farFRET: Extending the Range in Single-Molecule FRET Experiments

Jun 24, 2015 - Single-molecule Förster resonance energy transfer (smFRET) has become a powerful nanoscopic tool in studies of biomolecular structures...
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Letter pubs.acs.org/NanoLett

farFRET: Extending the Range in Single-Molecule FRET Experiments beyond 10 nm Georg Krainer,*,†,‡,§ Andreas Hartmann,†,§ and Michael Schlierf*,† †

B CUBE  Center for Molecular Bioengineering, Technische Universität Dresden, Arnoldstraße 18, 01307 Dresden, Germany Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Straße 13, 67663 Kaiserslautern, Germany



S Supporting Information *

ABSTRACT: Single-molecule Förster resonance energy transfer (smFRET) has become a powerful nanoscopic tool in studies of biomolecular structures and nanoscale objects; however, conventional smFRET measurements are generally blind to distances above 10 nm thus impeding the study of long-distance phenomena. Here, we report the development of farFRET, a technique that extends the range in single-molecule FRET (smFRET) measurements beyond the 10 nm line by enhanced energy transfer using multiple acceptors. We demonstrate that farFRET can be readily employed to quantify FRET efficiencies and conformational dynamics using double-stranded DNA molecules, RecA-filament formation on single-stranded DNA and Holliday junction dynamics. farFRET allows quantitative measurements of large biomolecular complexes and nanostructures thus bridging the remaining gap to superresolution microscopy. KEYWORDS: smFRET, large conformational changes, biomolecular complexes, confocal spectroscopy, RecA, Holliday junction

S

Here, we report the development of a single-molecule technique, termed farFRET, which extends the accessible range of smFRET measurements beyond 10 nm. FarFRET probes the energy transfer efficiency between a single-donor and multiple identical acceptor fluorophores in close proximity (Figure 1a). Mechanistically, the acceptors act as individual receivers that collect excitation energy from the single donor with independent probability, resulting in an enhanced total energy transfer (Supporting Information Figure S1). The total energy transfer rate kT of this parallel-acting “antenna system”14,15 has a strong impact on the donor lifetime τD(A) = 1/(kT + (1/ τD(0))) and can be used experimentally to read out the enhanced FRET efficiencies at the single-molecule level by measuring the donor lifetime in the presence (τD(A)) and absence (τD(0)) of acceptors according to τD(A) E=1− τD(0) (eq 1)

ingle-molecule techniques have rapidly developed as powerful tools for the analysis of structure and function of complex biomolecular processes.1−3 Methodological advances over past decades have made particularly singlemolecule Förster resonance energy transfer (smFRET) a popular technique reporting on molecular distances and conformational changes of biomolecules and in bionanotechnological applications.4−8 A typical smFRET experiment probes the energy transfer efficiency between a single donor−acceptor fluorophore pair within a distance of 2−8 nm, while being blind to distances above 10 nm.1,9 Nonetheless, numerous biological processes and nanotechnological assemblies rely on large and extended multicomponent complexes exceeding the length scales accessible by smFRET experiments. While recent advances in super-resolution microscopy have paved the way for measuring distances down to 20 nm in lateral resolution,10,11 there still remains a gap to directly resolve structures and interactions between 10 and 20 nm. Multicolor single-molecule FRET approaches have been developed allowing long-distance energy transfer by a FRET cascade encompassing several distinct fluorophores.12,13 However, these approaches are not easy to implement because of their technically challenging sample preparation, instrumentation, and complicated analysis as well as their reliance on fluorophores with spectrally compatible properties. Hence, to fully exploit the potential of smFRET in resolving heterogeneous and complex biological processes also in large systems, methods that overcome the relatively short-ranged distances in dual-color smFRET measurements are required. © XXXX American Chemical Society

Hence, an increasing number of acceptors leads to a gain in transfer efficiency and shifts the measured FRET distributions to higher efficiencies (Figure 1a,b). This can be employed to push the limit of smFRET measurements beyond 10 nm. To this end, we developed an approach using confocal spectroscopy to extract FRET efficiency histograms from single molecules using single-molecule fluorescence donor-lifetime measurements (Supporting Information Material and MethReceived: May 13, 2015 Revised: June 12, 2015

