Quantitative Measurement of Spatial Effects of DNA Origami on

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Quantitative Measurement of Spatial Effects of DNA Origami on Molecular Binding Reactions Detected using Atomic Force Microscopy Ping Zhang, Fei Wang, Wenjing Liu, Xiuhai Mao, Changchun Hao, Yi Zhang, Chunhai Fan, Jun Hu, Lihua Wang, and Bin Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 28, 2019

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ACS Applied Materials & Interfaces

Quantitative Measurement of Spatial Effects of DNA Origami on Molecular Binding Reactions Detected using Atomic Force Microscopy Ping Zhang,†,‡,‖ Fei Wang,#,‖ Wenjing Liu,†,‡,‖ Xiuhai Mao,# Changchun Hao,§ Yi Zhang,†,θ,* Chunhai Fan,†,# Jun Hu,θ,† Lihua Wang,†,θ,∥,* and Bin Li†,θ,* †Division

of Physical Biology & Bioimaging Centre, Shanghai Synchrotron Radiation Facility,

CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China ‡University

of Chinese Academy of Sciences, Beijing 100049, China

§Laboratory

of Biophysics and Biomedicine, College of Physics and Information Technology,

Shaanxi Normal University, Xi’an 710062, China #School

of Chemistry and Chemical Engineering, and Institute of Molecular Medicine, Renji

Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China θShanghai

Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China

∥Shanghai

Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry

and Molecular Engineering, East China Normal University, Shanghai, 200241, China

ACS Paragon Plus Environment

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KEYWORDS： DNA origami, spatial effect, atomic force microscopy, binding reaction, binding efficiency, binding rate ABSTRACT：The DNA origami is a ubiquitous nanostructure that can be used as a universal scaffold for constructing molecular motors, nanosensors, nanodrugs and optical devices. Understanding the inherent heterogeneity of DNA origami structures is crucial for optimizing the design of high-efficiency nanosized-devices. Here, we investigated the spatial effects of the DNA origami on binding reactions using atomic force microscopy. Protein complexes formed more efficiently at the vertex and rim than on the surface of the origami; surprisingly, the maximum difference in biotin-streptavidin binding efficiency was over 80%, and the change in binding rate was approximately 40-fold, suggesting the presence of distinct microenvironments at different locations on the DNA origami. Our findings are not only useful for the potential applications of the DNA origami, but also for clarifying differences in nanomaterials caused by non-uniform distribution or defects. 1. INTRODUCTION DNA origami has been widely used as a molecular platform in enzyme catalysis, plasmonics, microscopy, drug delivery and single-molecule analysis. Its excellent programmability allows precisely arranging various components such as nanoparticles, fluorophores, or biomolecules at the nanoscale1-7. To optimize the performance of DNA origami, a detailed understanding of location-related chemical and physical properties is necessary. Generally, DNA origami is assembled by folding a long, single-stranded scaffold DNA with several hundred complementary short staple strands8-14. Thus, the position of each nucleotide is well determined. The geometric locations on DNA origami could be divided into several parts, such as the surface, corner, vertex, and rim. The reaction properties may differ at these types of


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locations. For example, previous studies have shown that rectangular origami monomers could be linked through blunt-end stacking along the short edges. This could result in aggregation or uneven distribution in solution or on a surface, which is unwanted for developing DNA origamibased observing platforms15,

16.

Strategies such as omitting staple strands and adding four-

thymine hairpin loops to staple strands at the turning points have been employed to prevent this phenomenon16. In addition to the unwanted aggregation, lateral variations in protein-ligand reactions have tremendous effects on the overall performance of the constructed DNA origami and the obtained results17-21; and the placement and number of hydrophobic moieties coupled nanostructures have been shown to influence binding and insertion of those modified DNA nanostructures to lipid membranes22-24. In these cases, the local microenvironment of the DNA origami at different structural sites and the organization of the duplex within the DNA origami might be vital for the delicate construction of hierarchical superstructures, as well as for practical applications such as single-molecule chemistry, enzyme reactions, and quantum dot patterning2530.

