Programmed Switching of Single Polymer Conformation on DNA Origami Abhichart Krissanaprasit,†,§ Mikael Madsen,†,‡ Jakob Bach Knudsen,† Daniel Gudnason,† Werasak Surareungchai,§ Victoria Birkedal,† and Kurt Vesterager Gothelf*,†,‡ †
Center for DNA Nanotechnology (CDNA) at the Interdisciplinary Nanoscience Center (iNANO) and ‡Department of Chemistry, Aarhus University, DK-8000 Aarhus C, Denmark § School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkhuntien Campus, Bangkok 10150, Thailand S Supporting Information *
ABSTRACT: DNA nanotechnology offers precise geometrical control of the positioning of materials, and it is increasingly also being used in the development of nanomechanical devices. Here we describe the development of a nanomechanical device that allows switching of the position of a single-molecule conjugated polymer. The polymer is functionalized with short single-stranded (ss) DNA strands that extend from the backbone of the polymer and serve as handles. The DNA polymer conjugate can be aligned on DNA origami in three well-defined geometries (straight line, left-turned, and rightturned pattern) by DNA hybridization directed by single-stranded guiding strands and ssDNA tracks extending from the origami surface and polymer handle. We demonstrate switching of a conjugated organic polymer conformation between left- and right-turned conformations of the polymer on DNA origami based on toehold-mediated strand displacement. The switching is observed by atomic force microscopy and by Förster resonance energy transfer between the polymer and two different organic dyes positioned in close proximity to the respective patterns. Using this method, the polymer conformation can be switched six times successively. This controlled nanomechanical switching of conjugated organic polymer conformation demonstrates unique control of the shape of a single polymer molecule, and it may constitute a new component for the development of reconfigurable nanophotonic and nanoelectronic devices. KEYWORDS: nanomechanical switching, DNA origami, polymer−DNA conjugate, atomic force microscopy, Förster resonance energy transfer, strand displacement
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nanomaterials have been successfully aligned on DNA origami by hybridization between ssDNA conjugates of the materials and extended staple strands on the DNA origami. These materials include gold nanoparticles,8,12,13 silver nanoparticles,14,15 carbon nanotubes,16−18 and quantum dots.19,20 In addition, our group recently presented a soft and bendable DNA-functionalized polymer (APPV-DNA).21 The polymer is
he ability to precisely control the orientation of matter at the nanoscale is one of the main objectives of nanotechnology. Structural DNA nanotechnology and especially DNA origami1 provide the opportunity to form welldefined structures in two2,3 and three dimensions4−6 by selfassembly. Highly elaborate nanostructures can be formed via the self-assembly of a long single-stranded DNA (ssDNA) scaffold and a number of short synthetic oligonucleotides (staple strands) of designed sequences. The method enables organization and manipulation of nanomaterials,7,8 including biomolecules,9−11 with nanometer precision due to the unique positional addressability inherent to the method. Several © XXXX American Chemical Society
Received: November 1, 2015 Accepted: January 14, 2016
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Figure 1. Illustration of the procedure used for synthesizing poly(APPV-DNA). The partly deprotected APPV polymer is immobilized on a CPG solid support. Removal of the remaining silyl ether protection groups liberates hydroxyl groups that act as a starting point for solidphase oligonucleotide synthesis. The poly(APPV-DNA) is isolated after cleavage and deprotection in AMA (1:1 mixture of aqueous methylamine (40%) and ammonium hydroxide (30−33%)) followed by size exclusion chromatography. The method for purification of poly(APPV-DNA) is described in more detail in the Supporting Information.
molecular electronic circuitry and also for use in nanosensing. Additionally, the platform for the nanomechanical switching of conjugated polymers is based on self-assembly, and therefore, this work provides a new step toward the development of selfassembled electronic circuitry at the nanoscale.
