Eliminating Spurious Zero FRET States in Diffusion-based Single

Subscriber access provided by Service des bibliothèques | Université de Sherbrooke

Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Eliminating Spurious Zero FRET States in Diffusionbased Single-molecule Confocal Microscopy Sourav Kumar Dey, John R Pettersson, Andrea Z. Topacio, Subha R. Das, and Linda A. Peteanu J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b00362 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 25, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry Letters

Eliminating Spurious Zero-FRET States in Diffusion-based Single-molecule Confocal Microscopy Sourav K. Dey,† John R. Pettersson, Andrea Z. Topacio, Subha R. Das, Linda A. Peteanu* Department of Chemistry and Center for Nucleic Acids Science & Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA). AUTHOR INFORMATION Present Addresses †

Department of Pharmacology, Weill Cornell Medical College, New York, NY, 10065.

Corresponding Author *Linda A. Peteanu, Email: [email protected]

1 Environment ACS Paragon Plus

The Journal of Physical Chemistry Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Single-molecule Förster resonance energy transfer (smFRET) of freely diffusing biomolecules using confocal microscopy is a simple and powerful technique for measuring conformation and dynamics. However a spurious zero-FRET population can significantly distort the measured histograms and lead to incorrect results, particularly in measurements of intrinsically low-FRET systems. Using a model system consisting of duplex DNAs, we show that there are two important contributions to the zero-FRET state: (1) formation of a dark triplet state of the acceptor dye and (2) presence of donor-only strands due to incomplete hybridization between donor- and acceptor-labeled strands. The combined strategy of using Trolox as a tripletstate quencher and labeling the same DNA strand with donor and acceptor dyes effectively eliminates the zero-FRET population, even for constructs with intrinsically low FRET efficiencies. This strategy allows us to perform smFRET experiments in a simple confocal microscope.

TOC GRAPHICS

KEYWORDS: Single-molecule FRET (smFRET), confocal microscopy, dual-labeled DNA, zero-FRET population, Trolox.


Page 2 of 21

Page 3 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The conformational and dynamical fluctuations of biological molecules at the bulk1–3 and single molecule levels4–7 are frequently measured using Fӧrster Resonance Energy Transfer (FRET). In single-molecule FRET (smFRET) experiments, individual dye-labeled biomolecules are probed either while immobilized on a microscope slide surface or matrix or while diffusing in solution.6–10 Numerous proof-of-principle experiments have demonstrated the reliability of translating the energy-transfer efficiency (EFRET) values into distance-based models of biomolecular conformation and dynamics.7,9–11 Measuring EFRET values of freely-diffusing biomolecules in solution using confocal microscopy is relatively straightforward and avoids surface-induced artefacts that may occur when molecules are immobilized on the surface.4,6,12 The first use of this method was to determine the conformation of duplex DNA molecules having 7, 12, and 19 base pairs between the donor (D) and acceptor (A) dyes (TMR and Cy5, respectively).4 As these molecules are conformationally-restricted, a single population with an EFRET value associated with the D-A separation on the DNA strand would be expected. However, in all cases, a second peak with FRET efficiency centered near zero was observed in addition to the expected FRET distribution. As none of the DNA structures examined would be expected to show a zero-FRET state based on their expected degree of rigidity, the observed zero-FRET peak is considered spurious. This phenomenon has been observed in other dual-labeled DNA molecules labeled with a variety of dye pairs, indicating that it is fairly universal.13,14 The zeroFRET state interferes with the characterization of the ‘true’ EFRET value, especially for systems with maxima at small EFRET values and/or those with broad FRET distributions.15 Importantly, zero-FRET peaks represent a false positive in experiments that use the loss of FRET to follow a biochemical process such as a enzymatic cleavage.4 Such spurious zero-FRET states are not limited to measurements on DNA samples. For example, Schuler et al. observed zero-FRET