Indeed, some studies have suggested that the microenvironment at different positions on the

DNA origami is not exactly the same, resulting in differences, e.g., in the kinetics and yield of reactions31, 32. Therefore, it is of particular importance to have a full understanding of variable microenvironments and accessibility of functional moieties within DNA origami nanostructures. However, there remains a lack of simple and suitable methods to quantitatively analyse local variations on the DNA origami at the nanoscale level. Quantitative analysis of the binding performance and molecular recognition on DNA origami could provide a physical insight into the unique characteristics of molecule-directed self-assembly super-nanostructures, which could be useful for DNA nanotechnology.


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To address this issue, we used two different biological technical systems—biotin-streptavidin (SA) and antigen-antibody (IgG)—to investigate the spatial effects of DNA origami on the speed and efficiency of binding reactions at the single-molecule level. We chose a sharp triangular origami for the biotin-SA reaction, as it is a widely used as a reaction platform and it has common features including a surface, vertex, and a specific corner domain. Within the corner region, the edges of two sides form a nanoscale room linked by unpaired thymine residues (0–3 thymine residues for DNA strands to minimise the strain on each helix). This sharp triangular origami provides multiple sites to study the spatial effects of DNA origami nanostructures. Atomic force microscopy (AFM) has been proved to be an excellent tool for high-resolution imaging of molecules on surface33-35, and it could provide direct topography characterization DNA origami-based nanostructures. The assessment using AFM of streptavidin bound to DNA origami decorated with biotin has been extensively studied26, 36, 37. In a previous study, we have developed a time-lapse AFM method to record the formation of biotin-SA complexes and discovered the phenomenon of ‘molecule threading’ on the DNA origami38, 39. Here, by designing specific binding locations on the DNA origami, we studied spatial variation-induced reaction differences between biotin and SA. By recording the dynamic binding process and calculating the binding efficiency, as well as binding rate, at specific sites on the DNA origami, we obtained the location effects on the formation of biotin-SA complexes on the triangular DNA origamis. Biotin-SA and digoxin-IgG reactions were also carried out on a rectangular DNA origami to test the universality of location effects of DNA origami with different shapes. 2. RESULTS AND DISCUSSION


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2.1 Locations designed for protein complex binding on DNA origamis. To avoid steric hindrance from nearby protein complexes, and to simultaneously cover the maximum number of different areas on the origami surface, we modified the triangular origami40 to bind seven SA molecules on seven staple strands. The seven biotin sites were distributed on the triangular DNA origami (Fig. 1a) in one of two patterns: either at the vertex and corners (i.e. at seams where two sides merge to form trapezoid edges), or on the surface. SA is a tetramer with a size of ~4 × 5 × 6 nm3. The shortest distance between two biotin molecules was between sites 3 and 6 (~22 nm) which provided sufficient space to accommodate biotin-SA complexes. For the digoxin-IgG reaction, we modified the rim and surface of a rectangular DNA origami to bind eight IgGs on 16 digoxins at the 5’-end or in the middle of fifteen staple strands. IgG has a size of ~20 nm41, 42. The distances from the digoxin molecules to the rim-rim and rim-surface were ~25.0 nm and ~24.0 nm, respectively (Fig. 1b), to provide sufficient space to accommodate digoxin-IgG complexes. We also produced a biotinylated pattern on the rectangular DNA origami for the biotin-SA reaction. To avoid variations in action caused by the different modified locations on the staple strands, we chose to modify the 5’-ends of the staple strands. All of the biotinylated biotin molecules were located at the 5’-end, except site 7, which was bound to a site at the 3’-end of the staple strand. To detect digoxin-IgG reactions, six pairs of digoxin molecules were modified at the 5’-ends of the staple strand; two pairs of molecules were modified nonuniformly, in which, one digoxin was located at the 5’- end and the other was located in the middle of the staple strands on the rectangular DNA origami. In addition, to reduce the deflection of modified molecules from their designed sites, we did not use any additional linkers. The modified triangular and rectangular origamis were adapted according to ref. 8 and the nucleotide sequences of biotinylated/digoxin-labelled staple strands and biotin/digoxin sites are


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listed as the Supporting Information Table S1-4. In order to better identify the positions of the designed sites, we applied twenty-eight bases to middle of the staple strands on the triangular and rectangular origamis to form dumbbell hairpins as work marker (Table S5, S6).