furthermore conjugated and hence holds potential as an optical or electronic wire. The material is made from a conjugated (2,5dialkoxy)paraphenylenevinylene organic polymer backbone containing short ssDNA sequences extending from its backbone in a brush-type fashion. The poly(APPV-DNA) can be positioned on predesigned patterns in both two- and threedimensional DNA origami structures. Remarkably, versatile controlled shapes of poly(APPV-DNA) can be obtained. These include straight lines, 90° curves, U-shapes, circular shapes, helical shapes, and zigzag patterns. One of the great potentials of DNA nanotechnology is that it can be used for construction of programmable DNA nanodevices and controlled molecular dynamics of nanomaterial− DNA hybrids.22−30 Most such studies have focused on DNA walkers and nanomechanical DNA devices constructed of DNA only. However, in some cases, other materials, such as proteins, gold nanoparticles, or gold nanorods, have been involved in dynamic devices.9,25,26,28,30 Another level of control is to shape soft materials into well-defined conformations, and here we demonstrate control of the conformation of a conjugated organic polymer on DNA origami and switching between two predefined states. Conjugated organic polymers are interesting because they have the potential to function as optical wires, electronic semiconductors, or even metallic conductors.31,32 The nanomechanical conformational switching of poly(APPVDNA) demonstrated here is based on toehold-mediated strand displacements, and the different states are visualized using atomic force microscopy (AFM). By making use of the optical properties of poly(APPV-DNA), we realized a setup where switching between two different conformations of single polymer molecules on 2D DNA origami results in two different optical Förster resonance energy transfer (FRET) outputs. Multiple consecutive switching events can be performed, and hence, the design may have potential as an on/off switch in
RESULTS AND DISCUSSION The APPV-DNA polymer was prepared by first synthesizing the organic conjugated APPV backbone containing functional hydroxyl handles from each repeating unit as we have reported earlier.21 Then the polymer was immobilized on a controlled porous glass (CPG) solid support, allowing for the synthesis of oligonucleotides onto the polymer by automated DNA synthesis (Figure 1). The resulting poly(APPV-DNA) contains ssDNA extending from the majority of the repeating units, and the DNA serves as the polymer’s handle for hybridization with guiding ssDNA (gDNA) and ssDNA tracks on DNA origami. In this way, poly(APPV-DNA) can be immobilized in desired conformations on DNA origami based on sequence complementarity. We used rectangular DNA origami containing ssDNA tracks of 9 nucleotide (nt) extended staple strands serving to form predesigned patterns that can control the conformation of poly(APPV-DNA). For this purpose, we designed three geometrically distinguishable patterns of ssDNA tracks: (i) a straight line extending from the center of the edge to the center of the DNA origami orthogonal to the axis of the DNA helices of the origami, (ii) a 90° curve pattern with ssDNA track located on the left-hand side of the straight line (L-turned pattern), and (iii) approximate 140° curve pattern with extended ssDNA track placed on the right-hand side of the straight line design (R-turned pattern) (Figure 2). The sequences of the three ssDNA tracks are different. Only the straight line pattern of ssDNA has a sequence that is B
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Figure 2. Illustration of controlling the conformation of poly(APPV-DNA) on predesigned ssDNA tracks of staple strands extending from rectangular DNA origami. The polymer handle, extended short ssDNA (green), is complementary to the ssDNA track (light green) placed at the middle of the rectangular DNA origami. The left and right ssDNA tracks are represented in yellow and blue, respectively. Complementary sequences are shown in the same color and denoted as X and X*. For wiring the poly(APPV-DNA) on the L- or R-turned pattern, either LgDNA or RgDNA assists to control poly(APPV-DNA) conformation.