population in smFRET measurements on polyproline molecules of varying lengths labeled with Alexa488 and Alexa594 dyes.16 It has also been observed in other smFRET experiments with dual-labeled proteins.17–19 To minimize the impact of the spurious zero-FRET population in smFRET experiments, several experimental techniques have been developed. For example, Kapanidis et al. implemented an alternating laser excitation (ALEX) scheme in which the donor and acceptor dyes are excited alternately while in the confocal volume by rapidly switching between lasers that can excite the donor and acceptor dyes.20–22 The FRET histograms are then calculated exclusively from molecules that show both the donor and acceptor peaks in a single burst. Another variation of this technique, called Pulsed Interleaved Excitation (PIE), uses nanosecond pulses to excite the donor and acceptor alternatively and can separate the donor and acceptor emission both spectrally and temporally.23,24 This technique can not only identify molecules without acceptors but can also reduce the crosstalk between different spectral channels. Seidel et al. developed a multi-parameter-based smFRET measurement in which several other properties of single diffusing molecules, such as donor lifetime and anisotropy, are also calculated.25–27 The zero-FRET states can then be identified as those having a donor lifetime similar to donor-onlylabeled sample. Although these techniques are useful for smFRET studies, their implementation increases the complexity and cost of the required instrumentation. Moreover, they do not decrease the population of sample giving rise to zero-FRET peaks but only allow the spurious states to be identified and discounted. Several previously published results have suggested that the zero-FRET peak results when the acceptor dye undergoes a photo-physical transition to the triplet state. For example, the zeroFRET population for dual-labeled polyproline oligomers was found to increase with increasing


Page 4 of 21

Page 5 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


laser intensity.16 Moreover, use of an oxygen scavenger system or a triplet-state quencher such as Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid) reduces, though does not completely eliminate, the zero-FRET peak in both immobilized13,28,29 and freely-diffusing13,15 DNA molecules. Trolox is the most commonly used triplet-state quencher for smFRET experiments with both immobilized and freely-diffusing biomolecules.28–30 Populating the triplet state of the acceptor decreases the FRET efficiency both because it is a poor FRET acceptor and because it increases the ground state recovery time (triplet-state shelving).16 While different photo-protection strategies have been used to eliminate triplet-state shelving, little attention has been given to the effect of the dye-labelling strategies on the appearance of the zero-FRET state. In the majority of published smFRET experiments with nucleic acids, the donor and acceptor dyes are conjugated onto separate strands which are then hybridized to generate a dual-labeled nucleic acid.4,15,28,31 With this strategy, a zero-FRET contribution can also arise if a fraction of the sample contains only the donor dye due to incomplete hybridization. However if both the donor and acceptor dyes were to be introduced on the same strand of nucleic acid, the donor-only fraction would be eliminated regardless of hybridization efficiency. Here we show that labeling the same strand of DNA in a duplex, in combination with the use of Trolox as a triplet state quencher, eliminates the spurious zero-FRET state. As a result, more symmetric nearly-Gaussian histograms are measured, even for constructs that exhibit very low FRET values (~20%).



Scheme 1. Structures of the dual-labeled DNAs used for this smFRET study. The terminally(DNA NA) and internally- (DNA NB and NC) labeled duplex DNAs have the donor and acceptor dyes on the same strand. The duplex DNA ND contains the donor and acceptor dyes on separate strands. N represents the number of bases-pairs (bps) between the dyes and ‘t’ indicates the location of the triazole linkage between the DNA and the donor or acceptor dyes (see SI Figure S1 for details). Several dual-labeled DNAs were synthesized with varying donor-acceptor distances by conjugating both the donor (Alexa 488) and acceptor (Alexa 594) dyes to the same single strand of DNA (ssDNA) and then hybridizing them to their complementary strands (Scheme 1). In addition to the terminally-labeled DNAs (DNA NA), a series of internally-labeled DNAs were prepared (DNA NB and NC) to show the generality of the method (Scheme 1). A combination of NHS ester chemistry and a Cu(I) catalyzed azide alkyne cycloaddition (CuAAC) reaction was used to label the DNAs with the dye pairs (SI Figure S1). The CuAAC reaction, which is highly efficient and orthogonal to NHS ester chemistry, has been extensively used to introduce chemical modifications to both DNAs and RNAs.32–34 In our strategy, the donor dye (Alexa 488) is always conjugated first to the DNA and then the acceptor dye (Alexa594) is conjugated to the donoronly labeled DNA to obtain the dual-labeled ssDNA (SI Figure S1). The hydrophobicity of the Alexa594 acceptor dye causes a large shift in the elution time of the dual-labeled DNA compared