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DOI: 10.1021/acs.nanolett.5b01878 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 1. Concept of farFRET and proof-of-principle experiments with DNA rulers. (a) Compared with a single donor−acceptor FRET pair, the use of multiple acceptors enhances the FRET efficiency (E) for distances around the Förster radius (left) as well as for distances beyond 10 nm (right). (b) Schematic illustration of FRET efficiency distributions and the FRET efficiency versus dye distance for one (green) to four (red) acceptors. The theoretical calculations (Supporting Information eq S3) predict a shift of the location of the FRET efficiency distribution to larger values with increasing number of acceptors. (c,d) FRET efficiency histograms of the dsDNA20 ruler with ∼7.1 nm and dsDNA32 ruler with ∼10.5 nm minimal distance between the single donor and one to four acceptors (top to bottom) show a significant shift of the FRET efficiency distribution. Interfluorophore distances were calculated using accessible volume simulations.28 The Gaussian fit center values are given in the panels with a localization error of ±0.005.

the sample with four acceptors. Already the addition of a second acceptor elevated the average FRET efficiency to ⟨E⟩ = 0.039, thus further away from experimental detection limits. In conclusion, the close proximity of several acceptors enhances the FRET efficiency (Supporting Information Figure S2), illustrating that farFRET readily extends the accessible range beyond the often experienced 10 nm limit on the biological scaffold double-stranded DNA. In a next step, we showed that farFRET allows quantification of long distances in complex biological assemblies (e.g., protein−DNA interactions) using RecA filament formation on single-stranded (ss) DNA, a system that has been well studied with typical smFRET experiments.16,17 RecA nucleoprotein filaments are key players in DNA recombination and repair and form extended structures exceeding 10 nm with only seven monomers bound to ssDNA.18 We designed a DNAduplex with a 21 nucleotide (nt) 3′-ssDNA tail overhang (Figure 2a) that sequesters seven RecA monomers and forms in the presence of the slowly hydrolyzable ATP analogue ATPγS an extended and stable RecA-filament with a total filament length of ∼10.7 nm. To report on changes in the end-to-end distance of the ssDNA tail upon RecA filament formation, we labeled a DNA construct with a single donor (ATTO532) at the 3′-end of the ssDNA and either one (pdT21-A) or two (pdT21-AA) acceptors (ATTO647N) at the ss-dsDNA junction (Figure 2b, insets). Using the pdT21-A construct the FRET efficiency distribution of bare ssDNA was centered at ⟨E⟩ = 0.357 (Figure 2b, upper, white) and shifted upon RecA binding to a peak centered at ⟨E⟩ = 0.003 (Figure 2b, upper, green). Hence, the extended RecA-ssDNA filament is too long to quantify FRET efficiency changes using a single donor− acceptor pair. A FRET efficiency of ⟨E⟩ = 0.003 is typically

ods). The major advantage of donor-lifetime measurements in comparison with intensity-based FRET measurements is that only the donor reports on the FRET efficiency and any secondary effects of the acceptor (e.g., self-quenching that would lead to a reduced acceptor emission intensity) can be omitted. Furthermore, nonideality in energy transfer and deviations from the simplistic assumption of noninteracting receivers that might arise from affected acceptors (e.g., from interacting acceptors due to stacking and Dexter transfer) can be included in an empirical calibration as illustrated below. We first set up proof-of-principle experiments on a biological nanostructure to demonstrate the enhancement effect by the presence of multiple acceptors. To this end, we designed a series of rigid double-stranded DNA (dsDNA) molecules labeled with a single donor (ATTO532) and increasing numbers of acceptors (ATTO647N). Up to four acceptors were placed in close proximity on adjacent and opposite sides of the dsDNA. Two sets of DNA rulers were employed: a short ruler (dsDNA20) with a minimal donor−acceptor(s) separation of 20 bp (∼7.1 nm) that is well accessible by smFRET measurements and a long ruler (dsDNA32) with a minimal 32 bp donor−acceptor(s) separation, translating into an interfluorophore distance of ∼10.5 nm. With increasing number of acceptors, we observed, in both set of constructs, a pronounced shift in the FRET efficiency histograms resulting from the enhancement in energy transfer efficiency from a single donor to multiple acceptors (Figure 1c,d). The dsDNA20 ruler showed a shift in average FRET efficiency from ⟨E⟩ = 0.179 to ⟨E⟩ = 0.318 for one versus four acceptors, respectively. More importantly, the center position of the FRET efficiency distributions in the dsDNA32 ruler constructs shifted for the sample with one acceptor from ⟨E⟩ = 0.018 to ⟨E⟩ = 0.085 for B

