, we found no significant difference between the three different labeling positions (Figure

415

2C-D).


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Taken together, we spectroscopically evaluated the performance of both donor dyes in

417

experimental conditions that are typically used in confocal burstwise smFRET measurements.

418

Their performance was comparable, as was also expected from their high structural similarity.

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Although Alexa488 was slightly brighter at the laser powers we typically use for smFRET

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(100 µW), it did suffer slightly more from blinking. When attached to dsDNA, the addition of

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aged Trolox had a slightly positive effect on both dyes’ brightness, apparent diffusion constant

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and fluorescence lifetime, which we attribute to a photostabilizing effect.

423

Atto647N is the superior far-red dye

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Next, we compared the four different FRET acceptor probes Atto647N, Alexa647, Atto655 and

425

StarRed, attached to DNA (A1-4 in Figure 1D). Structurally, Atto647N and StarRed share a

426

similar basic skeleton (carbopyronine and rhodamine, respectively) (Figure S1A), but StarRed

427

contains an additional heterocyclic ring moiety, a fluorinated phenyl moiety and two sulfonyl

428

groups, rendering the dye considerably larger (Table S1). The cyanine Alexa647 contains a

429

long and elongated skeleton, while the oxazine Atto655, containing an extended ring structure,

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is the smallest of the four dyes (Table S1). The simulated accessible volume of the dyes when

431

attached to dsDNA correlated quite well with their overall size (Table S3). Alexa647 carries

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most charged moieties, and is the most negatively charged molecule (3-), followed by StarRed

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(1-), Atto655 (0) and Atto647N (+1).

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We again performed FCS experiments to compare the brightness and blinking behavior of the

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four acceptor dyes in the DNA(A36) construct (Figure 1E). At excitation powers typically used

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in smFRET for the direct excitation of the acceptor dye (50 µW, except for Atto655: 150 µW),

437

the four dyes exhibited quite a different µs blinking behavior (Figure 1E, left). While Atto647N

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revealed almost no blinking (τblinking ~10 µs, Fblinking ~0.05) (Figure S7C), Alexa647 exhibited

439

a strong, excitation intensity dependent blinking (τblinking ~5 µs, Fblinking ~0.3) (Figure S7D).

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Carbocyanines such as the commonly used dye cyanine 5 (Cy5) are known to exhibit cis-trans

441

photo-isomerization causing µs blinking37,52-53, and both Cy5 and Alexa647 have additionally

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been shown to exhibit (thiol-induced) longer-lived dark states.36,54-56 For Atto655, the

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correlation function could be well described without any blinking term (Figure S7E). However,

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when attached to DNA, Atto655 showed a complex blinking behavior (τblinking,1 ~6 µs, Fblinking,1

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~0.2, τblinking,2 ~60 µs, Fblinking,2 ~0.28), which showed only a weak dependence on laser power

446

(Figure S7E). StarRed-labeled DNA exhibited one blinking process in PBS (τblinking ~6 µs,

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Fblinking ~0.20), and two different blinking processes in PBS/Trolox (τblinking,1 ~6 µs, Fblinking,1 17 ACS Paragon Plus Environment


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~0.10, τblinking,2 ~30 µs, Fblinking,2 ~0.20) (Figure S7F) that scaled strongly with laser power.

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Trolox shifted the diffusion part of the Atto647N-DNA and StarRed-DNA correlation functions

450

rightward, indicating a photostabilizing effect, while for Atto655-DNA, Trolox had an adverse

451

effect, the observed apparent diffusion time was slightly smaller than in PBS, which might be

452

attributed to the presence of an additional blinking process on the diffusion timescale (data not

453

shown). In terms of apparent molecular brightness, all dyes (except Atto655) exhibited a

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>100-kHz brightness at the laser powers typically used for smFRET (50 µW), with Atto647N

455

the brightest (Figure 1E, right). Interestingly, the brightness of Atto655-DNA was much lower

456

than that of the free dye (Figure 1E, right). The relative brightness (εbright, corrected for the

457

presence of dark states, Eq. 6) between all dyes (except Atto655) (Figure S8C-F) compared

458

well to what we predicted for the used microscope (Table S1). However, the population of dark

459

states for Alexa647 and StarRed did render these dyes on average quite dimmer than Atto647N

460

(Figure 1E, right).