Page 6 of 21

Page 7 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


to the donor-only (Alexa488) labeled DNA during HPLC purification (SI Figure S2). This allows efficient purification of the dual-labeled DNA free from any donor-only-labeled DNA (SI Figure S2 and S3). The HPLC traces and mass spectrographs of the dual-labeled DNAs do not show any visible peaks for donor-only contaminant strands (SI Figure S3). Next, the dual-labeled ssDNAs were hybridized to unlabeled complementary strands to generate dual-labeled duplex DNAs (Scheme 1). For each of the DNA constructs shown in Scheme 1, three dual-labeled DNAs were prepared with N represented by 7, 14 and 21 base-pairs between the donor and acceptor dye such that the predicted FRET efficiencies ranged from 90% to 20%. Finally, for comparison we formed a dual-labeled dsDNA with the donor and acceptor dyes on separate strands (DNA ND) (Scheme 1).



Figure 1. Representative smFRET time traces of dual-labeled DNA 14A in presence and absence of 1mM Trolox (a) Burst data for DNA 14A in absence of Trolox shows show several peaks with donor only fluorescence (indicated by *). (b) Burst data for DNA 14A in presence of 1 mM Trolox. Laser power is 400 µW. The smFRET measurements on freely-diffusing DNA molecules were performed using a confocal microscope by exciting the donor dye (Alexa488) using a pulsed 485 nm diode laser (see SI for details) and collecting the donor and acceptor emission simultaneously using two different detection channels. A representative time trace from a smFRET experiment for DNA 14A in the absence and presence of Trolox is shown in Figure 1. In ~70% of the fluorescent bursts, the donor and acceptor intensities appear simultaneously whereas the remainder show only donor signal while the acceptor signal is close to the background level (indicated by * in 8 Environment ACS Paragon Plus

Page 8 of 21

Page 9 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1). The latter correspond to EFRET values close to zero. As our HPLC data indicates no donor-only-labeled DNA 14A is present (see caption SI Figure S3), the zero-FRET population in this construct must arise from a dark state of the acceptor dye. For DNA 14A, the burst data in presence of Trolox shows very few donor-only peaks (Figure 1b). This indicates that the addition of the Trolox depopulates the dark state of the acceptor dye thereby enabling energy transfer. Similar effects were observed for all other dual-labeled DNAs (SI Figure S4 and S5). Effective depopulation of the dark state by Trolox demonstrates that it corresponds to the acceptor triplet state. All the data shown here were obtained at powers for which the signal is linear in the presence of Trolox (400µW or a power density of 243 kW cm-2 at the focus). From data such as that in Figure 1, the EFRET value for each molecule was calculated yielding FRET histograms for DNA 14D and 14A in the absence or presence of Trolox (Figure 2). In the absence of Trolox, DNA 14D shows a large zero-FRET population along with the expected EFRET value (~40%) (Figure 2a). Addition of 1mM Trolox reduces the zero-FRET population though a significant contribution remains (Figure 2b). The FRET histogram of 14A, in which the donor and acceptor dyes are on the same strand, also shows a zero-FRET population in the absence of Trolox (Figure 2c). However, the addition of 1mM Trolox eliminates the zero-FRET population revealing a single nearly-symmetric Gaussian distribution (Figure 2d).