(Figures 3A,D and S2 and Table S2). It should be noted that since the AFM is recorded in solution, it is often only the part of the polymer that is immobilized on the origami by hybridization or on the mica surface that is visible, while the part of the polymer that extends from the origami into the solution is not visible. Due to the broad length distribution of poly(APPV-DNA), the relative percentage of L-turned and Rturned patterns was lower than that for straight line patterns. Yields of approximately 50 and 52% were observed for L-turned (Figure S3) and R-turned conformation (Figure S4), respectively. The total distance of straight line, L-turned, and R-turned ssDNA tracks have calculated values of 35, 60, and 70 nm, respectively, as expected from the DNA origami design. Having successfully established that poly(APPV-DNA) can be positioned on desired tracks on DNA origami by the use of guiding strands, we wanted to investigate the possibility of switching between the two conformations. For this purpose, toehold-mediated strand displacement was implemented via the use of guiding ssDNA (gDNA) and removing ssDNA (rDNA). During switching, the straight line pattern where the poly(APPV-DNA) is complementary to the staple strand extensions served as a hinge. The mechanism of the strand-displacementassisted switching is depicted in Figure 4A. The switching of polymer conformation occurs in two steps. First, the polymer is released from its track by hybridization of a remover strand with the guiding strand mediated by the 6 nt toehold. At this state, the polymer is only linked to the DNA origami via the straight line track that serves as a hinge. In the second step, poly(APPV-DNA) interacts with a new guiding strand that is added along with the remover strand. This guiding strand programs the polymer to bind to the other track and thereby facilitates switching. In this way, switching from the R-turned to the L-turned conformation and vice versa can be obtained by using two sets of guiding strands (LgDNA and RgDNA) and two sets of remover strands (removing strand of left and right guiding strand; LrDNA and RrDNA). In practice, the conformation was switched from the right-turned conformation
complementary to the polymer sequences. To wire the polymer on DNA origami with a straight line pattern, poly(APPV-DNA) was incubated at room temperature for 1 h with DNA origami and then characterized by AFM in solution. Due to the repulsion between the mica surface and the negatively charged phosphate backbone of both DNA origami and poly(APPVDNA), a Ni2+-containing buffer is required for DNA origami/ poly(APPV-DNA) adsorption and for obtaining clear visualization by AFM. For positioning of poly(APPV-DNA) on “L” or “R” patterns, additional guiding strands were required. Two different guiding strands, left guiding ssDNA (LgDNA) and right guiding ssDNA (RgDNA), were designed. These strands facilitate the binding to the L and R tracks, respectively. Both LgDNA and RgDNA consist of three main regions: (i) ssDNA track binding sequence, (ii) polymer binding sequence, and (iii) 6 nt toehold for poly(APPV-DNA) conformation switching based on toehold-mediated strand displacement (all DNA sequences are provided in Supporting Information Tables S4 and S6). Using these principles, we demonstrated control of the conformation of poly(APPV-DNA) with three different conformations following three tracks: straight line, L-turned, and R-turned tracks on predesigned DNA origami containing ssDNA extensions. To obtain binding to the “L” or “R” turn patterns, either a LgDNA or RgDNA is incubated with poly(APPV-DNA) and DNA origami before characterization. The resulting conformations of poly(APPV-DNA) were visualized by topography AFM, as shown in Figure 3A−C. After immobilization of DNA origami on mica, the tracks on the origami can be facing up or facing down. Due to the design of the DNA origami and the different angles of the R- and Lturned patterns, the face-up and face-down structures are easily distinguished from each other (Figure S1). In most cases, poly(APPV-DNA) aligned on DNA origami is facing up. For statistical analysis, both structures, facing up and facing down, were included. The percentage of well-aligned polymers that formed a straight line of a poly(APPV-DNA) pattern was 63% C
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Figure 3. Controlling the conformation of poly(APPV-DNA) immobilized on designed tracks on DNA origami. (A−C) Topography AFM images of poly(APPV-DNA) direction related to guiding sequences: (A) without guiding ssDNA, (B) with LgDNA, and (C) with RgDNA. For the straight line pattern, unbound polymer extending from the origami into the solution was not observed by AFM because of its high mobility in the solution. (D) Histogram of the percentage of poly(APPV-DNA) aligned on DNA origami in the presence of different linker strands. Data from the statistical analysis on these AFM measurements are shown in Table 2 in the Supporting Information. Scale bar is 100 nm.