461

Next, we quantified the fluorescence lifetimes and anisotropies of the acceptor dyes attached to

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DNA. Atto647N and StarRed showed similar fluorescence decays (Figure 1F) that could be

463

well described with a mono-exponential model, resulting in a fluorescence lifetime of around

464

4 ns, slightly larger than the corresponding manufacturer’s values. Alexa647 exhibited a 1.1-ns

465

lifetime, comparable to the manufacturer’s value, which did not change when attached to DNA

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(Figure 1F). The fluorescence decay for free Atto655, on the other hand, could only be

467

described well with a bi-exponential model (85% of 1.82 ns and 15% of 0.21 ns). When attached

468

to DNA, the fluorescence lifetime of Atto655 was reduced considerably, resulting in an

469

increased fraction of the short lifetime component (30% of 2.27 ns and 70% of 0.34 ns in PBS).

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(In Figure 1F, the species-averaged lifetime is displayed, the intensity-averaged lifetime (Eq.

471

8) was 1.77 ns). This is likely caused by quenching through oxazine-guanine photo-induced

472

electron transfer (PET), as has been observed before.53,57-58 In line with this, the observed

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fluorescence lifetime of Atto655 depended on the labeling position, whereby the attachment

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site surrounded by the most guanine residues exhibited the most pronounced quenching (Figure

475

S9B). All other dyes showed no change in fluorescence lifetime or position-dependent

476

quenching when bound to DNA (Figure S9B). We then analyzed the time-resolved anisotropy

477

decays of the labeled DNA molecules (Figure 2E). The rotational correlation time of the probes

478

attached to DNA approximately scaled with their size, being highest for Atto655 (~3 ns) and

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around 2-2.5 ns for the other dyes (Figure 2F-G). The anisotropy decayed completely only for

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molecules labeled with Alexa647 and StarRed (Figure 2E). On the other hand, Atto647N and 18 ACS Paragon Plus Environment

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Atto655 did exhibit residual anisotropies (rinf), indicating long-lived interactions with the DNA

482

molecule (Figure 2H). For Atto647N, rinf values where in the range of 0-0.03 and depended

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slightly on the labeling position (Figure 2H), while for Atto655 large rinf values up to 0.07 were

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observed that changed significantly with the position on the DNA. This suggests that Atto655

485

and, to a lesser extent, Atto647N, tend to stick to the DNA molecule. The absence of

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electrostatic repulsion of these two dyes by the negatively charged DNA (Table S1) is in line

487

with these observations.

488

Summarized, we spectroscopically evaluated four far-red dyes covalently attached to DNA

489

molecules under similar conditions as used for burstwise smFRET experiments. Atto647N

490

possessed the highest brightness and suffered the least from µs blinking, although a partial

491

sticking to the labeled molecule of interest was observed. In the context of DNA, Alexa647 and

492

StarRed each exhibited significant µs blinking, while Atto655 additionally suffered from

493

quenched fluorescence and sticking to the DNA. A clear positive effect of Trolox (aged or

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fresh) was not obvious in the performed experiments for any of the tested dyes, although Trolox

495

did seem to provide a slight photoprotection for all dyes except Atto655.

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All pairs exhibit heterogeneously distributed FRET histograms

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To test how the dyes behave as donor and acceptor in FRET experiments, we carried out a

498

comparative analysis of all DNA(D9A22) (11-bp distance) molecules using multi-parameter

499

single-molecule spectroscopy. For this, we diluted the samples to concentrations that allowed

500

single-molecule observation (typically 10 pM), and recorded 1-2-hour time traces per sample.

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After identifying single molecule bursts in the recordings, we calculated a number of burst-

502

averaged parameters for every single-molecule event: the intensity-based corrected (absolute)

503

FRET efficiency E (Eq. 10), the corrected stoichiometry parameter S (Eq. 11), the fluorescence

504

lifetime of the FRET donor, τD, and acceptor, τA, and the steady-state anisotropy of the FRET

505

donor, rD, and acceptor, rA. We filtered the data by selecting all bursts that contained

506

fluorescence from both FRET donor and acceptor and discarded bursts containing

507

photobleaching events (Figure S3). To be able to visually detect possible correlations between

508

parameters, we displayed the resulting data in a multi-dimensional histogram (Alexa488-

509

acceptor data in Figure 3, Atto488-acceptor data in Figure S10). The reader is referred to the

510

Materials and Methods section for more details on the multi-dimensional histogram.

511

Because the sub-ensemble dataset of each FRET pair supposedly consisted of 100% identical

512

molecules, uniform and centrosymmetrical distributions were expected in each of the plots in 19 ACS Paragon Plus Environment


Page 20 of 47

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the multidimensional histogram. Quite surprisingly, this was not observed for any of the

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samples. For those containing Atto647N as acceptor, two distinct subpopulations were obvious

515

in the multi-parameter plots, that each laid on the static FRET line in the E-τD space (Figure 3A

516

and Figure S10A), similarly to what has been observed before by Kalinin et al.29 Interestingly,

517

while the fluorescence lifetime of Atto647N (τA) was similar, we detected a higher burst-

518

averaged anisotropy (rA) for the low-FRET species (Figure S11). This suggests that the high-

519

FRET species is given by DNA molecules with a more freely rotating acceptor moiety, while

520

in the low-FRET species the dye is interacting with the DNA molecule. The low-FRET species

521

was slightly more populated, indicating that the equilibrium is in favor of the DNA bound state.

