A DNA Walker as a Fluorescence Signal Amplifier - ACS Publications

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A DNA walker as fluorescence signal amplifier Dongfang Wang, Carolin Vietz, Tim Schröder, Guillermo P. Acuna, Birka Lalkens, and Philip Tinnefeld Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b01829 • Publication Date (Web): 28 Jul 2017 Downloaded from http://pubs.acs.org on July 28, 2017

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A DNA walker as fluorescence signal amplifier Dongfang Wang, Carolin Vietz, Tim Schröder, Guillermo Acuna, Birka Lalkens, and Philip Tinnefeld* Institute for Physical & Theoretical Chemistry, and Braunschweig Integrated Centre of Systems Biology (BRICS), and Laboratory for Emerging Nanometrology (LENA), Braunschweig University of Technology, 38106 Braunschweig, Germany. * Email: [email protected]; phone: +49 531 391 55243.

ABSTRACT. Sensing nucleic acids typically involves recognition of a specific sequence and reporting by e.g. a fluorogenic reaction yielding one activated dye molecule per detected nucleic acid. Here, we show that after binding to a DNA origami track a bound DNA target (a “DNA walker”) can release the fluorescence of many molecules by acting as catalyst of an enzymatic nicking reaction. As the walking kinetics sensitively depends on the walker sequence, the resulting brightness distribution of DNA origamis is a sequence fingerprint with singlenucleotide sensitivity. Using Monte-Carlo simulations, we rationalize that the random selfavoiding walk is mainly terminated when steps to nearest neighbors are exhausted. Finally, we demonstrate that the DNA walker is also active in a plasmonic hotspot for fluorescence enhancement indicating the potential of combining different amplification mechanism enabled by the modularity of DNA nanotechnology.

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KEYWORDS. fluorescence, signal enhancement, DNA origami, single-molecule detection, DNA walker, single nucleotide mismatch

In recent years DNA nanotechnology has experienced dramatic advances. Complex structures with megadalton molecular mass are assembled with almost atomistic control that allow the arrangement of nano-objects into functional units including drug delivery systems,1 plasmonic signal enhancers2 and molecular force clamps.3 Dynamic DNA nanotechnology enables complex functions4, biochemical control circles5, 6 and even molecular computing.7, 8 Beautiful examples of dynamic aspects of DNA nanotechnology are DNA walkers that mimic natural molecular motors by biasing Brownian motion with chemical energy.9-11 An autonomous linear molecular motor moves along a track unidirectionally without intervention.12,

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The driving force of

artificial DNA based motors typically originates from additional base pairing offered by fuel strands or from DNA hydrolysis. The abilities of molecular motors that in first iterations were limited to a few steps have strongly improved with respect to speed, traveled distances and further functionalities.11, 14-16 Besides the processivity and range of the walkers, new biophysical tools for their study have yielded direct insight into their functioning using atomic force microscopy and optical singlemolecule detection.17, 18 In most cases, the molecular motors were studied as models of natural molecular motors or as fundamental building blocks of bottom-up nanotechnology such as in nanorobots,19 in programmable molecular assembly lines16 and recently also for the amplified detection of nucleic acids.10, 11, 20


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Figure 1. a) Scheme of the DNA walker on the DNA origami. The track for the DNA walker is formed by extended staple strands in the DNA origami and hybridizing quencher labeled stator strands on top of the DNA origami. b) Fluorescence amplification mechanism of the DNA walker. The start site is marked by first hybridizing a single start stator (step 1) followed by target binding (step 2). The preparation of the DNA origami is finalized by hybridizing the remaining stators with quenchers (step 3; here 4 additional stator strands). The fluorescence amplification experiment is launched with the addition of the nicking enzyme (step 4) that cleaves the stator strand at a position marked by the target/stator duplex. After nicking, the walker proceeds to the next stator via toehold mediated branch migration (step 5 and 6). The autonomous walking steps are repeated until the end of the track is reached (step 7). Inactivating the enzyme and adding the imager strand finalizes the experiment (step 8). The number of bright, unquenched fluorophores corresponds to the number of steps the walker has moved. c) In the absence of target, the imager strands can also bind to the stators but their fluorescence is quenched, as the quenchers were not removed in the walking process.