Figure 2. Effect of dye labeling and Trolox on the zero-FRET population of dual-labeled DNAs. FRET histogram of DNA 14D in (a) absence and (b) presence of 1mM Trolox. FRET histogram of DNA 14A in (c) absence and (d) presence of 1mM Trolox. Laser power is 400 µW. For labeled molecules with intrinsically low EFRET values, the FRET histogram can be significantly distorted if a large zero-FRET population is present. We measured the FRET histograms of several DNAs having expected EFRET of ~20% both with and without added triplet quenchers (Figure 3). The three different DNAs without Trolox show a large number of events with FRET values of zero which skews the FRET distribution towards the left (Figure 3a,b and c). However, when Trolox is used, a single nearly-symmetric Gaussian distribution that is undistorted by a zero-FRET contribution is attained, even for constructs having a mean FRET value as low as 20% (Figure 3d-f). Moreover, this is independent of whether the DNA is terminally- or internally-labeled. Notably, the width of the Gaussian fit to the FRET histogram agrees with the theoretically-predicted width from shot noise (SI Table S2).6,17,35 Similar results were also obtained for all the other DNAs synthesized for this study (SI Figure S5), for RNAs (data not shown), and when dithiothrietol (DTT, 2.5mM) is substituted for Trolox (SI Figure S6) as a triplet-state quencher. These data clearly show that both the use of a triplet-state quencher


Page 10 of 21

Page 11 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and the dye-labeling strategy described here are necessary to achieve the expected Gaussian distribution of FRET values through the elimination of the zero-FRET contribution.

Figure 3. Representative FRET histograms of dual-labeled DNAs with an expected EFRET value of 20% in presence and absence of Trolox. FRET efficiency histograms of DNA (a) 21A, (b) 21B and (c) 21C without Trolox and (d) 21A, (e) 21B and (f) 21C with 1mM Trolox. Laser power is 400 µW. The red lines shows a Gaussian fit to the FRET histogram. An increase in the intensity of the zero-FRET peak at high excitation power is expected due to the reduction in FRET efficiency while the acceptor is in its triplet state. Campos et al. recently showed that this limitation on the useable cw laser power and the data rates for smFRET measurements is significantly mitigated by the use of Trolox combined with a reactive oxygen scavenger.15 These additives also decreased the zero-FRET contribution though it is still clearly visible in the published histograms.13,15 The effect of Trolox on the power dependence of the donor and acceptor fluorescence and the overall FRET efficiency in our constructs is seen from



the measured donor and acceptor photon counts as a function of laser power for a concentrated sample of DNA 14A (2nM, Figure 4).

Figure 4. Bulk fluorescence properties of dual-labeled DNA 14A. Laser-power dependence of fluorescence intensity of the (a) donor and (b) acceptor channels for DNA 14A with and without Trolox. Time-resolved fluorescence decay of the (c) donor and (d) acceptor dyes with and without Trolox measured at 400 µW laser power. The donor intensity is linear with power both in the presence and absence of Trolox (Figure 4a) up to the highest power attainable by our laser. This indicates the absence of laser-powerdependent non-radiative deactivation processes for the donor dye (Alexa488). The acceptor intensity, however, shows a non-linear dependence on power in the absence of Trolox at relatively low laser powers (~100 µW, Figure 4b). In the presence of Trolox, the acceptor intensity is almost linear with laser power (Figure 4b) up to ~400 µW. The difference in acceptor intensity with and without Trolox increases with power which results in a corresponding increase in the zero-FRET population at high power (SI Figures S7 and S8).4,16 The above data demonstrate that addition of Trolox enables smFRET measurements to be performed at relatively high peak laser powers with a high signal to noise ratio. Furthermore, we 12 Environment ACS Paragon Plus

