âDNA Origami Traffic Lightsâ with a Split Aptamer Sensor for a Bicolor

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"DNA origami traffic lights" with split aptamer sensor for bicolor fluorescence readout Heidi-Kristin Walter, Jens Bauer, Jeannine Steinmeyer, Akinori Kuzuya, Christof M Niemeyer, and Hans-Achim Wagenknecht Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b00159 • Publication Date (Web): 02 Mar 2017 Downloaded from http://pubs.acs.org on March 3, 2017

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“DNA origami traffic lights” with split aptamer sensor for bicolor fluorescence readout Heidi-Kristin Walter,a Jens Bauer,b Jeannine Steinmeyer,a Akinori Kuzuya,c Christof M. Niemeyer,b and Hans-Achim Wagenknechta* a

Institute for Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany

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Institute for Biological Interfaces (IBG 1), Karlsruhe Institute of Technology (KIT), Hermannvon-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany

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Department of Chemistry and Materials Engineering, Kansai University, 3-3-35 Yamate, Suita, Osaka 564-8680, Japan

ABSTRACT: A split aptamer for ATP was embedded as recognition unit into two levers of a nanomechanical DNA origami construct by extension and modification of selected staple strands. An additional optical module in the stem of the split aptamer comprised two different cyanine-styryl dyes that underwent an energy transfer from green (donor) to red (acceptor) emission if two ATP molecules were bound as target molecule to the recognition module and thereby brought the dyes in close proximity. As a result, the ATP as target triggered the DNA

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origami shape transition and yielded a fluorescence color change from green to red as readout. Conventional atomic force microscopy (AFM) images confirmed the topology change from the open form of the DNA origami in the absence of ATP into the closed form in the presence of the target molecule. The obtained closed/open ratios in the absence and presence of target molecules tracked well with the fluorescence color ratios and thereby validated the bicolor fluorescence readout. The correct positioning of the split aptamer as the functional unit farest away from the fulcrum of the DNA origami was crucial for the aptasensing by fluorescence readout. The fluorescence color change allowed additionally to follow the topology change of the DNA origami aptasensor in real time in solution. The concept of fluorescence energy transfer for bicolor readout in a split aptamer in solution, and AFM on surfaces, were successfully combined in a single DNA origami construct to obtain a bimodal readout. These results are important for future custom DNA devices for chemical-biological and bioanalytical purposes because they are not only working as simple aptamers but are also visible by AFM on the single-molecule level.

KEYWORDS: energy transfer, dye, click chemistry, atomic force microscopy, nucleic acid, ATP

DNA nanotechnology applies self-assembly of DNA single strands to form molecular architectures in a highly programmed fashion.1-4 Especially the so-called “scaffolded DNA origami” method, in which long single-stranded DNA molecules are folded into designed nanostructures by short staple strands, is an increasingly powerful technique to create designed 2D and 3D DNA objects.5-9 In these objects, distinct sites are precisely addressable by chemically modifying the staple strands with functional units. This includes the incorporation of functional groups like amines, azides or thiols as well as binding sites for proteins like biotin,


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benzylguanine and chlorohexane which can be attached after the programmed self-assembly of the DNA origami structures.10-12 Embedding of functions, especially binding sites for small molecules into DNA origami yields custom DNA devices that combine the selective binding capability of aptamers with advanced analysis on the single-molecule level using atomic force microscopy (AFM) or electron microscopy.13-16 Komiyama and Kuzuya developed a nanomechanical DNA origami device that consists of two levers with ~170 nm length which are connected at a single and central fulcrum (Figure 1).17-19 Binding sites for metal ions, proteins, microRNAs, PNAs or small molecules can be embedded into both levers; the interplay and interactions of both binding sites with the target molecules trigger a shape transition of the DNA origami device to a fully closed or opened architecture, which can be visualized at molecular resolution by AFM. This first generation of constructs was equipped with fluorescent markers and quenchers to monitor the shape transition in real-time by fluorescence intensity changes at single emission colors, which always bears the risk of wrong readout due to non-specific fluorescence quenching. Recently, Dietz and coworkers developed DNA origamis with two emission color readout by energy transfer.20-22 Herein, we present a functional nanomechanical DNA origami device that combines a split aptamer as binding site for two molecules ATP with a wavelength-shifting fluorescent readout to follow the shape transition directly by a green-to-red fluorescence color change (Figure 1). The combination of two differently emitting fluorophores as covalent DNA labels and energy transfer pair follows our concept of “DNA and RNA traffic lights” that was firstly published based on a dye combination of thiazole orange and thiazole red as base substitutions23 and later significantly improved by photostable cyanine-styryl dyes as "clickable" 2'-modifications for optical modules of aptamers.24 This concept allows placing two fluorophores in very close proximity to each other (far below the Förster radius) without


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undesired self-quenching and with efficient energy transfer properties. These wavelengthshifting energy transfer systems do not respond to altered distances by simple quenching of fluorescence but rather, by large changes in fluorescence emission color with (red:green) contrasts of up to 60:1. Here, the implementation of the “traffic light” approach offers the advantage that target binding of the DNA origami aptamer can be followed simply by the emission color with enhanced signal-to-background ratios.