to left-turned conformation by simultaneous addition of RrDNA and LgDNA to the right-turned conformation followed by incubation for 1 h. Before characterization by AFM, excess ssDNA was removed by spin filtration. Initially, we attempted to perform conformational switching of poly(APPV-DNA) on DNA origami immobilized on mica; however, only a small number of L-turned patterns were observed. Figure S5C shows AFM images of poly(APPV-DNA) conformation shifting from the R-turned to the L-turned pattern. A large number of high-contrast dots were observed in the AFM image after switching. Further investigation of switching on mica could not be carried out due to the presence of high amounts of these dot-like structures arising from impurities. To overcome the issue of surface contamination during switching on the surface, focus was turned toward performing the nanomechanical switching of poly(APPVDNA) conformation in solution. At first, poly(APPV-DNA) was guided to align on the R-turned pattern and then characterized by AFM (Figure 4B and Figure S6). Then, RrDNA and LgDNA were introduced and incubated with the solution containing the R-turned pattern of poly(APPV-DNA) on DNA origami. Before AFM imaging, excess RrDNA and LgDNA was removed by a 100 kDa spin-filter unit, and a few microliters of the sample solution were drawn for conformational analysis by AFM. The remaining solution was used for further switching experiments. Figure 4C and Figure S7 show L-turned conformation (first switching) as characterized by AFM after the switching process from R-turned to L-turned pattern. The efficiency of switching conformation from R- to Lturned pattern is 45%, as shown in Figure 4F. In a second
switching step, the conformation was switched back from Lturned to R-turned pattern. AFM images of this switching are presented in Figure 4D and Figure S8. The percentage of poly(APPV-DNA) aligned with the R-turned pattern is around 40%. A third switching was also carried out. However, only a small number of well-formed constructs were observed, and the sample size was insufficient for statistical analysis (Figure S9). An AFM image of poly(APPV-DNA) aligned with L-turned conformation at the third round of switching is shown in Figure 4E and Figure S9. More detailed information on statistical analysis of polymer switching is presented in Table S3. During the experiments, the sample was filtered to retain the origami after each step, and it is evident from the AFM images that only a very small amount of free polymer is carried through the switching series. Moreover, the amount of polymer removed by spin filtration was quantified using agarose gel electrophoresis. As shown in Figure S10A,B, approximately 85% of free polymer is removed during purification by spin filtration. In experiments where only RrDNA is added to polymers bound in the rightturned conformation, the polymers remain bound to the hinge region as shown by AFM (Figure S11). We thus argue that it is indeed the same polymer permanently linked to the linear hinge region that is switching. The decreasing amount of constructs observed by AFM characterization after successive rounds of switching is attributed to the dilution caused by the successive spin filtrations. This in turn resulted in the fact that only two successive rounds of switching could be characterized with statistical significance. To overcome the dilution effect, we decided to study the dynamic nanomechanical switching of poly(APPV-DNA) D
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Figure 4. Nanomechanical switching of poly(APPV-DNA) on DNA origami based on toehold-mediated strand displacement. (A) Schematic illustration of guiding/removing ssDNA-assisted switching of poly(APPV-DNA) conformation. Complementary sequences are shown in the same color and denoted as X and X*, respectively. The arrowheads represent the 3′ end of DNA. (B−E) Topography AFM images showing the switchable conformation of poly(APPV-DNA). Inset images show zoomed-in AFM images of L-turned and R-turned polymer conformation. (F) Percentage of polymer aligned on the different patterns during the switching process as characterized by AFM. Data from the statistical analysis on these AFM measurements are shown in Table 3 in the Supporting Information. Scale bars are 500 nm.
Alexa Fluor 594 is denoted “acceptor 1”, and the right corner staple strand with Alexa Fluor 647 is denoted “acceptor 2”. The precise positions of the acceptor fluorophores are shown in Figure S18. To obtain a high FRET signal, the acceptor was modified at the 5′ end of the extended staple strand that was positioned in close proximity to the polymer. The distance between acceptor and polymer backbone is approximately 2−3 nm (Figure S19). In order to avoid excitation of acceptor 1 when exciting poly(APPV-DNA), the polymer was not excited at its maximum excitation wavelength (525 nm). Instead, it was excited at 475 nm, where acceptor 1 is not excited. The emission spectrum of poly(APPV-DNA) when excited at 475 nm is shown in Figure 5A (left). For switching conformation of polymer in solution, the polymer was first aligned in the Lturned pattern. To switch from L- to R-turned conformation, a small volume of the mixture of concentrated LrDNA and RgDNA was added in order to avoid significant dilution effects and the consequent decrease in fluorescence intensity. When positioned in the L-turned conformation, poly(APPV-DNA) is
conformation using FRET. For this, we utilized the inherent fluorescent properties of poly(APPV-DNA) that can be efficiently excited at wavelengths between 230 and 290 nm and also at wavelengths between 450 and 550 nm, while its emission is strongest from 550 to 650 nm. The excitation and emission spectra of poly(APPV-DNA) are shown in Figure S12. We have previously demonstrated that FRET can occur between the polymer and an acceptor dye, incorporated into DNA origami using fluorescently modified staple strands.21 Here the optical properties of poly(APPV-DNA) along with the ability to precisely position dyes on DNA origami enable us to study nanomechanical switching using FRET. To achieve this, experiments were designed where poly(APPV-DNA) serves as the donor fluorophore while two small-molecule fluorophores (Alexa Fluor 594 and Alexa Fluor 647) with distinguishable emission serve as acceptor fluorophores. Two extended staple strands positioned at the left and right corner of rectangular DNA origami, respectively, were labeled with one of the acceptor fluorophores each. The left corner staple strand with E
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Figure 5. Characterization of polymer switching using FRET. (A) Left: emission spectrum of poly(APPV-DNA) (red solid line) and emission spectra of acceptors 1 (green dashed line) and 2 (purple dashed line). Poly(APPV-DNA) aligned on L-turned pattern (middle) and on Rturned pattern (right) is represented in orange and blue, respectively. Decomposed emission spectra of acceptors 1 and 2 are shown in green and purple, respectively. The excitation wavelength of poly(APPV-DNA) is 475 nm, and emission is measured from 500 to 800 nm. (B) Timedependent FRET measurement of nanomechanical switching. For FRET experiments, the initial state of poly(APPV-DNA) was the L-turned pattern. Fluorescence intensities of acceptor 1 measured at its maximum emission wavelength, 618 nm (yellow line), and acceptor 2 measured at its maximum emission wavelength, 670 nm (blue line), were used to follow polymer switching through energy transfer from the polymer to the acceptors. (C) FRET measurement of nanomechanical switching showing six successive events of programmed poly(APPV-DNA) switching.