DOI: 10.1021/acs.nanolett.5b01878 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 2. Application of farFRET to protein−DNA interactions with large conformational changes and long-distance structural dynamics. (a) In the presence of ATPS, RecA forms an elongated filament on single-stranded DNA. Seven RecA monomers stretch ssDNA already by 10.7 nm according to the crystal structure18 and thus exceeding the typical FRET detection limit. (b) FRET efficiency histograms of bare (white) and RecA decorated ssDNA (colored) for pdT21-A (top) and pdT21-AA (bottom). For pdT21-A, the stabilized RecA filament resulted in a FRET efficiency of ⟨E⟩ = 0.003 (top, green), bearing limited information. Two acceptors shift the average FRET efficiency of the RecA-decorated ssDNA to ⟨E⟩ = 0.042 and thus allowed monitoring the nucleation of seven RecA monomers (bottom, orange). (c) Structural dynamics of a four-way Holliday junction (4WJ) between two conformers (isoI and isoII). (d) In the isoI conformation, the donor and the acceptor are far apart from each other, resulting in a low FRET efficiency of ⟨E1⟩ = 0.039, whereas in the isoII conformation the reduced distance results in an average FRET efficiency of ⟨E2⟩ = 0.275 (construct 4WJ-A, top). One additional acceptor shifts the average FRET efficiencies of both conformations to ⟨E1⟩ = 0.046 and ⟨E2⟩ = 0.378, respectively (construct 4WJ-AA, bottom). (e) farFRET efficiency histograms of the pdT21-AA construct at different MgCl2 concentrations were reproduced with a kinetic model (black line, Supporting Information Note S1) to extract interconversion rates between isoI and isoII (top three). The rates, k12 and k21, of the 4WJ-AA decreased with increasing MgCl2 concentration (bottom, black open triangles and circles, respectively), while the ratio of the rates k12/k21 and the changes in Gibbs free energy, ΔG°, remained constant (bottom, red closed circles). Lines connecting the symbols are guides to the eye. The Gaussian fit center values are given in the panels with a localization error of ±0.005.

dynamics strongly depend on Mg2+ concentration. The 4WJ contained again a single donor (ATTO532) and either one (4WJ-A) or two (4WJ-AA) acceptors (ATTO647N) (Figure 2c). To report on long-range conformational changes, the position of the acceptor fluorophores was chosen such that the FRET efficiency in the isoI conformer translates into an interfluorophore distance around 10 nm, whereas in the isoII conformation the distance is reduced, resulting in a higher FRET efficiency. The FRET efficiency histogram of the 4WJ-A construct (Figure 2d, upper) at 10 mM MgCl2 shows a poorly resolved bimodal distribution indicating the two interconverting conformers. Using the 4WJ-AA construct at the same conditions (Figure 2d, lower), the addition of a second acceptor shifted the center positions of both conformers toward higher FRET efficiencies. Notably, the FRET efficiency of the conformer isoII was shifted more pronounced due to the nonlinear energy transfer dependency, leading to a separation of the two FRET distributions and an increase of the dynamic range of smFRET experiments. Reducing the Mg2+ concentration in a series of experiments with the 4WJ-AA construct lead to a marked acceleration in stacking conformer transitions (Figure 2e). We quantified the increase in conformational switching using a modified probability distribution analysis21,22 (PDA) algorithm for FRET efficiency histograms determined by the donor lifetime (Supporting Information Note S1). With the decrease of the MgCl2 concentration from 10 to 1 mM, the