Figure 1. Principle design of “DNA origami traffic lights” with a split aptamer sensor (marked in green and red). Both parts of the aptamer carry one fluorescent dye of an energy transfer pair. The DNA origami aptamer binds two molecules ATP (yellow) and thus changes its shape and its fluorescence color from green (open, no energy transfer, green star) to red (closed, with energy transfer, red star) at the same excitation wavelength. Thus, visualization of successful target binding can be achieved by both topographic readout and steady-state fluorescence spectroscopy in solution. To test our concept (Figure 1), three different origami structures were designed (A-C, in Figure 2), all of which contained the energy donor dye 1 and acceptor dye 2 tethered to suited staple strands farthest away from the fulcrum (#66s and #178s, see SI). Construct A lacked the split aptamer strands but contained two extended variants of staples #74s and #170s to enable actuation of the origami by simple hybridization and strand exchange experiments. Two DNA origami constructs, B and C, were designed and contained the fluorophor-tagged strands


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connected directly to the split-aptamer (#66s-apt and #178s-apt, see SI). Construct C contained a second split-aptamer for ATP as modified staple strands at a different position of the levers (#74s-apt and #170s-apt, see SI).

Figure 2. DNA origami structures A-C with marked positions and sequences of the modified extensions of their staple strands (s) at positions #66s, #74s, #170s, and #178s, and structures of dye-anchor cU and of dyes 1 and 2. If the staple strands are equipped with the split aptamer they marked by “s-apt”; the split aptamer is shown in the annealed form according to the closed forms of the DNA origamis.


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To initially investigate the implementation of the wavelength-shifting “DNA traffic lights” concept and to validate that the two-color readout is observable as a result of the shape transition by closing the DNA origami, construct A was assembled in solution. Since, the overhanging ends of the extended staples #74s and #170s were complementary to each other, they spontaneously annealed and, therefore, construct A was in a closed conformation. As expected for the closing of DNA origami A, the fluorescence readout measured in solution by excitation of dye 1 at 435 nm showed an efficient energy transfer and thus a significant amount of the red fluorescent of acceptor dye 2 at 608 nm (IA) and only little contribution of the green fluorescence of donor dye 1 at 522 nm (ID) (Figure 3). The resulting red:green fluorescence intensity ratio IA/ID was 3.2(±0.5):1. Indeed, the corresponding AFM images, taken as topological readout on a mica surface, showed a large excess of the closed form of origami A. Statistical analysis indicated that about 81(±2)% were in a closed and 19(±2)% in an open form. This corresponds to a ratio of 4.3(±0.6):1 and tracks well with the observed fluorescence color contrast IA/ID as readout in solution. This quantitative comparison of the two different readout is valid because the recorded fluorescence spectra were corrected against both lamp output and detector sensitivity. Next, we tested whether an opening can be achieved by strand exchange and how this conformational change affects the spectroscopic properties. After addition of the single-stranded oligonucleotide “cs” to DNA origami A opened up and the energy transfer between the dyes was no longer possible due to the increased distance between the fluorophores. This counterstrand is complementary to the extended sequences of staple strands #66s and #74s; it anneals first with the overhanging 3’-ends (toeholds) of these two staple strands and induces the opening of their duplexes with the staple strands #178s and #170s. More than 5 equiv. cs were needed to effectively open DNA origami A. Upon addition of 15 equiv. cs, the fluorescence showed nearly


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exclusively the green fluorescence of dye 1 and only little red fluorescence; the recorded ID/IA (green:red) contrast as fluorescence readout was 5.1(±0.5):1. The corresponding AFM images showed 89(±2)% open and 11(±2)% closed form. This represents a ratio of 7.8(±1.1):1 as topological readout and again tracks reasonably well with the fluorescence color contrast that was obtained in solution. Furthermore, agarose gel electrophoresis of DNA origami A additionally supported the shape transition from the closed to the open form because a gel mobility shift was obtained upon addition of cs. These results showed that the energy transfer in this type of DNA origami devices works well and that the two different conformations of the DNA origami constructs can be simply distinguished by their fluorescence color (red=closed, green=opened).