522

Moreover, as two distinct species were observed, the overall dwell time in each state must be

523

longer than the burst duration (~1 ms). The presence of aged Trolox in the measurement buffer

524

approximately doubled (from 21% to 43%) the percentage of (clearly identifiable) double-

525

labeled molecules (i.e. the ratio of the D/A bursts to the total number of detected bursts, rescaled

526

to 100%), while the percentage of D-only (from 24% to 14%) and A-only (from 21% to 11%)

527

molecules decreased. The total number of bursts per second (at the same overall dilution)

528

increased by about a factor of two in the presence of aged Trolox. Omitting aged Trolox from

529

the buffer additionally increased the overall FRET population width (Figure S12A, left and

530

middle) and increased the measured D/A distance slightly (Figure S12A, right). The effect of

531

Trolox was not related to the known distance-dependent FRET histogram broadening29, but

532

more likely to the agent reducing photobleaching and –blinking, as also concluded from the

533

ensemble experiments.

534

Samples with Alexa647 as the FRET acceptor exhibited narrower FRET distributions (Figure

535

3B and Figure S10B), showing one main population that was slightly stretched towards low

536

FRET efficiencies. Here, the presence of aged Trolox again increased the percentage of double-

537

labeled molecules (from 16% to 24%), decreased the percentage of D-only (from 22% to 14%)

538

and A-only (26% to 21%) molecules, increased the total number of bursts, and had a similar

539

effect on the shape and mean of the FRET histograms (Figure S12B).

540

For pairs with Atto655 as acceptor, the corrected FRET efficiency was much lower compared

541

to the other acceptor dyes (Figure 3C and Figure S10C) due to the smaller Förster radius (Table

542

1). Data also appeared much noisier because of the low acceptor brightness. A variety of long-

543

lived (>> 1-ms) emissive (i.e. not dark) states of the Atto655 dye, each exhibiting a different

544

fluorescence lifetime, was obvious in the E-S, E-τA and rA-τA plots, as has been reported

545

before.57 Contrary to the other acceptor dyes, the addition of aged Trolox seemed to have an 20 ACS Paragon Plus Environment

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546

adverse effect. The percentage of clearly identifiable D/A bursts decreased (from ~18% to

547

~8%), the percentage of D-only bursts increased (from 6% to 28%), and the percentage of A-

548

only bursts decreased from 33% to 5%. Trolox also increased the overall spread on the data,

549

and seemed to give rise to even more different emissive states of the dye (Figure S12C).

550

Finally, samples with StarRed as acceptor exhibited a densely populated, well-defined high

551

FRET state, together with a long tail of different minor states extending to almost zero FRET

552

efficiency (Figure 3D and Figure S10D). The burst-averaged lifetime of the StarRed dye in all

553

these states, however, was invariant, suggesting the dye switches between bright and dark

554

(rather than quenched) states. Aged Trolox increased the percentage of D/A bursts slightly

555

(from ~24% to ~29%), decreased the percentage of D-only bursts (from 22% to 12%) and A-

556

only bursts (from 18% to 10%) but had no effect on the shape of the FRET histograms (Figure

557

S12D).

558

For all studied molecules, the data laid on the static FRET line in the E-τD space, however some

559

deviation was observed for Atto655 due to the existence of multiple acceptor states (Figure

560

3and Figure S10). The center values of the burstwise lifetime histograms of all FRET donors

561

and acceptors matched the values obtained from ensemble analysis (for Atto655 it compared

562

well to the intensity-weighted average ensemble lifetime, Eq. 8). The rD-τD and rA-τA plots,

563

showing the steady-state anisotropy and Perrin equation overlay (Eq. 16), additionally showed

564

a relatively high rotational mobility of all dyes on the DNA, except for the Atto655 and

565

Atto647N, in line with the ensemble experiments.

566

In summary, in confocal smFRET experiments on freely diffusing molecules all dye pairs

567

showed heterogeneous FRET distributions with multiple populations. Trolox increased the

568

percentage of D/A bursts considerably for all dye combinations, and reduced the width of the

569

FRET efficiency distributions, except when Atto655 was the acceptor. Both FRET donors

570

performed well in smFRET experiments. Alexa647 was the better FRET acceptor with respect

571

to the population homogeneity: molecules exhibited one main FRET species with only a lowly

572

populated low-FRET tail. Neither Atto655 nor StarRed seemed to provide good smFRET data.