the sample was not filtered between each step to avoid dilution issues, it cannot be ruled out that polymers in solution absorb and desorb at the origami and also contribute to part of the fluorescence signal. After ∼30 min, a complete toeholdmediated strand displacement switching of the polymer between the two conformations in solution was observed. Switching can be performed repeatedly, and it was possible to observe six successive controlled conformational switching events of poly(APPV-DNA) (Figure 5C). Photobleaching of poly(APPV-DNA) and of acceptors 1 and 2 is responsible for the observed decrease in fluorescence intensity and one of the factors limiting the number of possible subsequent switching events.
within FRET distance of acceptor 1 and energy can be transferred from poly(APPV-DNA) to acceptor 1. In this case, the fluorescence emission of acceptor 1 is observed in the range from 600 to 630 nm (Figure 5A, middle). Similarly, significant fluorescence intensity of acceptor 2 can be observed from 650 to 670 nm when poly(APPV-DNA) is aligned in the R-turned conformation (Figure 5A, right). In order to quantify the emission intensities of acceptor 1 and 2, the fluorescence spectra of nanomechanical switching constructs were decomposed using emission spectra of poly(APPV-DNA), acceptor 1, and acceptor 2 as components (see Supporting Information, Figures S13 and S14). The positioning of the acceptor dyes on the origami was varied to obtain a clear FRET signal (see Supporting Information, Figures S15−S17). Switching dynamics were investigated by recording the timedependent fluorescence signal of acceptors 1 and 2 excited through energy transfer from the polymer after the addition of switching strands (Figure 5B). In the initial state, poly(APPVDNA) was positioned in the L-turned conformation. The observed fluorescence intensities were high for acceptor 1 and low for acceptor 2 as expected. At the first switching, the timedependent FRET signal showed that, after addition of LrDNA and RgDNA, the fluorescence intensity of acceptor 1 significantly decreased, while the intensity of acceptor 2 increased gradually. This indicates that the polymer switched from the L-turned to the R-turned conformation. The fast fluorescence intensity decrease is likely due to the excess LrDNA compared to the initial LgDNA concentration. Subsequent addition of LgDNA and RrDNA resulted in a second switching step where the fluorescence intensities of acceptor 2 decreased, while that of acceptor 1 increased. Here the fluorescence intensity decay and rise times are comparable, indicating switching of the polymer on the DNA origami. As
CONCLUSION The poly(APPV-DNA) was positioned in well-defined patterns on DNA origami as programmed by ssDNA guiding strands. With this system, we performed controlled conformational switching of poly(APPV-DNA) on DNA origami based on toehold-mediated strand displacement. The switching was characterized with both AFM and FRET experiments, and we thereby demonstrate the first single-molecule nanomechanical switch based on conjugated organic polymers. We exploited the conjugated polymer as the donor fluorophore in FRET experiments and used this to demonstrate six successive switching processes of poly(APPV-DNA) conformation on DNA origami. This is an example of a nanomechanical switch that is capable of guiding the excitation energy of the polymer to either of two acceptors based on the programming. The conjugated polymer has the potential to function as an optical or electronic wire, and the ability to switch the conformation of such wires in a controlled fashion at the nanoscale is crucial for the development of functional nanodevices. Our study provides F