interpreted as any interdye distance beyond 10 nm, thus bearing only limited information. The addition of a second acceptor at the ss-dsDNA junction (pdT21-AA) shifted the average FRET efficiency of the bare ssDNA overhang to ⟨E⟩ = 0.482 (Figure 2b, lower, white) and that of the matured RecA filament to ⟨E⟩ = 0.042 (Figure 2b, lower, orange). Thus, the second acceptor pushed the upper limit of smFRET beyond the 10 nm line, and conformational changes into previously undetectable distances became quantifiable, thereby extending the dynamic range of dual-color smFRET experiments. FarFRET experiments also allow the quantification of distances from measured smFRET efficiencies. We calibrated the FRET efficiency-distance dependence of multiple acceptors using the above-described dsDNA rulers (Supporting Information Figure S3). For the RecA filament on the pdT21-AA sample an ⟨E⟩ = 0.042 was determined, which can be converted to a distance of RfarFRET = 10.6 nm and is thus in excellent agreement with the distance Rcrystal = 10.7 nm extrapolated from the crystal structure.18 To explore the ability to use farFRET in assessing longdistance structural dynamics, we probed the conformational switching in a four-way DNA Holliday junction (4WJ), the central structural element in DNA recombination, which has also been applied as a nanometronom. 19,20 The 4WJ predominantly assumes two antiparallel stacked X-structures (isoI and isoII) whose milli- to submillisecond interconversion C

DOI: 10.1021/acs.nanolett.5b01878 Nano Lett. XXXX, XXX, XXX−XXX

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Nano Letters relaxation rate kobs = k12 + k21 of interconversion dynamics raises from 3.3 to 1725 s−1, while the ratio of the interconversion rates, and hence also the change in Gibbs free energy, remained constant (Figure 2e, lower). Taken together, by using an enhanced energy transfer mechanism based on multiple acceptors, farFRET can be readily used to monitor structures and conformational changes in biomolecular systems that surpass the conventional range in dual-color smFRET measurements. Our experiments showed that the attachment of already two acceptors increased the energy transfer to a level that allows quantification of FRET efficiencies and conformational dynamics for distances greater than 10 nm with an enhanced dynamic range. Thus, farFRET opens a new avenue for directly observing long-distance dynamics and conformational transitions in biomolecular complexes (e.g., filament-forming proteins, spliceosome, signaling cascades) and structural information on bionanotechnological assemblies. farFRET can become particularly useful in studying in vitro processes on extended structures of nucleic acids because of advanced labeling methods currently available for DNA and RNA. Nevertheless, an intelligent fluorophore bunch design (e.g., using dendritic multidomain architectures) with only one reactive group could also open new possibilities for efficient attachments at functional groups on proteins to reduce labeling reactions. Moreover, together with recently developed internalization approaches (e.g., microinjection,23 electroporation24) of dye-labeled biomolecules into living cells, farFRET can also prove valuable in studying long-range phenomena in cell-based experiments. In conclusion, singlemolecule fluorescence-based methods including photoinduced energy transfer (PET),25,26 protein-induced fluorescence enhancement (PIFE),27 single-molecule FRET, and now farFRET allow tunable distance sensitivity from subnanometers to distances bridging the current gap to super-resolution microscopy.



experiments, Anastasiia Vlasiuk for help with accessible volume simulations and Mario Avellaneda for creating the table of contents graphic artwork. We are grateful for insightful comments by Taekjip Ha, Sandro Keller, and Stefan Diez. This work was supported by the German Federal Ministry of Education and Research BMBF 03Z2EN11 and 03Z2ES1 (M.S.) and a scholarship by the Stipendienstiftung RheinlandPfalz (G.K.).



ASSOCIATED CONTENT

* Supporting Information S

Material and Methods, Note S1, Figures S1−5, and Table S1. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b01878.



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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (G.K.). *E-mail: [email protected] (M.S.). Author Contributions §

G.K. and A.H. contributed equally to this work. G.K. conceived of the idea. G.K., A.H., and M.S. designed experiments. G.K. and A.H. performed experiments. A.H. developed analytical tools and performed simulations for kinetic measurements. A.H. and G.K. analyzed data. All authors discussed results, wrote the manuscript and have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank all group members of the Schlierf group for helpful discussions, in particular, Philip Gröger for initial simulations. We want to thank Mengfei Gao for assistance in preliminary D

DOI: 10.1021/acs.nanolett.5b01878 Nano Lett. XXXX, XXX, XXX−XXX