Page 12 of 21

Page 13 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


show that it is possible to obtain high-quality histograms even if the number of photons is insufficient to produce high-quality fluorescence lifetime curves for single molecules. This is particularly useful if the EFRET value is low and therefore the expected decrease in lifetime is small or if the donor exhibits more than one emission decay component. As an illustration, bulk emission lifetime results for DNA 14A show there is only a modest decrease in the donor decay time with versus without Trolox (Figure 4b) that is due to enhanced energy transfer. As expected, Trolox has a negligible effect on the fluorescence lifetime of the acceptor (Figure 4d). The use of confocal microscopy for smFRET measurement of freely-diffusing molecules is a simple and powerful technique to detect sub-populations in a multi-component system. However the accuracy of such measurements is diminished by unwanted photo-physical processes that result in a zero-FRET peak. This is especially problematic when the intrinsic FRET values are low. Although previous studies have suggested different origins for this zero FRET state,6,17,36 we show clearly that the zero FRET population for this dye pair is due to population of the acceptor triplet state upon excitation by the donor combined with incomplete labeling of the biomolecules. When a triplet state quencher such as Trolox or DTT is included in the imaging buffer and the donor-acceptor pair is placed on the same DNA strand, the zero-FRET state is eliminated, even at relatively high excitation power. The use of dual-labeled DNAs with the donor and acceptor dyes on the same strand is shown to be essential to eliminating the unwanted zero-FRET contribution. With the advent of new and orthogonal labelling strategies, dual labeling of single DNA/RNA strands has become more readily accessible.34,37,38 This will lead to improved accuracy in measurements of lowFRET structures and conformations and decrease false positives in monitoring enzymatic processes such as cleavage reactions with the use of a simple confocal microscope.



EXPERIMENTAL METHODS Synthesis of dual-labeled DNAs: The precursor DNA strands for making the dual-labeled ssDNAs contained a 5'-C6-NH2 modification and either a terminal 3'-O-Propergyl modifications (for DNA NA) or an internal 2'-O-Propargyl modification (DNA NB and NC). These were synthesized using regular phosphoramidite chemistry and then deprotected following standard protocols. Then the donor (Alexa488) and acceptor (Alexa594) dyes were conjugated to the DNA using either NHS ester chemistry or CuAAC as required (see SI Figure S1 for details). The dual-labeled ssDNAs were purified using HPLC and characterized using MALDI mass spectrometry (SI Table S1). Then the dual-labeled ssDNAs were hybridized with the respective complementary strand (without any dye) by heating at 90˚C for two minutes and then at 65˚C for ten minutes and then cooling to room temperature over 1 hour. The hybridization reaction mixture contained 100 µL of 1 µM dual-labeled ssDNA and 1.5 µM of the complementary strand in 1X hybridization buffer (50 mM Tris.HCl (pH 7.5), 50 mM NaCl and 0.005% TritonX-100). Confocal microscope for smFRET experiments: The confocal microscope for the smFRET measurement is built on an inverted microscope base (Olympus IX-71) using epi-illumination with the addition of a confocal pinhole, and two-color time-resolved detection. A 485 nm pulsed diode laser (Picoquant LDH P-C 485) is used as an excitation source for fluorescent molecules. The laser beam is directed to the objective (Olympus oil immersion, 100x, NA1.3) using a 500 nm dichroic mirror. To eliminate the out-of-focus light from the confocal excitation volume a 75 µm pinhole is placed at the focal point of the emission. Following this, emission from the donor and acceptor is separated using a 580 nm dichroic mirror and then passed through bandpass filters (531/46 nm for donor and 641/75 nm for acceptor) to further reduce the background.