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pattern; DNA origami (20 μL, 10 nM in 1× TAE-Mg2+ buffer), poly(APPV-DNA) (10 μL, 2 μM in Milli-Q water), and RgDNA (10 μL, 8 μM in 1× TAE-Mg2+ buffer) were mixed and incubated at RT for 1 h. For characterization of polymer conformation, 5 μL of sample was taken and visualized by tapping mode AFM in liquid. Further switching experiments were performed using the remaining sample. In order to switch direction of polymer from R-turned to L-turned pattern, RrDNA (20 μL, 8 μM) and LgDNA (20 μL, 8 μM) were added into the remaining sample and subsequently incubated at RT for 1 h before purification and characterization. Excess ssDNA was removed using spin filtration by washing two times with 1× TAE-Mg2+ buffer using 0.5 mL centrifugal filters units, 100 kDa cutoff. Again, 5 μL of the sample was taken for characterization, and the rest of the sample was used for further experiments. To switch the direction from an L-turned to an R-turned pattern, LrDNA (20 μL, 8 μM) and RgDNA (20 μL, 8 μM) were added and then incubated at RT for 1 h before purification and characterization. Estimation of the Amount of Poly(APPV-DNA) Remaining after Spin Filtration. Poly(APPV-DNA) was aligned on DNA origami in the R-turned pattern (initial state). After that, the conformation of poly(APPV-DNA) was switched from R-turned to L-turned pattern followed by purification by spin filtration (first switching state). Samples of poly(APPV-DNA) in the initial and first switching states were then analyzed by agarose gel electrophoresis (1.5% agarose gel, 35 V, 3.50 h). Gel was prestrained with SYBR Safe. After electrophoresis, the gel was scanned on a Typhoon scanner (Amersham Biosciences). The fluorescence intensities of the scanned gels were quantified on an ImageQuant TL 7.0 (GE Healthcare). Characterization of Polymer Alignment on DNA Origami Using AFM. The sample (2 μL) was deposited on freshly cleaved mica and left for 1 min to promote strong absorption on the mica surface. Then 400 μL of imaging buffer (1× TAE-Mg2+ containing 5 mM NiCl2, 1× TAE-Mg-Ni) was added into the liquid cell. DNA origami was visualized by tapping mode AFM (Agilent AFM series 5100) using silicon nitride cantilevers (Olympus). All recorded AFM images were processed and analyzed by Gwyddion software (available online at http://gwyddion.net/). FRET Experiment. A sample of 80 μL of polymer aligned on DNA origami (20 μL of DNA origami (10 nM), 10 μL of poly(APPV-DNA) (2 μM), 10 μL of LgDNA (8 μM), and 40 μL of 1× TAE-Mg2+ buffer) was incubated at RT for 1 h. The sample was added to a 60 μL fluorescence quartz cuvette (Hellma Analytics). The excitation wavelength of poly(APPV-DNA) is 475 nm, and the emission spectrum is observed from 500 to 800 nm. In the initial state, the polymer is aligned in the L-turned pattern. To switch polymer conformation from L- to R-turned pattern, the mixture of LrDNA (2 μL, 100 μM) and RgDNA (2 μL 100 μM) was added to the cuvette and incubated at RT for 30 min before fluorescence emission spectra were recorded. For further switching from the R- to the L-turned conformation, the mixture of RrDNA and LgDNA was then added and incubated for 30 min. Fluorescence emission spectra were recorded with 0.5 s integration time and 1 nm wavelength interval at 25 °C. All of the fluorescence measurements were performed on a scanning spectrofluorimeter (Fluoromax 3, HORIBA Scientific). FRET data were analyzed using the freeware program “a|e”.33
proof of concept for a single-molecule polymer nanomechanical switch, which may be used in future self-assembled molecular optical and electronic circuitry.