573

Atto655 suffered from very low brightness, and the FRET data for both dyes exemplified

574

multiple states.

575

Accuracy and precision over the 35-90-Å range

576

With the dye photophysics and overall behavior in confocal burstwise smFRET characterized,

577

we next investigated how well distances could be determined from the measured FRET 21 ACS Paragon Plus Environment


Page 22 of 47

578

efficiency distributions. We estimated the expected distances between the dyes from accessible

579

volume simulations on the modeled DNA structures (the reader is referred to the Materials and

580

Methods section for details on the simulations). Measured distances were subsequently

581

determined in two ways: (i) by visually estimating E (as illustrated in e.g. Figure 4A, left) and

582

(ii) by photon distribution analysis (as illustrated in e.g. Figure 4A, middle and right). We chose

583

the first method over the more commonly used Gaussian fitting, since the latter only performs

584

well for unimodal and normally distributed data. The visual approach, however, only works for

585

samples exhibiting one well-defined state, and is subject to user bias. The PDA method should

586

in principle be sensitive to the different observed FRET states. As mentioned previously,

587

Atto647N bearing samples exhibited two FRET states, most pronounced for distances around

588

R0, i.e. DNA(D19-11A122), which we could also detect by PDA analysis (Figure 4A). For

589

samples carrying either FRET donor with Atto647N as the acceptor, PDA managed to recover

590

the inter-dye spacing within 10 Å of the simulated data (at least when using the higher FRET

591

state). The visual approach matched the PDA derived distances, however for low values of E

592

significant deviations between the two methods were observed (Figure 4B,C). For all Alexa647

593

bearing samples, and irrespective of the used FRET donor, a minimum of two states was

594

required for a proper PDA fit, to account for the low-E tail in the FRET histogram (Figure 4D).

595

Both PDA and the visual approach slightly overestimated the inter-dye distance relative to the

596

expected distances by up to 1 nm, except for >70 Å distances (Figure 4E,F). For Atto655,

597

visually estimating the mean FRET efficiency seemed the better approach (Figure 4G-I), likely

598

because too many FRET states were present in the samples (see f.e. Figure S12C) to allow for

599

efficient PDA analysis. For StarRed, the visual approach allowed a good estimation of distance

600

(< 5-Å difference between experiment and simulation for most samples), while PDA analysis

601

performed increasingly worse as distance increased (Figure 4J-L).

602

In terms of relative distance precision, changes in D/A distance down to 2 nucleobases (=6.8

603

Å, not taking any rotation of the dyes around the DNA helix into account) were easily picked

604

up by the analysis over the whole distance range of 35-90 Å for all FRET pairs except the ones

605

carrying Atto655. For the Atto488/Alexa647 bearing molecules, we estimated the error of the

606

determined distances based on the uncertainty of the donor quantum yield and the refractive

607

index (Figure 4F, y error bar). Additionally, we determined the error of the simulated distances

608

based on different structures for the DNA double helix Figure 4F, x error bar). The reader is

609

referred to the Materials and Methods section for details. Taking the uncertainty into account,

610

experiment and simulation matched reasonably well for most D/A distances. 22 ACS Paragon Plus Environment

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611

Together, we determined that inter-dye distances can be quantified over the complete 35-90-Å

612

distance range with < 1-nm accuracy (< 5 Å for any donor plus either Atto647N or StarRed).

613

For the analysis, we focused on the major FRET species of each of the nine possible FRET

614

molecules for a given dye pair. Both Atto647N, Alexa647 and StarRed could easily pick up

615

differences in D/A distance with a precision better than 2 nucleobases (i.e. better than ~6.8 Å).

616

If different FRET states in a single sample were closely spaced, PDA analysis could generally

617

recover the distances better.

618

Alternating laser excitation can affect the acceptor quantum yield

619

Because of the observed deviation between simulated and measured distances, we next

620

investigated a possible bias in the burstwise smFRET experiments induced by active FRET

621

acceptor excitation via PIE. We performed smFRET experiments on mixtures of DNA

622

molecules with a different D/A separation, varying the 635-nm laser power from 0-50 µW

623

(0-150 µW for Atto655 due to the lower brightness). We calculated the apparent FRET

624

efficiency, here called proximity ratio, based on background-corrected photon counts and

625

correction for direct excitation without correcting for crosstalk (Figure 5A-D, vertical

626

histograms). The proximity ratio directly reveals changes in the apparent acceptor quantum

627

yield, since crosstalk and direct excitation are independent of the acceptor excitation power.