Figure 1. Schematic illustration of the locations designed for protein complex binding on DNA origamis. (a) Biotin-streptavidin (SA) complexes on a sharp triangle-shaped DNA origami. The DNA origami was designed with three sides (A, B, and C). Side A was modified with a marker (grey rectangle) for reference. Seven biotin molecules were attached to form two patterns. The first pattern was at the vertex (dashed pink rectangular) and three corners (dashed blue rectangular) connecting two adjacent sides of the origami: five biotin molecules were fixed to the end of five DNA helices (numbered 1–5, where sites 1 and 5 are on the vertices and sites 2–4 are on the corners where a difference of three to one unpaired thymine (dT) exists). The second pattern was placed on the surface of the central areas of sides B and C (purple); from the outside to the inside of the triangular sides, biotin molecules were bound to the second DNA helix at sites 6 and 7, respectively. (b) Eight digoxin-IgG complexes were bound on the surface (dashed purple rectangle) and rim (light blue rectangle) of a rectangular DNA origami.

2.2 Available individual biotin molecules at different locations of the triangular DNA origami surface adsorbed on a mica surface. To verify whether biotin molecules bound to the DNA origami adsorbed onto the mica surface were available for reactions, an excess of SA was added to the liquid cell, after which AFM imaging was immediately performed. The topography


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of the biotinylated DNA origami in the absence of SA is shown in Fig. 2a. We identified thirtyfour intact DNA origamis; most had the expected triangular shape. The interior and exterior angles were 60° the lengths of the inner and outer edges were 44 and 132 nm, respectively; and the width of the sides was 30 nm, with a loop at the edge of side C. A bright dot corresponding to the marker was on the surface of side A. The structural features of the DNA origami corresponded well with the designed geometry (Fig. 2a1). The height of the DNA origami adsorbed onto mica was ~2.0 nm (Fig. 2a2), which is similar to the estimated diameter of double-stranded DNA. Individual biotin molecules on one side of the origami could not be detected by AFM owing to their small size (244 Da). When an excess of SA was added to the imaging cell (Fig. 2b), we observed as many as eight dots on some origamis one on side A corresponding to the marker, and the others at the designed locations of the conjugated biotin molecules (Fig. 2b1). There were four to seven dots at the biotin sites on most of the origamis, with heights ~3.6 nm (Fig. 2b2), which is consistent with the size of SA, as reported in previous studies43-45. We therefore concluded that the dots were biotinSA complexes. The fact that most of the biotin sites (including sites 6 and 7) on the DNA origami appeared as bright dots immediately after incubation with a high concentration of SA indicated that each biotin site was able to bind SA, and that the most of biotin molecules were indeed directed towards the solution, and not the mica surface. The face-up origamis (i.e., those with the biotin-modified origami surface facing the solution, green arrow in Fig. 2a) allowed for the quantitative analysis of the effect of biotin location on the interactions between biotin and SA on the mica surface. Thus, all the following data were based on the face-up origami unless it was mentioned.


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Figure 2. Atomic force microscopy (AFM) images of biotinylated DNA origamis adsorbed onto a mica surface before and after adding streptavidin (SA). (a) Topographical features of DNA origami before adding SA. The facing-up (green circle) and facing-down (pink circle) origamis adsorbing on mica surface. (a1) Enlarged view of the area in the white square in (a). (a2) Height profile of the DNA origami along the red dashed line shown in (a1). (b) Topographical features of the DNA origami and biotin-SA complexes after adding SA. (b1) Enlarged view of the area in the white square in (b). (b2) Height profile of biotin-SA complexes along the red dashed line shown in (b1).