Figure 3. AFM images of DNA origami A without (top left) and in the presence of 15 equiv. counterstrand cs (bottom left), agarose gel electrophoresis (top right) of closed (1) and open (2)


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form and corresponding fluorescence spectra (20 °C, bottom right) of titration with the complementary counterstrand cs. The white spots presumably are precipitations of salts or other buffer components. Next, we wanted to study if the fluorescence color change as optical readout can be obtained by a DNA origami as aptasensor. Out of the various ATP-specific DNA aptamers,25-27 we chose the central recognition module for the DNA origami aptasensor B as derived from a 27-mer sequence as adenosine and ATP binding DNA aptamer by Huizenga and Szostak (with dissociation constant for the non-split aptamer KD=6±3 µM).25 According to NMR-structural studies by Lin and Patel, this DNA aptamer binds two molecules ATP.26 In order to embed this aptamer into the two levers of the origami it was cut between nucleotide 14 and 15 resulting in a split aptamer,24 one strand on each side of the DNA origami levers. Binding of two target molecules ATP should cause closing of this origami and increasing the red fluorescence of dye 2 by energy transfer from dye 1. Initial hints for the ATP-induced shape transition were obtained by agarose gel electrophoresis of DNA origami B, i.e. a shift in gel electrophoretic mobility between the band in the absence of ATP and the band in the presence of 1 mM ATP. As expected, the spectra of DNA origami B in solution in the absence of ATP showed solely the green fluorescence of dye 1 (IA/ID=0.4(±0.15)) suggesting the presence of mostly open structures (Figure 4). Indeed, this result was confirmed by AFM images which revealed 93(±2)% open conformation of B on the mica surface. The addition of 1 mM ATP led to an increase in the red acceptor fluorescence, thus suggesting formation of closed DNA origami B. The observed IA/ID ratio (red:green) in solution was 1.2(±0.3):1 and corresponding AFM images on surface showed 56(±3)% of closed structures, indicating a closed:open ratio of 1.3(±0.2):1. Moreover, the closing procedure of DNA origami B can be followed in real time by the IA/ID ratio (red:green)


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as fluorescence readout. Biexponential fitting of the fluorescence readout reveals comparably fast initial rates for the fluorescence color change of k1=0.44±0.14 min-1 at 20 °C and of k1=0.32±0.05 min-1 at 30 °C that were assigned to the process of DNA origami closing because they were accompanied by an increase of acceptor fluorescence intensity. At 30 °C, the closing is efficient enough to nearly reach the IA/ID contrast value of 1.2 that was obtained under strict temperature control at 37 °C. The calibration of the fluorescence color contrast IA/ID with the isolated DNA aptamer of DNA origami B revealed that it is sensitive in a concentration range between 0.10 mM and 1.00 mM ATP (see Fig. S23 in Supporting Information). Although the melting temperature of the aptamer was estimated as approximately 42-45 °C in the absence of ATP,24 we avoided higher temperatures than 37 °C to not adversely affect the hybridization of the two and only partially complementary oligonucleotides. In order to demonstrate that DNA origami aptasensor B is not only sensitive but also selective for ATP as the target, the fluorescence was recorded in the presence of GTP as a non-complementary target, but with high structural similarity to ATP. As expected, the corresponding emission of aptasensor B in solution was nearly identical to that in absence of ATP and thus provoked no shape transition into the closed form by GTP as wrong target.


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Figure 4. AFM images of DNA origami B without target (top left) and with ATP (bottom right), fluorescence of DNA origami B in absence of the target and in the presence of ATP as right and GTP as wrong target (20 °C, top right), and the corresponding kinetics of the IA/ID fluorescence intensity ratio (red:green) recorded before (IA/ID=0.22 at 20 °C, 0.24 at 30 °C) and after 1 mM ATP addition at 20/30 °C (bottom right). The white spots on the AFM images presumably are precipitations of salts or other buffer components. It is important to mention that we also varied the position of the split aptamer strands inside additional DNA origami constructs D-F (see Supporting Information). The experiments with DNA origamis B, D, and E showed that positioning of the dyes nearer to the fulcrum resulted in less substantial changes in fluorescence readout properties, presumably due to a more pronounced, unwanted hybridization of the two strands of the split aptamer in the absence of ATP because of a closer distance of the two strands and a lower force exerted by the two levers.