628

Additionally, the quenching of the donor lifetime provides a readout of the FRET efficiency

629

that is independent of the acceptor quantum yield (Figure 5A-D, horizontal histograms). For

630

molecules carrying Atto647N (Figure 5), we observed no significant changes of the τD and E

631

distributions. In line with this, Atto647N did not show significant triplet state population

632

(Figure S7C) and the employed laser powers were below saturation (Figure S8C). As such, it

633

performs well as an acceptor in the performed PIE experiment. The smFRET data obtained for

634

Atto655 showed no change in the donor lifetime as a function of acceptor laser power (Figure

635

5C), but conversely, an apparent shift to low proximity ratio was observed in the absence of

636

acceptor excitation, which is likely an artifact of incomplete removal of donor-only labeled

637

molecules. For molecules carrying StarRed, we observed a systematic shift of the proximity

638

ratio as the acceptor excitation power increased (Figure 5D). StarRed showed a large blinking

639

fraction that increased with laser power (Figure 1E and S2F). We also observed a smaller

640

increase in the brightness of StarRed compared to Atto647N and a reduction of its quantum

641

yield with increasing laser power (Figure S8F and Figure S5F), which we attribute to the higher

642

population of dark states. Interestingly, the donor lifetime was insensitive to the used acceptor

643

excitation regime, indicating that the dark states of StarRed still function as FRET acceptors 23 ACS Paragon Plus Environment


Page 24 of 47

644

with similar Förster radius, i.e. similar absorption spectrum. For samples carrying Alexa647,

645

we also observed an effect of the acceptor laser power on the proximity ratio, albeit weaker as

646

for StarRed. Compared to no acceptor excitation, we measured a small decrease of the

647

proximity ratio, which showed no further change upon increasing the laser power from 20 µW

648

to 50 µW (Figure 5B). Different than for StarRed, the fraction of dark states of Alexa647

649

decreased slightly with increasing laser power (Figure S7D), however its quantum yield still

650

showed a minor decrease (Figure S5D). Surprisingly, we observed a small increase of the donor

651

lifetime in the absence of direct excitation of Alexa647, indicating a lower FRET efficiency.

652

Since direct acceptor excitation reduces the dark state fraction of Alexa647, the FRET

653

efficiency to the dark state must thus be larger. In other words, the Förster radius for the dark

654

state must be smaller than for the bright state due to a change of the acceptor absorption

655

spectrum. To see if we could correct for the observed quantum yield changes of Alexa647 and

656

StarRed in the presence of direct acceptor excitation, we determined the γ-factor for every

657

measurement individually and calculated the accurate FRET efficiencies (Figure S13). For

658

Alexa647, the FRET efficiency histograms overlaid, however, StarRed still showed differences

659

even after γ-correction.

660

In summary, Atto647N proved to be a good acceptor for PIE experiments since its apparent

661

quantum yield was not affected by the direct acceptor excitation. Atto655, as before, produced

662

low quality data, but was also invariant under PIE excitation. The blinking of Alexa647 and

663

StarRed had only a marginal effect on the donor lifetime, indicating that the dark states still act

664

as FRET acceptors. Both Alexa647 and StarRed showed a changing quantum yield when

665

directly excited, however for Alexa647 the effect could be negated when accurate FRET

666

efficiencies are calculated using intrinsically determined γ-factors. We conclude that both

667

Atto647N and Alexa647 are suitable to be used in FRET experiments under PIE excitation.

668

Active Alexa647 excitation reduces photophysics-dependent FRET dynamics

669

On a burst-averaged level, we found that alternating excitation of Alexa647 affects its quantum

670

yield, but also has an effect on the FRET efficiency as measured from the donor lifetime. Since

671

Alexa647 shows a large population of dark states, we hypothesized that the blinking behavior

672

at the different acceptor laser powers was the cause of this behavior, whereby the Förster radius

673

for the dark state is lower. To investigate this effect in more detail, we calculated the

674

autocorrelation functions for the donor signal and the cross-correlation functions between the

675

donor and FRET-sensitized acceptor signals, as well as between donor and direct acceptor

676

excitation signals, at increasing acceptor excitation powers (Figure 6). If the Förster radius of 24 ACS Paragon Plus Environment

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677

the acceptor changes between the bright and dark state, the acceptor blinking should be visible

678

in the donor autocorrelation function. Additionally, donor and FRET-sensitized acceptor

679

signals should anti-correlate on the time scale of acceptor blinking. As a negative control, we

680

used samples with Atto647N as the acceptor since it showed the least amount of blinking in the

681

ensemble experiments (Figure 1E and Figure S7C). Although the FRET donor exhibited

682

slightly more blinking when Atto647N was present (Figure 6A, red/blue versus black), no

683

donor-acceptor anti-correlation was detected (Figure 6A, cyan/magenta). We also could not

684

detect a significant effect of the acceptor excitation rate on the donor autocorrelation functions