Page 14 of 21

Page 15 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Finally, the donor and acceptor emission is focused onto the active area of two single photon avalanche diodes (SPAD, Microphoton Devices) using two separate micro lenses. The signal generated in the SPAD detectors due to the fluorescent photons is first directed into a 4 channel router and then the output is fed into a TCSPC board (Picoquant). The smFRET data collected using this confocal microscope is analyzed using commercially available software (Symphotime 64, Picoquant) to generate lifetime data and FRET histogram. See supporting information for more detailed description. Measurement of bulk FRET data and smFRET histograms: For the bulk measurement, the duallabeled duplex DNAs were diluted to 2 nM concentration in 1X buffer (50 mM Tris.HCl (pH 7.5), 50 mM NaCl and 0.005% TritonX-100). Then 400 µL of the sample is placed in a homebuilt chamber consisting of a glass coverslip (22x22 mm, #1.5) at the bottom. The laser beam is then focused onto the solution and the bulk data is collected for 1 min. Following this, the solution of dual-labeled DNA in the chamber is diluted 40 times to reach a final concentration of 50 pM at which the smFRET measurements were performed. A typical smFRET measurement is performed for 10 mins which yielded 5000-10000 single bursts. The burst data is then analyzed using the Symphotime 64 software to calculate the FRET histograms.



ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publication Website and includes experimental methodology, detailed synthesis protocols for dual-labeled DNAs HPLC purification and MALDI characterization, instrumental setup, smFRET data on additional constructs including power dependences, and the effect of DTT on the zero-FRET peak intensity, and calculation of the expected smFRET distribution widths due to shot noise. AUTHOR INFORMATION Corresponding author *Email: [email protected] Present Addresses †

Department of Pharmacology, Weill Cornell Medical College, New York, NY, 10065.

Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS The authors thank Dr. Eric Wu for helpful technical discussions. This work was supported by NIH grant R01GM110414 and this is gratefully acknowledged.

REFERENCES (1)

Stryer, L.; Haugland, R. P. Energy Transfer: A Spectroscopic Ruler. Proc. Natl. Acad. Sci.


Page 16 of 21

Page 17 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


U. S. A. 1967, 58, 719–726. (2)

Murchie, A. I. H.; Clegg, R. M.; von Kitzing, E. ; Duckett, D. R.; Diekmann, S.; Lilley, D. M. J. Fluorescence Energy Transfer Shows That the Four-Way DNA Junction Is a RightHanded Cross of Antiparallel Molecules. Nature 1989, 341, 763–766.

(3)

Selvin, P. R. The Renaissance of Fluorescence Resonance Energy Transfer. Nat. Struct. Biol. 2000, 7, 730–734.

(4)

Deniz, A. A.; Dahan, M.; Grunwell, J. R.; Ha, T.; Faulhaber, A. E.; Chemla, D. S.; Weiss, S.; Schultz, P. G. Single-Pair Fluorescence Resonance Energy Transfer on Freely Diffusing Molecules: Observation of Förster Distance Dependence and Subpopulations. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 3670–3675.

(5)

Ha, T.; Enderle, T.; Ogletree, D. F.; Chemla, D. S.; Selvin, P. R.; Weiss, S. Probing the Interaction between Two Single Molecule S: Fluorescence Resonance Energy Transfer between a Single Donor and a Single Acceptor. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 6264–6268.

(6)

Deniz, A. A.; Laurence, T. A.; Dahan, M.; Chemla, D. S.; Schultz, P. G.; Weiss, S. Ratiometric Single-Molecule Studies of Freely Diffusing Biomolecules. Annu. Rev. Phys. Chem. 2001, 53, 233–253.

(7)

Roy, R.; Hohng, S.; Ha, T. A Practical Guide to Single-Molecule FRET. Nat. Methods 2008, 5, 507–516.

(8)

Moerner, W. E.; Fromm, D. P. Methods of Single-Molecule Fluorescence Spectroscopy and Microscopy. Rev. Sci. Instrum. 2003, 74, 3597–3619.



(9)

Okamoto, K. Introduction of FRET Application to Biological Single-Molecule Experiments. Int. J. Biophys. 2013, 3, 9–17.