In this work, we converted a nicking enzyme-assisted DNA walker15,

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fluorescence signal amplifier that can improve the detection of target molecules such as nucleic acids. From this perspective, the walker itself represents the target that catalyzes an amplification reaction by walking. The walker randomly moves on a rectangular DNA origami substrate while


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consuming the track with every step (self-avoiding walk). For each step, a quenching molecule is released so that after binding of fluorescently labeled imager strands, the final brightness of the DNA origami in a population is a measure of the path length travelled. The system might therefore also be referred to as step counter or fluorescence integrator. As progression probability for each step is dependent on the correct sequence the final brightness distribution is characteristic for the walker sequence with single-nucleotide sensitivity. Finally, we show that the linear signal amplifier is compatible with additional nanophotonic fluorescence enhancement in a plasmonic hotspot formed by two 80 nm gold nanoparticles. A two-dimensional, rectangular DNA origami22 of 100 nm length and 70 nm width was folded from one M13mp18-derived scaffold strand and 192 staple strands and served as platform for the walker’s track (see scheme in Figure 1 and AFM images in Figure S1a). One staple strand is extended by a specific DNA sequence at the 3’ end for indicating the starting position of the DNA walker. The principle of the walker and its realization as a linear fluorescence amplifier is shown in Figure 1 with the DNA origami represented as a gray bar, and the starting position as red bar (see supporting information for experimental details and sequences). For a track of four walking steps, four further staple strands are extended with identical sequences for the positioning of the track stators (Figure 1b, black extensions). After binding of the starting stator strand the walker/target is bound (steps 1 and 2 in Figure 1b). Next, the other stators are bound to the DNA origami. All stators carry a quencher (BBQ650) at their 5’ end indicated as black sphere in Figure 1. The differentiation of starting position and the other stators is necessary to proof processive walking without intermediate dissociation or binding of two target molecules. In applications for nucleic acid detection, the differentiation is not necessary, as it is not critical where on the origami a putative walker molecule binds and initiates processive walking. In


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addition, several steps for preparing the walking reaction could be automatized to reach similar simplicity as isothermal PCR reactions. After these preparatory steps, the DNA walker starts upon the addition of a nicking enzyme. The walker DNA-stator duplex contains a recognition sequence (5’GCAGTGNN3’, N is any nucleotide) that is nicked by the nicking enzyme yielding a short 6-nt duplex on one side (see step 4 in Figure 1b). Due to thermal instability after cleavage, the 6-nt oligo carrying the quencher is released from the stator. The target DNA then moves to an adjacent stator through toehold-mediated branch migration (steps 5 and 6 in Figure 1b). The newly formed target stator hybrid is again cleaved by the nicking enzyme so that the walking cycle is autonomously repeating along the track (step 7 in Figure 1b). After walking, the nicking enzyme is inactivated by paraformaldehyde. To visualize the successful walking a dye-labeled (ATTO647N) imager strand is added and hybridizes to the stator sequence. Only when the quencher is removed by the walking process, the ATTO647N of the imager strand can emit. In case that no target has bound, the imager strands also bind but fluorescence is suppressed by the presence of the quenchers.23 The processivity and functioning of the nicking enzyme assisted DNA walker is demonstrated by placing the track stators in different geometries on the DNA origami surface. In a first experiment, the stators are separated by 6 nm (Figure 2a) and in a second experiment, the stators are separated by 36 nm (Figure 2c). The experiments were performed on DNA origamis immobilized on a cover slip and therefore forced to be planar.24 This does not alter the linear distances between the stator strands. Due to the length of the DNA stators and the stiffness of the DNA origami, processive walking should only be facilitated for a stator separated by 6 nm.25 The experimental procedure depicted in Figure 1b was carried out for both preparations after origami immobilization by six biotinylated strands (yellow marks in Figure 2a,c) on cover slips