685

(Figure 6A, red versus blue). For Alexa647, the donor also showed more blinking presence of

686

the acceptor (Figure 6B, black versus red). The donor blinking fraction, however, decreased

687

with increasing acceptor excitation rate (Figure 6B, red/green/blue data), similar to the behavior

688

observed for Alexa647 in the ensemble experiments (Figure S7D). More strikingly, a clear anti-

689

correlation was observed when donor and acceptor signals were cross-correlated (Figure 6B,

690

cyan/magenta). Thus, we show that the blinking of Alexa647 induces FRET-sensitized blinking

691

of the donor. This further supports the hypothesis that the Förster radius for the dark state of

692

Alexa647 must be different. Interestingly, this effect was minimized at higher acceptor

693

excitation power.



Page 26 of 47

694

Discussion

695

In this paper we assessed the performance of different dyes (Figure S1A, Table S1, Table S3)

696

and FRET pairs (Table 1) via confocal burstwise smFRET experiments (Figure S2) on freely

697

diffusing single- and double labeled dsDNA molecules (Figure S1B and Table S2), as a model

698

system for molecules that do not exhibit conformational dynamics on the milliseconds

699

timescale.29,59-61

700

Characterizing smFRET systems via combined ensemble and single molecule analysis

701

We followed a thorough approach to characterize each dye and FRET pair. We performed both

702

ensemble and single-molecule analyses using the same confocal microscope, equipped with

703

nanosecond alternating donor/acceptor excitation and with single photon sensitive lifetime- and

704

anisotropy-resolved detection (Figure S2). We characterized all dyes in the context of single-

705

labeled DNA molecules using ensemble correlation, lifetime and anisotropy analyses (Figur e1

706

and Figure 2). These results helped to find the optimal experimental conditions for each dye

707

with respect to laser power and buffer conditions. Additionally, they revealed dye or labeling-

708

position dependent characteristics (Figure 2 and Figure S9), such as blinking (Figure S7),

709

photostability, dye rotation, apparent molecular brightness (Figure S8) and quantum yield

710

(Figure S5), information that can be important when interpreting single-molecule smFRET data

711

in terms of absolute distances. We then performed single-molecule burst analysis of double-

712

labeled molecules with all combinations of donor and acceptor fluorophores (Figure 3 and

713

Figure S10). We showed that no single dye or dye pair is perfect, at least in the confocal

714

burstwise smFRET experiments performed here. Good agreement was found between lifetimes

715

and anisotropies determined on the ensemble level compared to the molecule-wise

716

distributions.

717

The multidimensional parameter space available in the single-molecule analysis can help with

718

identifying the cause of heterogeneity in FRET-dependent parameters. We could e.g. correlate

719

the presence of two distinct Atto647N FRET states to a difference in the anisotropy of the

720

acceptor dye in both states (Figure 3A, Figure S10A and Figure S11). This, in turn, suggests

721

the dye is interacting (‘sticking’) to DNA, as has been reported before.29,62 The effect might be

722

due to hydrophobic or electrostatic (the positive charge on the dye’s iminium moiety is

723

delocalized over the dye’s conjugated system) interactions with the hydrophobic core or the

724

negatively charged phosphate backbone of the DNA, respectively. Dyes with an overall similar

725

skeleton to Atto647N are known to stick to DNA.29,35,39,62-64 On the other hand, Atto647N has


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726

also been shown to exhibit spontaneous intensity switching, independent of molecular

727

interactions, resulting in different spectra and fluorescence lifetimes.39,65

728

In general, a combined ensemble and single-molecule analysis is a robust tool for characterizing

729

a particular smFRET system. On the one hand, we find it an excellent intrinsic methodological

730

control: as ensemble and single-molecule data report on the same molecular properties via

731

different analysis algorithms they should be mutually confirmatory. Providing a fail-proof

732

quantification of smFRET experiments is especially important when FRET is used for accurate

733

structural studies on more complex macromolecules such as proteins.

734

Burstwise smFRET on DNA molecules

735

While the results presented here have been obtained using dsDNA oligonucleotide molecules,

736

the performance evaluation is expected to relate to other, e.g. protein systems as well. However,

737

since the local physicochemical environment of the dye can be different on a protein surface,

738

this might affect the spectroscopic properties of the dye, and thus its performance. Additionally,

739

quenching may occur through photoinduced electron transfer (PET) between the fluorophore

740

and certain amino-acid side chains such as tryptophan, similar to what we observed for Atto655

741

on DNA here.66 In general, weak-force (hydrophobic, Van der Waals, electrostatic, H-bonding)

742

interactions or energy transfer (e.g. FRET or PET) processes of dyes with their local

743

environment can be different depending on the labeled molecule of interest, and have to be