2.3 The dynamic process of biotin-SA formation on different locations of triangular DNA origami adsorbed on mica surface. It has been reported that the binding rate of SA to biotin is controlled by diffusion46. However, the current temporal resolution of AFM is not sufficiently high to capture the precise moment of binding. To record the dynamic process of biotin-SA complex formation on different biotin sites, we imaged continuously while adding SA solution at a molar ratio of ～1:15 (biotin:SA) to a liquid cell. We took precautions to


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ensure that the SA solution just touched the edge of the imaging buffer in the liquid cell to allow the SA solution to mix with the imaging buffer by diffusion. To record the process of biotin-SA complex formation in situ in real time, images were taken approximately every 4 min, and continued for 180 min (Fig. S1). As shown in Fig. 3a, time-lapse imaging showed an increase in the number of dots appearing at the biotin sites of the origamis over time. The rate of biotin-SA complex formation varied according to biotin location. The first biotin-SA complex was formed after ~52 min at site 5 (pink circle) on an DNA origami where the biotin molecule was located at the vertex between sides B and C. The second complex was detected after ~68 min at site 2 (black circle) at the inside angle between sides B and C. Subsequently, four complexes were observed at sites 1 (green circle), 3 (dark blue circle), 4 (light blue circle), and 6 (red circle); no binding occurred at site 7 (purple circle) after incubation for 180 min. As shown in Fig. 3b, the binding efficiency increased over time, and reached a maximum of ~90% at vertex sites 1 and 5; ~40–70% at corner sites 2, 3, and 4; and ~10% at surface site 6. Binding events were rarely detected until 40 min; thereafter, binding efficiency rapidly increased and reached a plateau at ~120 min for sites 1 and 5. Other groups showed a gradual increase at ~110 min, and some sites seemed reached a plateau after ~160 min. The observed variations in the binding efficiency (Fig. 3b, 3c) indicate that the location of the biotin molecule was critical for its reaction with SA. Sites 1 and 5 had more surrounding space than the other sites, and we clearly observed that the binding efficiency at sites 1 and 5 differed by ~10% for the first 90 min and after 120 min. The first difference was likely due to the difference in freedom in the two dimensions parallel to the origami surface: 300° for site 5, and 60° for site 1 (Fig. 3b). The second difference was likely caused by the effects of the AFM tip, as


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the position of site 5 was more vulnerable to scanning forces, which could potentially displace SA. Actually, as determined by Strauss et al., variations in the binding efficiency at different locations may also result from different incorporation probabilities depending on the location in the staple strand (ref.7). To determine in detail the differences between the interactions at sites 1 and 5, sites 9, 10, 11 on the three rims of the triangular DNA origami were also modified (Fig. S2). Ranking of the resulting binding efficiency was found to be 1>5>rim (9, 10, 11). The mechanism underlying these resulted needs to be explored further. Interestingly, the surface position sites 6, 7, and 8 also showed differences in binding efficiency, but not as we expected. Ranking of the observed binding efficiencies was 6>7>8, and not 6>8>7 (Fig. S2 c, right). Both the corner and seam effects of the DNA origami would contribute to this result. For the surface site 7, which is near the seam, the electrostatic effect may be different because of the relatively sparse DNA helices near the seam compared to the surface sites 8 and 6. It should be noted that we recorded the binding events were sites 1 after 2 during the first 80 minutes (Fig. 3a). We thought these binding events showed a reasonable fluctuation for a bulk reaction system. Importantly, the final binding efficiency presented as a function of time (Fig. 3b) indicated a tendency of binding to site 2 after site 1.


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Figure 3. Dynamic formation of individual biotin-SA complexes at different locations of the DNA origami adsorbed on a mica surface. (a) Representative AFM images of biotin-SA complexes formed on the origami after incubation for 3 h at room temperature. The first SA-biotin complex formed among the seven biotin sites are marked by coloured circles and shown in the enlarged images, respectively. (b) Binding efficiency between biotin and SA as a function of time. (c) Schematic of the position effect of the seven biotin sites on the efficacy of formation of biotin-SA complexes on the triangular-shaped DNA origami. Higher detection binding efficiencies (‘hotspot’, marked in red) generally occurred at the corners and vertex of the structure, and lower detection efficiencies (marked in bottle-green) were seen towards the centres of the DNA origami. (n = 22 DNA origamis)