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coated with biotinylated bovine serum albumin (BSA) and neutravidin. Three green dye molecules (ATTO532, indicated as green spots in Figure 2a,c) were additionally incorporated in the DNA origamis to visualize the origami locations on the cover slip. We used two-color imaging with a single-molecule sensitive home-built confocal microscope for reading out the results of the DNA walking (see ref.26 and experimental section for details of the imaging). A 532 nm excitation pulsed laser was used to image the green ATTO532 origami marker to indicate the positions of the DNA origamis in Figure 2a,c. Excitation at 640 nm was used for visualizing the red ATTO647N imager strands. Yellow spots in Figure 2b,d indicate colocalization of DNA origamis and imager strands. It is immediately visible that more and brighter yellow spots occur for the DNA origamis with 6 nm step size (Figure 2b) compared to the 36 nm step size (Figure 2d). The fluorescence intensity of ATTO647N was analyzed from each colocalized spot and plotted as histograms in Figure 2e. For the 36 nm step length, the histogram has a peak at ~300 counts corresponding to single unquenched ATTO647N molecules. One imager strand can also bind and emit unquenched for the 36 nm sample as the imager strands can displace the target at the initial target binding position where the nicking enzyme cleaved off the oligonucleotide with the quencher. In addition, a minor contribution to fluorescent spots in Figure 2d arises from missing quencher molecules, prebleached/inactive quencher molecules or imperfect quenching.23 The number and brightness of the yellow spots of the 6 nm sample is substantially higher with a peak at about 700 counts. Alternatively, we determined the number of unquenched imager strands by placing individual DNA origamis in the laser focus and recording fluorescence transients until complete photobleaching occurred. Counting the number of bleaching steps of transients such as shown in Figure 2f yielded the histograms presented in Figure 2g. Zero photobleaching events indicate origamis with no


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ATTO647N molecule as the spots for taking the transients were selected based on the signal on the green ATTO532 channel. Clearly, a distribution with on average ~2.9 dye molecules (excluding the peak at zero) is obtained that reflects the multiplied probabilities of successful stator binding, successful walking and successful imager binding as well as the influence of premature photobleaching of molecules. In contrast, walking is suppressed for the 36 nm sample as indicated by the peak at one photobleaching step. Up to five steps (representing 5 dyes) are achieved for the 6 nm sample as exemplified in Figure 2f.

Figure 2. DNA walker on origami with different step length. a), c) Scheme of the DNA origami indicating starting position (red), stators (purple) and ATTO532 reference dyes (green) for step length of 6 nm (a) and 36 nm (c). b), d) False-color images after walking and hybridizing the imager strands. Green spots indicate DNA origami position, red spots show unspecific imager binding and yellow spots indicate colocalization of DNA origami and imager strands. e) Imager intensity histograms of colocalized spots from images such as in b) and d). The histogram is


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plotted as imager intensity versus frequency count. f) The fluorescence transients from DNA walker on DNA origami with different walking step size with laser intensity of 5.5 µW. g) Histograms of photobleaching steps from DNA walker with different walking step size.

In control experiments without nicking enzyme or without target DNA (Figure S2), we observed very few colocalized fluorescent spots of low intensity. The importance of the proximity of the stators demonstrates the processivity of the DNA walker and supports the suggested walking mechanism.15 These results also indicate the success of visualizing walking activity by cumulated fluorescence signals with the number of fluorescent molecules per origami being representative for the number of steps travelled by the walker. Next, we studied the extent of amplification that can be reached by the DNA walker on a single DNA origami. To this end, we designed the rectangular DNA origami with one starting point for the DNA walker (marked by a red dot in Figure 3a) and 183 stators as track (purple dots in Figure 3a). Three further stators were hybridized with ATTO550 labelled oligonucleotides for DNA origami identification (see green marks in Figure 3a). The target sequence can perform a random walk while consuming the track on the DNA origami. As only steps to adjacent stator strands are likely, the walker can come to a standstill once it has no unused stator in its immediate environment. Only green spots were detected before the walking had started by addition of the nicking enzyme in the presence (Figure 3b) and absence (Figure 3d) of target DNA, respectively. After walking, deactivating the enzyme and adding the imager strand, we observed bright yellow-red spots in the presence of target DNA (Figure 3c) whereas only a few yellow spots appeared in the absence of target DNA (Figure 3e). To estimate the number of ATTO647N molecules per spot we normalized the spot intensities to those of DNA origamis with only one ATTO647N molecule incorporated.27 The resulting histograms in Figure 3f show that the brightness of the DNA origamis in the absence of target has a typically fluorescence intensity corresponding to ~2 dye molecules. The brightness distribution in the