744

taken into account in quantitative analyses. Another important aspect with respect to more

745

complex systems is the influence of the dye on the structure and dynamics of the biomolecule,

746

which can only be addressed when FRET is combined with complimentary structural

747

methods.67

748

With respect to the employed smFRET method, it is worth mentioning, again, that due to the

749

intrinsically imperfect D/A labeling of smFRET samples, burst analysis using PIE in principle

750

also allows carrying out detailed analysis on the D-only and A-only populations via sub-

751

ensemble molecule selection, albeit only at laser powers that allow single-molecule burst

752

identification in the raw data trace. This is useful since information on three different species

753

(D, A and DA) can be obtained from a single measurement. Secondly, characteristic for

754

burstwise experiments is a short observation time per molecule of ~1-10 milliseconds. In

755

surface-immobilized smFRET experiments (confocal or TIRF-based), molecules are observed

756

for seconds to minutes at frame rates on the millisecond timescale (if camera-based detection

757

is used), which makes photobleaching, longer-time scale blinking (>>ms) and total photon 27 ACS Paragon Plus Environment


Page 28 of 47

758

yield, dye properties that were not quantified in the present work, a limiting factor in those

759

experiments. Photobleaching and -blinking can be suppressed by a combination of oxygen

760

removal and reducing and oxidizing agents (such as Trolox) to rescue the fluorophores from

761

long-lived triplet states39, however the exact composition of the stabilizing buffer has to be

762

optimized for each fluorophore. As many experimental variables affect the FRET process or

763

quantification thereof, a detailed analysis of the applied dye combination via the methods

764

employed here can be a useful first step, regardless of the smFRET methodology that is used

765

for e.g. FRET guided structural biology. In a next step, individual molecules can be studied

766

with nanosecond time-resolution using confocal smFRET on immobilized molecules, or with

767

ms time-resolution using TIRF-based smFRET, the latter method providing the additional

768

advantage that many molecules can be sampled simultaneously, thereby considerable

769

decreasing the total measurement duration.

770

Insights in acceptor performance are necessary

771

Actively exciting the FRET acceptor via PIE has defined advantages: changes in the acceptor’s

772

quantum yield can be detected through its lifetime,19 optical sorting of double- from single

773

labeled molecules is straightforward,16 correlations between FRET and acceptor properties can

774

be directly investigated (Figure 3 and Figure S10), and artifacts such as photobleaching can be

775

corrected for (Figure S3).19 Via the stoichiometry and acceptor lifetime we could show that the

776

properties of Atto655 changed drastically as a function of labeling position (Figure S14A and

777

B), confirming our prior ensemble observations (Figure 2G-H and Figure S9B). Secondly, dyes

778

that suffer from excitation dependent blinking can exhibit a different burst-averaged quantum

779

yield, as we observed for both Alexa647 and StarRed (Figure 5B and D). This effect can be

780

accounted for by determining the relative detection efficiency correction parameter γ under the

781

excitation conditions used for the smFRET experiments (Figure S13, Figure S4 and Figure

782

S15).

783

We showed that, for Alexa647 and StarRed, the dark states still act as FRET acceptors, since a

784

change in the population of the dark state did not result in a significant change of the donor

785

lifetime. However, for Alexa647, the situation proved more difficult because the Förster radii

786

are different between the dark and the bright state, resulting in different FRET efficiencies.68-69

787

The overall blinking fraction decreased slightly when the dye was excited more efficiently

788

(Figure S7D). At the same time, the burst-averaged FRET donor fluorescence lifetime

789

decreased, i.e. the FRET efficiency increased (Figure 5B). In other words, when the dye is less

790

in a dark state, it becomes a better FRET acceptor, indicating that the Förster radius for the dark 28 ACS Paragon Plus Environment

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791

state is smaller. Absorption properties that depend on the dye’s emissive state have been

792

observed before for Alexa647.70-71 Moreover, when acceptor blinking changes the dye’s

793

absorptive properties, donor and FRET-sensitized acceptor signals will give rise to anti-

794

correlating FRET dynamics (Figure 6B). One should be careful not to misinterpret this effect

795

as molecular conformational dynamics. A general rule-of-thumb for experiments on e.g.

796

proteins would be to always verify this effect on a reference molecule that is expected to show

797

no conformational dynamics. In the case of Alexa647, we found that direct acceptor excitation

798

reduces the magnitude of false FRET dynamics.