(10)

Sasmal, D. K.; Pulido, L. E.; Kasal, S.; Huang, J. Single-Molecule Fluorescence Resonance Energy Transfer in Molecular Biology. Nanoscale 2016, 8, 19928–19944.

(11)

Joo, C.; Balci, H.; Ishitsuka, Y.; Buranachai, C.; Ha, T. Advances in Single-Molecule Fluorescence Methods for Molecular Biology. Annu. Rev. Biochem. 2008, 77, 51–76.

(12)

Deniz, A. A.; Laurence, T. A.; Beligere, G. S.; Dahan, M.; Martin, A. B.; Chemla, D. S.; Dawson, P. E.; Schultz, P. G.; Weiss, S. Single-Molecule Protein Folding: Diffusion Fluorescence Resonance Energy Transfer Studies of the Denaturation of Chymotrypsin Inhibitor 2. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 5179–5184.

(13)

Grunwell, J. R.; Glass, J. L.; Lacoste, T. D.; Deniz, A. A.; Chemla, D. S.; Schultz, P. G. Monitoring the Conformational Fluctuations of DNA Hairpins Using Single-Pair Fluorescence Resonance Energy Transfer. J. Am. Chem. Soc. 2001, 123, 4295–4303.

(14)

Dietrich, A.; Buschmann, V.; Meller, C.; Sauer, M. Fluorescence Resonance Energy Transfer (FRET) and Competing Processes in Donor-Acceptor Substituted DNA Strands: A Comparative Study of Ensemble and Single-Molecule Data. Rev. Mol. Biotechnol. 2002, 82, 211–231.

(15)

Campos, L. A.; Liu, J.; Wang, X.; Ramanathan, R.; English, D. S.; Muñoz, V. A Photoprotection Strategy for Microsecond-Resolution Single-Molecule Fluorescence Spectroscopy. Nat. Methods 2011, 8, 143–146.

(16)

Schuler, B.; Lipman, E. A.; Steinbach, P. J.; Kumke, M.; Eaton, W. A. Polyproline and


Page 18 of 21

Page 19 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The “Spectroscopic Ruler” Revisited with Single-Molecule Fluorescence. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 2754–2759. (17)

Merchant, K. A.; Best, R. B.; Louis, J. M.; Gopich, I. V; Eaton, W. A. Characterizing the Unfolded States of Proteins Using Single-Molecule FRET Spectroscopy and Molecular Simulations. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 1528–1533.

(18)

Mukhopadhyay, S.; Krishnan, R.; Lemke, E. A.; Lindquist, S.; Deniz, A. A. A Natively Unfolded Yeast Prion Monomer Adopts an Ensemble of Collapsed and Rapidly Fluctuating Structures. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 2649–2654.

(19)

Ferreon, A. C. M.; Gambin, Y.; Lemke, E. A.; Deniz, A. A. Interplay of Alpha-Synuclein Binding and Conformational Switching Probed by Single-Molecule Fluorescence. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 5645–5650.

(20)

Kapanidis, A. N.; Lee, N. K.; Laurence, T. A.; Doose, S.; Margeat, E.; Weiss, S. Fluorescence-Aided Molecule Sorting: Analysis of Structure and Interactions by Alternating-Laser Excitation of Single Molecules. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 8936–8941.

(21)

Lee, N. K.; Kapanidis, A. N.; Wang, Y.; Michalet, X.; Mukhopadhyay, J.; Ebright, R. H.; Weiss, S. Accurate FRET Measurements within Single Diffusing Biomolecules Using Alternating-Laser Excitation. Biophys. J. 2005, 88, 2939–2953.

(22)

Kapanidis, A. N.; Laurence, T. A.; Nam, K. L.; Margeat, E.; Kong, X.; Weiss, S. Alternating-Laser Excitation of Single Molecules. Acc. Chem. Res. 2005, 38, 523–533.