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presence of target shows the same peak at ~2 dye molecules and an additional distribution at higher values with a peak at ~20 dye molecules but extending to ~60 molecules. We attribute the distribution at ~2 dye molecules to DNA origamis that did not bind a target molecule and exhibit residual fluorescence due to imperfect quenching and missing or photobleached quenchers. Importantly, this background fluorescence is not significantly higher than unavoidable impurity and unspecifically bound molecules on the surface. Target binding and walking, on the other hand, yield DNA origamis that are brighter and exhibit a distribution clearly separated from the distribution of DNA origamis without target or without enzyme as depicted in Figure 3g. This indicates that low abundant DNA could be detected by single-origami signal amplification. The path of the DNA walker resembles that of a self-avoiding walk with some obstacles (staple strands without stators used for biotin binding or for binding the green reference dye corralled to the area of the DNA origami. We carried out Monte-Carlo simulations to rationalize the intensity distribution obtained in Figure 3f (for details, see supporting information, Figure S4a). Assuming that the DNA origami walker can proceed to up to six stators in its immediate proximity the reaction would terminate when no stator is in the walker’s nearest environment. The number of steps until that happens depends on the specific path taken and a broad distribution of step numbers is expected. The simulations yield the step number distribution shown in Figure S4a with an average of 32 walking steps for a 100% yield of walking (walking probability is defined as the ratio of the average walking rate constant in the presence of at least one neighboring stator divided by the sum of the walking rate constant and any additional rate constant that can terminate the walking including e.g. thermal dissociation.) and perfect labeling. Considering that the labeling yield with imager strands is not quantitative, the simulations


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reproduce the experimental values qualitatively well and might also indicate that walking steps bigger than 6 nm might rarely occur to yield brighter distributions15 (see Figure S4a).

Figure 3. DNA walker on rectangular origami with 184 stators. a) DNA walker design with one starting point (red spot) and 183 stators (purple spots). Three capture strands were hybridized with ATTO550 for co-localization (green spots). Six yellow spots represent staples 5’ labelled with biotin and not used for the DNA walker track. b), c) Confocal fluorescence images before and after walking. Image size is 10 µm × 10 µm. d),e) Reference images without target DNA. Image size is 10 µm ×10 µm. f) Count versus number of dye histogram of DNA walker with or without target DNA as determined from intensity histograms. The number of dyes on each origami is determined by normalizing the imager intensity to origami with a single dye. g) Average number of dyes (normalized imager intensity to origami with single dye) for the target DNA as well as in the absence of target DNA or enzyme. Standard errors (N(-E+T)=162, N(+E-T)=205, N(+E+T)=144) are given.

While other signal enhancement mechanisms6,

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such as the polymerase chain reaction

(PCR) can produce exponential signal growth compared to our linear fluorescence amplification there is an interesting notion about the linear signal amplification. PCR is very specific but as


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soon as an error e.g. in case of single-nucleotide variation occurs, this error propagates exponentially. In contrast, the DNA walker should interrogate the correctness of the sequence with every step. As the DNA walker uses toehold mediated migration, the length and binding efficiency of the toehold from the target DNA plays a key role during the walking. We therefore carried out similar experiments as shown in Figure 3 but varied the sequence of the walker by one to three nucleotides (see SI for sequences used).

Figure 4. Mismatch experiment on rectangular DNA origami with 184 stators. Average number of unquenched dyes (normalized imager intensity to origami with single dye) on DNA origami with perfect complementary target DNA, one, two and three mismatches of nucleotides of target DNA, no target DNA. Error bars represent standard deviation (SD, black) from three independent measurement and standard errors (SE, red) from the measurement with largest standard deviation.