799

Trolox improves the quality of smFRET data considerably

800

Aged Trolox, containing both the reductant Trolox and oxidant Trolox-quinone, is known to

801

rescue some dyes from long-lived dark states from which photobleaching can occur.40 In our

802

experiments, Trolox exhibited a clear photoprotective effect for the used FRET donor dyes, as

803

evidenced by a larger FCS brightness (Figure 1B) and correlation time scale (data not shown),

804

and longer ensemble fluorescence lifetimes (Figure 1C), although the agent increase the µs

805

blinking tendency of the dyes considerable (Figure S7). The beneficial effect of Trolox

806

translated itself, at least in part, into a higher percentage of double-labeled molecules and lower

807

percentage of D-only molecules in the smFRET experiments (Figure 3 and S7), as well as in a

808

lower overall spread on the smFRET data (Figure S12), except when Atto655 was the acceptor.

809

At the ensemble level, fresh Trolox had overall similar effects, suggesting that mainly Trolox

810

itself (and not its quinone form) is the beneficial agent for these dyes.40 For the acceptor dyes,

811

the effect of Trolox was less well-pronounced: although a rightward shift of the FCS

812

correlations was obvious when measurements were performed in Trolox (data not shown), the

813

FCS brightness and fluorescence lifetimes were largely insensitive to Trolox. The percentage

814

of A-only bursts did significantly decrease in Trolox (except when Atto655 was the acceptor),

815

corroborating that the agent does also protect most acceptor dyes from long-live dark states or

816

photobleaching, as has been observed before for.39,72 On a more general note, as the presence

817

of Trolox increased the total number of detected bursts by a factor of two, and the percentage

818

of D/A bursts, it can be used in smFRET experiments to reduce the total measurement time.

819

Moreover, as it generally improved dye behavior, Trolox decreased the overall spread on the

820

smFRET data (Figure S12).

821

Towards atomic scale distance measurements with smFRET

822

All FRET pairs showed deviations from the predicted distances for all nine probed distances.

823

There might be many reasons for this. Experimental FRET efficiencies, at least when measured 29 ACS Paragon Plus Environment


Page 30 of 47

824

and corrected for via a standardized procedure (Hellenkamp et al., submitted) can safely be

825

assumed to be correct. Transforming E to distance requires the Förster radius R0, which in turn

826

depends on a number of parameters (orientation factor κ2, refractive index n, donor emission

827

spectrum and quantum yield φ (Figure S5), acceptor spectrum and extinction coefficient), each

828

of which has a certain uncertainty associated to it.48 We propagated the uncertainty of some

829

parameters to estimate the precision of the obtained distances (Figure 4F, y error bars).

830

Especially for small distances, the discrepancy between experiment and simulation was quite

831

large (up to 1 nm). We do not expect that the dyes come into physical contact even at the

832

smallest spacing (8 bp). Due to the highly non-linear (6th power) scaling of R with E, small

833

errors in E are, however, magnified when calculating distances. The distance simulations, on

834

the other hand, were based on many assumptions. Although generally assumed to be rigid and

835

linear, previous smFRET experiments have shown that dsDNA molecules may not be

836

completely rigid and can assume bent conformations.59,61 In this respect, DNA origamis might

837

provide a better reference sample for smFRET experiments.13,60 We attempted to compensate

838

for the partially unknown DNA structure by using a linear and a bent dsDNA structure to

839

determine expected distances (Figure S1C and Figure 4F, x error bars). Secondly, the simulated

840

distances were obtained by accessible volume simulations26,73, which are only a rough

841

approximation of the actual average space occupancy by the dyes. Full molecular dynamics

842

(MD) simulations to account for possible electrostatic (or other) interactions of the dye with

843

their labeled substrate, are tempting now that force fields for many dyes have appeared in

844

literature74, although obtaining the time-averaged behavior of dyes would require MD

845

simulations up to the milliseconds timescale.

846

Summarized, although discrepancies between the simulated and measured data were observed

847

up to 1 nm, relative changes on the scale of 2 nucleobases (6.8 Å) could easily be resolved.

848

Future attempts on finding reference samples for smFRET with absolutely known R0 and known

849

(FRET-averaged) D/A spacing would be beneficial for anyone setting up or applying smFRET

850

in daily routine.

851

Conclusion

852

In this work, we showed that both Atto488 and Alexa488 are suitable donors for confocal

853

burstwise single-molecule FRET analysis, which is positive in view of the many literature

854

reports where these dyes were used. In ensemble confocal spectroscopy experiments, Atto647N

855

was the superior far-red dye, although it did suffer from sticking to the DNA. Atto655 30 ACS Paragon Plus Environment

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856

performed worst owing to low brightness, the presence of many emissive states of the dye and

857

a large position dependent quenching on DNA. On the other hand, confocal burstwise smFRET

858

experiments showed heterogeneous data for every used FRET pair, especially for distances

859

around R0. However, the obtained accuracy (

Evaluation of Blue and Far-Red Dye Pairs in Single-Molecule FRET

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