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The topographical differences of the DNA origami between sites 2, 3, and 4 were not clearly visible through AFM imaging, but the observed variations in binding rate and efficiency were clear, possibly owing to the number of unpaired thymine molecules in the corner areas. Site 4 had only 1-dT, less than sites 2 and 3 (3-dT and 2-dT, respectively), resulting in a slightly less flexibility or/and smaller hole on the origami. To provide quantitative information on the binding reaction rate of biotin-SA, after SA was injected into the centre of the liquid cell using a syringe, AFM images were collected immediately (Fig. S3). We calculated the second order reaction rate (kb) using a well-established method: the Simbiology Toolbox in Matlab. The site-related kb was shown in Fig. 4. The sites on the vertex (for example: No. 1) demonstrated the fastest kb (7.1 × 105 M-1s-1), while the site on the surface (for example: No. 7) exhibited a greatly reduced kb (1.8 × 104 M-1s-1). A difference of ~40-fold was observed (Fig. 4a). The distribution of the kb on the triangle-shaped DNA origami was plotted in Fig. 4b, and generally followed the trend of vertex>corner>surface. Furthermore, a difference of ~2 fold was found between sites 6 and 7 (Fig. 4c). Both sites were in the centre of one side of the triangular-shaped DNA origami surface, but with a difference of ~10 nm relative to the triangle centre (although the seam effect on the site 7 may cause the difference as mentioned above). In addition, up to a ~1.5–2 fold difference was observed between sites 2, 3 and 4, while the number of dT in the corner differed by 3, 2 and 1, respectively, as mentioned above (Fig. 4d), indicating a local environment influence on the binding reaction.


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Figure 4. Binding kinetics is strongly related to the site of biotin on the surface of the DNA origami. (a) Distribution of binding rate for seven biotin sites. A difference as large as ~40-fold was observed. (b) Outline of the biotin-SA binding rate on the triangular DNA origami. (c) A ~2 fold difference was found on the DNA origami surface with a differing distance of ~10 nm relative to the triangular centre. (d) A ~1.5–2 fold difference was found with a difference of dT molecules in the corner.

2.4 Binding behaviour of SA to biotin attached at different locations of triangular DNA origami in solution. To test whether the differences in binding behaviour would also be visible on a DNA origami in solution, we chose typical sites representing the surface and corners of the DNA origami to conduct further biotin-SA reactions in solution. We indeed observed similar behaviour. More dots were observed on site 2 than on site 6. The differences in binding efficiency were maintained over a wide range of concentrations of SA; differences were observed at a range of molar ratios of biotin to SA of between ~1:10and ~1:160 (Fig. S4).


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To determine the binding constant in solution, affinity experiments using biotin-SA complexes were conducted at sites 1, 3, and 7, which represent the locations of vertex, corner and surface, respectively. The dissociated constants (Kd) which is 1/Ka, for the three sites were all similar (~5.0 nM, ~5.0 nM and ~6.3 nM, respectively) (Fig. S5), indicating only minor differences in binding strength between them. 2.5 Binding behaviour of SA to biotin attached at different locations of the rectangular DNA origami on mica surface and in solution. To further test the contribution of modification sites on the DNA origami in the binding behaviour between the surface and vertex of the DNA origami for the targeted molecules, biotin-SA binding reactions were also conducted on the rectangular DNA origami on the mica surface and in solution. SA was added to the biotinylated DNA origami adsorbed onto a mica surface or to the biotinylated DNA origami in solution. AFM images showed the formation of a single biotin-SA complex on the designed sites of the DNA origami after reacting with SA (Fig. S6, S7). Statistical data showed a higher biotin-SA binding efficiency at biotin molecules fixed on the rim and vertex compared to those on the surface of the DNA origami (Fig. 5). The results were similar to those of the biotin-SA reaction on the triangular DNA origami, providing further evidence that the site of modified moieties affects the reaction behaviour.


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Figure 5. Binding efficiency of SA to biotin molecules at different locations on the DNA origami adsorbed on a mica surface and in solution. (a) Schematic representation of sites modified with biotin on rectangular DNA origami. (b) Binding efficiency at the vertex, rim and surface of DNA origami on a mica surface. (n = 101 DNA origamis). (c) Binding efficiency at the vertex, rim and surface of DNA origami in solution (n = 89 DNA origamis). Error bars presented standard deviations.