Figure 4 shows the mean values of the intensity distributions obtained for the different walker sequences after 2 hours reaction time. In addition, the standard deviations reflecting the reproducibility and standard errors reflecting the precision of the average values are provided. To test whether the intensity differences between the DNA walker sequences is a result of slower walking kinetics we carried out analogous measurements for the perfect match, the 1 mismatch and the 3 mismatch DNA sequence for different reaction times of 0.5 h, 2 h and 4 h (see Figure S5). While the reaction for the perfect match is almost finished after two hours, saturation is not


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reached for the mismatched samples after 4 hours indicating that the differentiation between the DNA sequences is to some extent dependent on the reaction time. We used the Monte-Carlo simulations to also estimate the rate constant of walking assuming a single rate constant for steps to each of active neighboring stators (see SI for details). We obtained a walking rate constant of 0.0024 s-1 for the DNA walker. With a single mismatch, the rate constant is reduced to 0.0013 s-1 and three mismatches yield a value smaller than 10-4 s-1. With three mismatches, the DNA walker is very slow and the rate determination is imprecise by the effective binding at the starting position that yields one fluorescent dye and very slow fluorescence increase in the following. The data clearly show that although the differentiation between DNA sequences exploits the kinetics of the reaction it is not very sensitive to the specific point in time and the populations can be distinguished over minutes to hours. For single-nucleotide variation detection, each DNA origami represents essentially a distinct sample of a large population, thus providing a sufficient amount of data in one single experiment for reliable statistical analysis. We next elucidated whether the sensitivity of a DNA walker experiment could be increased by combining it with another signal enhancing mechanism such as plasmonic fluorescence enhancement. Plasmonic fluorescence enhancement can be achieved in the hotspot of an optical antenna. This was recently accomplished by creating optical antennas with docking sites using a pillarshaped DNA origami equipped with two 80-100 nm gold nanoparticles.2, 31 In this work, we modified a recently published optical antenna32 and incorporated the starting sequence and four track stators for the combination of linear signal amplification by walking and plasmonic fluorescence enhancement (see Figure 5a for sketch of the antenna equipped for walking and experimental section and SI for details on the optical antenna). In the center between the


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nanoparticles, five capture strands protruding from the original staple strands were inserted to bind the starting and track stators for the DNA walker. The distance between the stators is ~7 nm.

Figure 5. DNA walker in a DNA origami nanoantenna. a) Sketch of DNA walker in the plasmonic hotspot of a selfassembled dimer nanoantenna formed by two 100 nm gold nanoparticles. b) Confocal image of DNA walking in the plasmonic hotspot. Image size is 10 µm x 10 µm. Colocalized spots were selected and further excited with 640 nm laser to obtain fluorescence transients. c) Scatter plot of fluorescence enhancement versus lifetime of ATTO647N. The lines indicate the separation between nanoparticle dimers (τ < 1.0 ns), monomers (1.0 < τ < 3.0 ns) and no nanoparticle (τ > 3.0 ns). The enhancement is relative to one ATTO647N dye bound to DNA origami. d) Fluorescence transient of the red spot indicated in c) with the laser intensity of 1 µW. Four bleaching steps indicate successful walking in the hotspot.

For the DNA origami with walker in analogy to the walkers discussed in Figure 2 and 3, we obtained fluorescence images after walking as shown in Figure 5b. Again bright yellow-red spots appear indicating colocalization of ATTO647N (red) and ATTO542 (green) which was inserted to indicate origami positions. In addition, a fraction of green intensity is related to scattering from gold nanoparticle aggregates. We placed all colocalized spots representing ATTO647N nanoantennas successively in the laser focus and recorded fluorescence transients. Only spots


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that exhibited clear photobleaching signatures were used for further analysis. The intensity versus fluorescence lifetime histogram (Figure 5c) of colocalized spots that were additionally filtered by the quality of fluorescence transients reveals the three populations of dye only (fluorescence lifetime τ > 3 ns), single nanoparticle bound (1 ns < τ < 3 ns) and nanoparticle dimer (τ < 1 ns) (see Figure S7a,b for reference measurements and assignment of populations). Typical fluorescence enhancement values relative to fluorescence transients with one bleaching step are up to 15 fold and in individual cases up to more than 40 fold. Due to the dispersion of fluorescence enhancement values the mere intensity is not directly reporting on the walker’s activity in the plasmonic hotspot.2 Fluorescence transients were therefore analyzed for the number of bleaching steps and their fluorescence lifetime. The transient depicted in Figure 5d, for example, exhibits fluorescence enhancement of 14.1 fold and 4 bleaching steps clearly indicating walking. In addition, the fluorescence lifetime is 0.44 ns (see supporting Figure S8) indicating the presence of two nanoparticles. Together, these data demonstrate that walking in the hotspot of an optical antenna occurs and the amplification mechanism might be synergistically applied fully exploiting the modularity of DNA nanotechnology. We turned a DNA walker into a linear fluorescence amplifier that counts the number of walking steps. From a biosensing point of view, we converted a target that in common assays creates a single-dye signal by being a binding partner into a catalyst that creates many signaling molecules. In our random DNA walking on a rectangular DNA origami with a defined number of possible walking steps, the walker performs typically between 20 and 60 steps. Monte-Carlo simulations revealed that the walker is highly processive and that walking likely terminated when no intact stator strands are within immediate environment of the DNA walker. As the walker sequence is interrogated for each walking step the walking speed is dependent on the