(23)

Müller, B. K.; Zaychikov, E.; Bräuchle, C.; Lamb, D. C. Pulsed Interleaved Excitation.



Biophys. J. 2005, 89, 3508–3522. (24)

Hendrix, J.; Lamb, D. C. Pulsed Interleaved Excitation: Principles and Applications. Methods Enzymol. 2013, 518, 205–243.

(25)

Widengren, J.; Kudryavtsev, V.; Antonik, M.; Berger, S.; Gerken, M.; Seidel, C. A. M. Single-Molecule Detection and Identification of Multiple Species by Multiparameter Fluorescence Detection 1. Anal.Chem. 2006, 78, 2039–2050.

(26)

Sisamakis, E.; Valeri, A.; Kalinin, S.; Rothwell, P. J.; Seidel, C. A. M. Accurate SingleMolecule FRET Studies Using Multiparameter Fluorescence Detection. Methods Enzymol. 2010, 475, 455–514.

(27)

Sindbert, S.; Kalinin, S.; Nguyen, H.; Kienzler, A.; Clima, L.; Bannwarth, W.; Appel, B.; Muller, S.; Seidel, C. A. M. Accurate Distance Determination of Nucleic Acids via Förster Resonance Energy Transfer: Implications of Dye Linker Length and Rigidity. J. Am. Chem. Soc. 2011, 133, 2463–2480.

(28)

Rasnik, I.; McKinney, S. A.; Ha, T. Nonblinking and Long-Lasting Single-Molecule Fluorescence Imaging. Nat. Methods 2006, 3, 891–893.

(29)

Ha, T.; Tinnefeld, P. Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging. Annu. Rev. Phys. Chem. 2012, 63, 595–617.

(30)

Cordes, T.; Vogelsang, J.; Tinnefeld, P. On the Mechanism of Trolox as Antiblinking and Antibleaching Reagent. J. Am. Chem. Soc. 2009, 131, 5018–5019.

(31)

Sabanayagam, C. R.; Eid, J. S.; Meller, A. Long Time Scale Blinking Kinetics of Cyanine Fluorophores Conjugated to DNA and Its Effect on Förster Resonance Energy Transfer. J. 20 Environment ACS Paragon Plus

Page 20 of 21

Page 21 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Chem. Phys. 2005, 123, 1–7. (32)

El-Sagheer, A. H.; Brown, T. Click Chemistry with DNA. Chem. Soc. Rev. 2010, 39, 1388–1405.

(33)

Paredes, E.; Das, S. R. Click Chemistry for Rapid Labeling and Ligation of RNA. ChemBioChem 2011, 12, 125–131.

(34)

Mack, S.; Fouz, M. F.; Dey, S. K.; Das, S. R. Pseudo-Ligandless Click Chemistry for Oligonucleotide Conjugation. In Current Protocols in Chemical Biology; John Wiley & Sons, Inc., 2016; pp 83–95.

(35)

Gopich, I.; Szabo, A. Theory of Photon Statistics in Single-Molecule Fӧrster Resonance Energy Transfer. J. Chem. Phys. 2005, 122, 1–18.

(36)

Best, R. B.; Merchant, K. A.; Gopich, I. V; Schuler, B.; Bax, A.; Eaton, W. A. Effect of Flexibility and Cis Residues in Single-Molecule FRET Studies of Polyproline. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 18964–18969.

(37)

Paredes, E.; Evans, M.; Das, S. R. RNA Labeling, Conjugation and Ligation. Methods 2011, 54, 251–259.

(38)

Rinaldi, A. J.; Suddala, K. C.; Walter, N. G. Native Purification and Labeling of RNA for Single Molecule Fluorescence Studies. In Methods in Molecular Biology; 2015; Vol. 1240, pp 63–95.


Eliminating Spurious Zero FRET States in Diffusion-based Single

Recommend Documents