2.6 Binding behaviour of IgG reacting with its epitopes modified on rectangular DNA origami in solution. To investigate the correlation between surface heterogeneity and the behaviour of other biomolecules, the binding of digoxin with its IgG antibody was explored using DNA origami as the supporting scaffold; small digoxin molecules (780 Da) were positioned at the designed sites as shown in Fig. 6a. AFM images showed the formation of individual digoxin-IgG complexs on the surface and at the rims of the DNA origami after the reaction with IgG (Fig. 6b). The measured distance between two Fab domains in an enlarged image (white square in Fig. 6b) was 11.0 nm (Fig. 6b1), consistent with the designed distance. Cross-sectional profile analysis showed that the height of the Fab domains was 3.1 nm (Fig. 6b1). The sizes of IgG spots were consistent with the spots obtained by AFM in solution. Almost every complex formed at the digoxin-modified sites on the DNA origami. Generally speaking, a higher IgG binding efficiency was found at digoxin molecules fixed on the rims compared to those on the surface of the DNA origami. The binding efficiency of IgG molecules on the rims was approximately double that of the binding efficiency on the surface of the DNA origami under this designed digoxin pattern (Fig. 6c, d, and Fig. S8).


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Figure 6. Formation of digoxin-IgG complexes on a rectangular DNA origami in solution. (a) Outline of the locations of digoxin on the surface (purple rectangle) and rim (light blue rectangle) of the DNA origami. (b) AFM topography images of IgG and the DNA origami. (b1) Enlarged AFM image marked with a white square in (b), and the cross-sectional profile of two Fab domains in a single IgG molecule. Inset image shows the measured height line along the IgG molecule. (c) Statistical data on the binding efficiency of digoxin-IgG complexes on the surface and rims of the DNA origami. (d) Heatmap of the detected binding efficiency in (c). Error bars presented standard deviations (n = 50 DNA origamis).

It is important to note that there were several major differences between the biotin-SA and digoxin-IgG systems, such as the molecular size, the complexity of the molecular structures, and the isoelectric points (pI) of IgG (~8.0) and SA (~6.0) in a near-neutral environment; however, the modification sites on the DNA origami did commonly contribute to differences in the binding behaviour between the surface and edge of the DNA origami for the targeted molecules in solution.


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Differences in behaviour among the vertex, rim, corner and surface/interface due to nonuniform distribution or defects have been reported in many nanomaterials47,

48.

However, no

model system currently exists for the quantitative analysis of these differences at a nanoscale resolution. The self-assembling DNA origami technology provides a programmed and reproducible way to create well-defined nanostructures. Their highly specific Watson-Crick base pairing, where each pair is separated by 0.34 nm in a double helix, could be applied to precisely design and construct binding sites for target molecules (here biotin and digoxin). Therefore, the programmable DNA origami provides a versatile platform for exploring specific molecular events in a defined nanoscale area, within which reactions can be contained. Using this method, we present the inherent features of nanometre-scale objects. The useful information will allow us to improve the overall performance of nanomaterials for the development of nanodevices such as nanobiosensors, nanorobots, and nanodrugs.

3. CONCLUSION In summary, the spatial organisation of DNA helices at the vertices, rims, corners, and surface of the DNA origami is non-uniform, resulting in possible distinct local environments. In this study, we explored the effects of local sites in a two-dimensional DNA origami nanostructure through molecular recognition reactions on a mica surface and in solution at the single-molecule level. We found that the binding efficiency varied based on the location of the molecule on the DNA origami for both the biotin-SA and digoxin-IgG reactions; the calculated binding kinetics of the formation of biotin-SA on the interface was also location-dependent. By taking advantage of the DNA origami, the programmable designed distribution sites of biotin molecules at nanoscale precision enabled us to simultaneously record the dynamic process of binding reactions at various locations in real-time and in situ with sufficiently high spatiotemporal resolution by AFM. Thus, even slight differences in binding behaviour at the origami vertex,