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correct sequence and the rate constant for walking decreases by almost a factor of 2 for a single mismatch. Due to the repeated interrogation of the walker’s sequence, the obtained intensity pattern after a defined reaction time report on the correct sequence with single nucleotide sensitivity. Further unique features of the assay are that many experiments are carried out in parallel – essentially every DNA origami is one sample of a large population or can be seen as an ultra-small pixel of a DNA biochip. With many identical DNA origamis in the sample good statistics is obtained from a single experiment. Moreover, we demonstrate that the amplification mechanism is applicable in conjunction with plasmonic fluorescence amplification. The fact that each DNA origami walker creates a signal significantly higher than the background makes the approach appealing for sensitive DNA detection with single-nucleotide discrimination taking DNA walkers on DNA nanostructures one step further from academic models of molecular motility to useful nano-devices.

AUTHOR INFORMATION Corresponding Author * Email: [email protected]; phone: +49 531 391 55243. Notes The authors have filed a provisional patent application, EP16183555.8, on the described method of DNA-walker based signal amplification. ACKNOWLEDGMENT We thank J. Bohlen for AFM imaging. This work was funded by the BMBF (Pocemon) and the Deutsche Forschungsgesellschaft (AC 279/2-1 and TI 329/9-1). We acknowledge funding of


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the state ministry for research of Lower Saxony in the frame of the "Quantum- and Nanometrology" (QUANOMET) strategic research area. Quanomet is part of the LUH-TUBS research alliance. CV is grateful for a scholarship of the Studienstiftung des Deutschen Volkes. ASSOCIATED CONTENT Supporting Information Details on materials and methods including the preparation of the DNA origami samples, DNA walker assembly, the functionalization of gold nanoparticles, confocal measurement and analysis, Monte Carlo simulations, and supplementary Figures S1-S13. A list of all the staple strands and other DNA are included in Table S1-S5. REFERENCES (1) Douglas, S. M.; Bachelet, I.; Church, G. M. Science 2012, 335, 831-834. (2) Acuna, G. P.; Möller, F. M.; Holzmeister, P.; Beater, S.; Lalkens, B.; Tinnefeld, P. Science 2012, 338, 506-510. (3) Nickels, P. C.; Wünsch, B.; Holzmeister, P.; Bae, W.; Kneer, L. M.; Grohmann, D.; Tinnefeld, P.; Liedl, T. Science 2016, 354, 305. (4) Kuzyk, A.; Yang, Y.; Duan, X.; Stoll, S.; Govorov, A. O.; Sugiyama, H.; Endo, M.; Liu, N. Nat. Commun. 2016, 7, 10591. (5) Seelig, G.; Soloveichik, D.; Zhang, D. Y.; Winfree, E. Science 2006, 314, 1585-1588. (6) Zhang, D. Y.; Turberfield, A. J.; Yurke, B.; Winfree, E. Science 2007, 318, 1121-1125. (7) Nangreave, J.; Han, D.; Liu, Y.; Yan, H. Curr. Opin. Chem. Biol. 2010, 14, 608-615. (8) Qian, L.; Winfree, E. Science 2011, 332, 1196. (9) Omabegho, T.; Sha, R.; Seeman, N. C. Science 2009, 324, 67. (10) Yehl, K.; Mugler, A.; Vivek, S.; Liu, Y.; Zhang, Y.; Fan, M.; Weeks, E. R.; Salaita, K. Nat. Nanotechnol. 2016, 11, 184-190. (11) Jung, C.; Allen, P. B.; Ellington, A. D. Nat. Nanotechnol. 2016, 11, 157-163. (12) Yin, P.; Yan, H.; Daniell, X. G.; Turberfield, A. J.; Reif, J. H. Angew. Chem., Int. Ed. 2004, 43, 4906-4911. (13) Bath, J.; Green, S. J.; Turberfield, A. J. Angew. Chem., Int. Ed. 2005, 44, 4358-4361. (14) Liber, M.; Tomov, T. E.; Tsukanov, R.; Berger, Y.; Nir, E. Small 2015, 11, 568-575. (15) Wickham, S. F. J.; Endo, M.; Katsuda, Y.; Hidaka, K.; Bath, J.; Sugiyama, H.; Turberfield, A. J. Nat. Nanotechnol. 2011, 6, 166-169. (16) Gu, H.; Chao, J.; Xiao, S.-J.; Seeman, N. C. Nature 2010, 465, 202-205. (17) Tomov, T. E.; Tsukanov, R.; Liber, M.; Masoud, R.; Plavner, N.; Nir, E. J. Am. Chem. Soc. 2013, 135, 11935-11941.