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Especially in DNA origami F in which the aptamer is placed nearest to the fulcrum, the direct excitation of dye 2 at 535 nm yields a lower fluorescence intensity than that by energy transfer due to excitonic interactions between the dyes by the more pronounced unwanted hybridization.28 Taken together, these results showed that the positioning of the functional unit of this type of DNA origamis significantly influences the fluorescence readout. This works best, if the split aptamers were placed furthest away from the fulcrum, as it is the case in construct B. Despite the best positioning, the fluorescence of DNA origami B in the presence of 1 mM ATP showed still some of the green donor emission at 520 nm which was assigned to the open form. In order to enforce the ATP-driven closing, an additional DNA origami C was constructed derived from DNA origami B, that bears a second split aptamer as replacement of staple strands #74s-apt and #170s-apt. Those staple strands were not modified by dyes to avoid undesired excitonic interactions as discussed in the paragraph above.28 Remarkably, addition of 1 mM ATP to DNA origami C in solution yielded less green and more red fluorescence, thus an improved red:green fluorescence ratio of IA/ID=1.6(±0.3):1 (compared to 1.2(±0.3):1 for B). The corresponding closed:open ratio in the AFM image was 3.1(±0.7):1. The fluorescence and AFM imaging in the absence of ATP showed already a small amount of non-specifically closed structures of the DNA origami C, presumably due to the presence of the second split aptamer as extension of staple strands #74s and #170s that are nearer to the fulcrum (see Figure 2). In conclusion, we showed that a specific aptasensor for ATP can be embedded into a DNA origami construct and combined with the “DNA traffic light” concept in order to obtain a fluorescence color change in addition to conventional topological probing by AFM. The central recognition module for the aptasensor was derived from a known 27-mer sequence as adenosineand ATP-binding aptamer and embedded into the two levers of the nanomechanical DNA


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origami device by cutting it into a split aptamer (or: two single-strands). An additional optical module in the stem of the aptamer comprised two different dyes that underwent an energy transfer from green (donor) to red (acceptor) emission if two ATP molecules were bound as target molecule and thereby brought the dyes in close proximity. As a result, the ATP as target triggered the DNA origami shape transition and yielded the fluorescence color change as readout. Conventional AFM images confirmed the topology change from the open form of the DNA origami in the absence of ATP into the closed form in the presence of the target molecule. The obtained closed/open ratios in the absence and presence of target molecules tracked well with the fluorescence ratios IA/ID and thereby validated the fluorescence color readout. Moreover, the fluorescence color change allowed to follow the topology change of the DNA origami aptasensor in real time in solution. The correct positioning of the split aptamer together with the optical module as the functional unit into this type of DNA origamis farest away from the fulcrum was crucial for the aptasensing by fluorescence readout. Therefore, both methodologies, fluorescence energy transfer with a concomittant color change and AFM as topological readout were successfully combined within a single DNA origami construct to generate a novel type of sensor for target binding to a split aptamer. Typical aptamers, which can nowadays be produced for almost arbitrary targets, hold a great potential for bioanalytical applications.27,29 However, due to their relatively small size, they do not permit detection of target binding by atomic force- or electron microscopy, which offers highest sensitivity down to the level of single molecules. We therefore believe that our concept will be particularly useful for bimodal sensor platforms, wherein large sample numbers and volumes can be pre-screened with highest throughput by fluorescence to identify samples for in depth analysis with ultimate sensitivity, similar as previously described for label-free RNA hybridization assays.30


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Preparation of modified oligonucleotides as staple strands; Images of HPLC analyses and MALDI-TOF MS analyses; Preparation and characterization of the DNA origamis; List of staple strands; Staple strand combinations in DNA origamis A-F; Additional fluorescence results for DNA origamis C-F (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The project was funded by the Deutsche Forschungsgemeinschaft (GRK 2039/1). ACKNOWLEDGMENT


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Financial support by the Deutsche Forschungsgemeinschaft (GRK 2039) and KIT is gratefully acknowledged. HKW thanks the Karlsruhe House of Young Scientists for financial support of her research stay in Japan. CMN acknowledges financial support by the Helmholtz programme BioInterfaces in Technology and Medicine. We thank PD Dr. Patrick Weis (Institute of Physical Chemistry, KIT) for measuring ESI-MS. REFERENCES (1)

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GRAPHICAL ABSTRACT


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âDNA Origami Traffic Lightsâ with a Split Aptamer Sensor for a Bicolor

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