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rims, corner, and surface could be quantitatively described. Our findings are valuable for a wide range of self-assembling nanostructures and provide a suitable method for studying the heterogeneous properties of nanomaterials at the single-molecule level. 4. EXPERIMENTAL SECTION 4.1 DNA origami nanostructure construction. The DNA origami nanostructure was constructed as previously described8, 40. Briefly, circular M13mp18 DNA (New England Biolabs, Ipswich, MA, USA) and ~200 short staple strands (purified by HPLC)—including those labelled with biotin or digoxin (Shanghai Sangon Bioengineering Co., Shanghai, China)—were mixed in a Tris-acetic acid-EDTA (TAE)/Mg2+ buffer composed of 40 mM Tris, 2.0 mM EDTA, and 12.5 mM MgCl2 (pH 8.0) at a molar ratio of 1:10, and then annealed in a thermal cycler (Eppendorf, Hamburg, Germany) from 95 °C to 20 °C at a rate of 0.1 °C per 10 s. This self-assembly step yielded biotinylated/digoxin-labelled DNA origamis. The product was purified for subsequent binding reactions by filtration (GE Healthcare, Aurora, OH, USA) with TAE/Mg2+ buffer to remove excess staple strands. After modified origami purified, the sample was measured with NanoPhotomer-P330 (IMPLEN, Munich, Germany) to determine the concentration of DNA origami. 4.2 AFM image collection and analysis. AFM images were collected in 30 µl TAE/Mg2+ buffer in a liquid cell, after a 1–3-µl drop of the DNA origami sample was placed on freshly cleaved mica and incubated for 3–5 min for adsorption. For the reaction on the mica surface, SA (Sigma Aldrich, St. Louis, MO, USA) in TAE/Mg2+ buffer was added to the liquid cell for detection by AFM without washing. For the reaction in solution, the biotinylated/digoxinlabelled DNA origami and SA/ IgG were incubated for 30 min before acquiring AFM samples. All experiments, imaging was performed by tapping-mode AFM (VIII-Multimode; Veeco


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Digital Instruments, Santa Barbara, CA, USA) with a probe spring constant of 0.7 N∙m−1 (Bruker, Bremen, Germany) and a scanning speed of 1 Hz. Data were analysed using NanoScope Analysis software (Veeco Digital Instruments). 4.3 Binding efficiency, kinetic and binding constant calculation. For quantitative analysis of binding efficiency, the intact DNA origami detected by AFM imaging was used as a reference for the location and number of biotin molecules. If the location and structure of a bright dot corresponded to the topography of a single biotin-SA/digoxin-IgG complex on the DNA origami, it was considered evidence of binding. The binding efficiency was calculated as the ratio of the number of complexes to the number of biotin/digoxin molecules at the same location on the DNA origami. A curve of the binding efficiency as a function of time were drawn and fitted by Origin software. A heatmap for the binding events on the surface of DNA origami was drawn based on the final detected binding efficacy of each biotin sites. To quantitatively describe the rate constant of biotin-SA complex formation for each biotin site in the complex system, kinetic measurements were performed in reactions for 20 min, in which SA concentrations of 17.5 nM with a final concentration of 0.2 nM origami concentration were used. Real-time AFM imaging was collected after SA injected into the centre of the liquid cell using a syringe to reduce the diffusion effect on the reaction. Data were plotted and fitted using the well-established Simbiology method, the Toolbox of Matlab software. To quantify the binding reaction, the affinity experiments of biotin-SA complexes were conducted in tubes, in which the concentration of the DNA origami in the reaction was kept in below 0.2 nM and SA was titrated over 0.5 nM to 33.3 nM. AFM images were collected after SA mixed with biotinylated origami for incubating 30 min to establish equilibrium.

SUPPORTING INFORMATION


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The supporting information is available free of charge on the ACS Publication website at DOI: Details of DNA sequence, results and figures.

AUTHOUR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]; [email protected]. Author Contributions ‖These

authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (nos. 31670871, 11674344, U1532260, and 21675167), the Key Research Program of Frontier Sciences, CAS (no. QYZDJ-SSW-SLH019), the Open Large Infrastructure Research of Chinese Academy of Sciences, and the LU JIAXI International team program supported by the K.C. Wong Education Foundation and CAS..


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Quantitative Measurement of Spatial Effects of DNA Origami on

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