METHODS Materials. M13mp18 scaffolds were purchased from New England Biolabs, Inc. Unmodified oligonucleotides were purchased desalted from Sigma-Aldrich. Fluorophore-tagged oligonucleotides (purified by HPLC) were purchased from Integrated DNA Technologies. All oligonucleotides were used as received without any further purification. Water used for all experiments involving DNA was purified on a Milli-Q Biocel system by Millipore. Amicon Ultra 0.5 mL centrifugal filters units, 100 kDa, were purchased from Millipore (USA). The concentrations of DNA and poly(APPV-DNA) were measured by a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Cambridge, MA). DNA origami and polymer immobilized on DNA origami were visualized by AFM (Agilent AFM series 5500). FRET measurements were performed using a fluorimeter (FluoroMax3, HORIBA Scientific). Polymer Synthesis. Poly(APPV-DNA) was obtained by oligonucleotide synthesis on immobilized PPV-polymers as described by Knudsen et al.21 Self-Assembly of DNA Origami. For self-assembly of DNA origami, ssDNA scaffold strand (M13mp18, 100 nM) and ssDNA staple strands (10 equiv) were mixed in 1× TAE-Mg2+ buffer (40 mM Tris-acetate, 1 mM EDTA, and 12.5 mM MgCl2, pH 8.3) to a final concentration of 10 nM. The structures were then self-assembled in a thermocycler (Eppendorf Mastercycler) using slow cooling according to the following procedure: (i) from 80 to 55 °C at −0.3 °C per 12 s, (ii) from 55 to 4 °C at a rate of −0.5 °C per 48 s, and (iii) kept at 4 °C. To purify DNA origami, excess staple strands were removed using spin filtration by washing twice with 350 μL of 1× TAE-Mg2+ buffer using Amicon Ultra 0.5 mL centrifugal filters units, 100 kDa cutoff. For AFM characterization, DNA origami was assembled without fluorophore-functionalized staple strands. DNA origami structures containing fluorophores were used for observing switching in FRET experiments. The positioning of fluorophore-tagged staple strands is depicted in Figure S18. Estimation of Polymer Concentration. The concentration of poly(APPV-DNA) was measured using a Nanodrop instrument. The concentration is based on absorbance at 260 nm and a calculated extinction coefficient of the polymer handle strand (9 nt). Assembly of the Polymer on Designed Tracks on DNA Origami. For wiring polymer in a straight line pattern on DNA origami, 20 μL of DNA origami (10 nM in 1× TAE-Mg2+ buffer) was mixed with 10 μL of poly(APPV-DNA) (2 μM, dissolved in Milli-Q water) and incubated at room temperature (RT) for 1 h before characterization by AFM. To control polymer conformation in the L-turned pattern on DNA origami, 20 μL of DNA origami (10 nM), 10 μL of poly(APPV-DNA) (2 μM), and 10 μL of LgDNA (8 μM, dissolved in 1× TAE-Mg2+ buffer) were incubated at RT for 1 h, and the sample was then visualized by tapping mode AFM in solution. Similarly, RgDNA was added instead of LgDNA in order to wire the polymer in an R-turned conformation. Switching of Conformation of Poly(APPV-DNA) on a Mica Surface. Poly(APPV-DNA) aligned in the R-turned pattern (2 μL) was dropped and left to immobilize on a freshly cleaved mica surface for 1 min. The poly(APPV-DNA) aligned on DNA origami was visualized by AFM. To switch the polymer pattern from R-turned to Lturned pattern, 10 μL of 8 μM RrDNA and LgDNA strands was added to the mica surface and incubated on the mica surface for 1 h. After the incubation, the mica surface was washed twice with 400 μL of 1× TAE-Mg2+ buffer to remove excess ssDNA, and 400 μL of 1× TAEMg2+-Ni2+ (40 mM Tris-acetate, 1 mM EDTA, 12.5 mM MgCl2, and 5 mM NiCl2, pH 8.3) was then added as an imaging buffer for visualization by AFM. Switchable Direction of Polymer in Solution. In the initial state, the polymer was first aligned on DNA origami in the R-turned