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(18) Masoud, R.; Tsukanov, R.; Tomov, T. E.; Plavner, N.; Liber, M.; Nir, E. ACS Nano 2012, 6, 6272-6283. (19) Lund, K.; Manzo, A. J.; Dabby, N.; Michelotti, N.; Johnson-Buck, A.; Nangreave, J.; Taylor, S.; Pei, R.; Stojanovic, M. N.; Walter, N. G.; Winfree, E.; Yan, H. Nature 2010, 465, 206-210. (20) Qu, X.; Zhu, D.; Yao, G.; Su, S.; Chao, J.; Liu, H.; Zuo, X.; Wang, L.; Shi, J.; Wang, L.; Huang, W.; Pei, H.; Fan, C. Angew. Chem., Int. Ed. 2017, 56, 1855-1858. (21) Wickham, S. F. J.; Bath, J.; Katsuda, Y.; Endo, M.; Hidaka, K.; Sugiyama, H.; Turberfield, A. J. Nat. Nanotechnol. 2012, 7, 169-173. (22) Rothemund, P. W. K. Nature 2006, 440, 297-302. (23) Holzmeister, P.; Wunsch, B.; Gietl, A.; Tinnefeld, P. Photochem. Photobiol. Sci. 2014, 13, 853-858. (24) Schmied, J. J.; Raab, M.; Forthmann, C.; Pibiri, E.; Wünsch, B.; Dammeyer, T.; Tinnefeld, P. Nat. Protoc. 2014, 9, 1367-1391. (25) Teichmann, M.; Kopperger, E.; Simmel, F. C. ACS Nano 2014, 8, 8487-8496. (26) Acuna, G. P.; Bucher, M.; Stein, I. H.; Steinhauer, C.; Kuzyk, A.; Holzmeister, P.; Schreiber, R.; Moroz, A.; Stefani, F. D.; Liedl, T.; Simmel, F. C.; Tinnefeld, P. ACS Nano 2012, 6, 3189-3195. (27) Schmied, J. J.; Gietl, A.; Holzmeister, P.; Forthmann, C.; Steinhauer, C.; Dammeyer, T.; Tinnefeld, P. Nat. Methods 2012, 9, 1133-1134. (28) Dirks, R. M.; Pierce, N. A. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 15275-15278. (29) Nallur, G.; Luo, C.; Fang, L.; Cooley, S.; Dave, V.; Lambert, J.; Kukanskis, K.; Kingsmore, S.; Lasken, R.; Schweitzer, B. Nucleic Acids Res. 2001, 29, e118. (30) Li, J. J.; Chu, Y.; Lee, B. Y.-H.; Xie, X. S. Nucleic Acids Res. 2008, 36, e36-e36. (31) Puchkova, A.; Vietz, C.; Pibiri, E.; Wünsch, B.; Sanz Paz, M.; Acuna, G. P.; Tinnefeld, P. Nano Lett. 2015, 15, 8354-8359. (32) Vietz, C.; Lalkens, B.; Acuna, G. P.; Tinnefeld, P. New J. Phys. 2016, 18, 045012.

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