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b06894. Additional results, Figures S1−S19, and Tables S1−S6 (PDF)
AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. G
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ACKNOWLEDGMENTS The authors acknowledge financial support from the Danish National Research Foundation to Center for DNA Nanotechnology (DNRF 81). A.K. and W.S. acknowledge the National Research University Project of Thailand’s Office of the Higher Education Commission. V.B. acknowledges support from the Danish Council for Independent Research. We thank Rasmus P. Thomsen for providing nonmodified staple strands for DNA origami structures. REFERENCES (1) Rothemund, P. W. K. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440, 297−302. (2) Woo, S.; Rothemund, P. W. K. Programmable Molecular Recognition Based on the Geometry of DNA Nanostructures. Nat. Chem. 2011, 3, 620−627. (3) Rajendran, A.; Endo, M.; Sugiyama, H. Single-Molecule Analysis Using DNA Origami. Angew. Chem., Int. Ed. 2012, 51, 874−890. (4) Andersen, E. S.; Dong, M.; Nielsen, M. M.; Jahn, K.; Subramani, R.; Mamdouh, W.; Golas, M. M.; Sander, B.; Stark, H.; Oliveira, C. L. P.; Pedersen, J. S.; Birkedal, V.; Besenbacher, F.; Gothelf, K. V.; Kjems, J. Self-Assembly of a Nanoscale DNA Box with a Controllable Lid. Nature 2009, 459, 73−76. (5) Douglas, S. M.; Dietz, H.; Liedl, T.; Högberg, B.; Graf, F.; Shih, W. M. Self-Assembly of DNA into Nanoscale Three-Dimensional Shapes. Nature 2009, 459, 414−418. (6) Han, D.; Pal, S.; Nangreave, J.; Deng, Z.; Liu, Y.; Yan, H. DNA Origami with Complex Curvatures in Three-Dimensional Space. Science 2011, 332, 342−346. (7) Jones, M. R.; Seeman, N. C.; Mirkin, C. A. Programmable Materials and the Nature of the DNA Bond. Science 2015, 347, 1260901. (8) Kuzyk, A.; Schreiber, R.; Fan, Z.; Pardatscher, G.; Roller, E.-M.; Hogele, A.; Simmel, F. C.; Govorov, A. O.; Liedl, T. DNA-Based SelfAssembly of Chiral Plasmonic Nanostructures with Tailored Optical Response. Nature 2012, 483, 311−314. (9) Douglas, S. M.; Bachelet, I.; Church, G. M. A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads. Science 2012, 335, 831−834. (10) Busuttil, K.; Rotaru, A.; Dong, M.; Besenbacher, F.; Gothelf, K. V. Transfer of a Protein Pattern from Self-Assembled DNA Origami to a Functionalized Substrate. Chem. Commun. 2013, 49, 1927−1929. (11) Derr, N. D.; Goodman, B. S.; Jungmann, R.; Leschziner, A. E.; Shih, W. M.; Reck-Peterson, S. L. Tug-of-War in Motor Protein Ensembles Revealed with a Programmable DNA Origami Scaffold. Science 2012, 338, 662−665. (12) Shen, X.; Song, C.; Wang, J.; Shi, D.; Wang, Z.; Liu, N.; Ding, B. Rolling Up Gold Nanoparticle-Dressed DNA Origami into ThreeDimensional Plasmonic Chiral Nanostructures. J. Am. Chem. Soc. 2012, 134, 146−149. (13) Sharma, J.; Chhabra, R.; Cheng, A.; Brownell, J.; Liu, Y.; Yan, H. Control of Self-Assembly of DNA Tubules Through Integration of Gold Nanoparticles. Science 2009, 323, 112−116. (14) Pal, S.; Deng, Z.; Ding, B.; Yan, H.; Liu, Y. DNA-OrigamiDirected Self-Assembly of Discrete Silver-Nanoparticle Architectures. Angew. Chem., Int. Ed. 2010, 49, 2700−2704. (15) Schreiber, R.; Do, J.; Roller, E.-M.; Zhang, T.; Schuller, V. J.; Nickels, P. C.; Feldmann, J.; Liedl, T. Hierarchical Assembly of Metal Nanoparticles, Quantum Dots and Organic Dyes Using DNA Origami Scaffolds. Nat. Nanotechnol. 2014, 9, 74−78. (16) Maune, H. T.; Han, S.-P.; Barish, R. D.; Bockrath, M.; Goddard, W. A., III; Rothemund, P. W. K.; Winfree, E. Self-Assembly of Carbon Nanotubes into Two-Dimensional Geometries Using DNA Origami Templates. Nat. Nanotechnol. 2010, 5, 61−66. (17) Mangalum, A.; Rahman, M.; Norton, M. L. Site-Specific Immobilization of Single-Walled Carbon Nanotubes onto Single and H
DOI: 10.1021/acsnano.5b06894 ACS Nano XXXX, XXX, XXX−XXX
