Spatial Regulation of Biomolecular Interactions with a Switchable

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Functional Nanostructured Materials (including low-D carbon)

Spatial Regulation of Biomolecular Interactions with a Switchable Trident-Shaped DNA Nanoactuator Chao Xing, Yuqing Huang, Junduan Dai, Lin Zhong, Huimeng Wang, Yuhong Lin, Juan Li, Chun-Hua Lu, and Huang-Hao Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10761 • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018

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ACS Applied Materials & Interfaces

Spatial Regulation of Biomolecular Interactions with a Switchable Trident-Shaped DNA Nanoactuator Chao Xing, Yuqing Huang, Junduan Dai, Lin Zhong, Huimeng Wang, Yuhong Lin, Juan Li, Chun-Hua Lu*, Huang-Hao Yang*. MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350116, P.R. China. KEYWORDS: DNA nanostructure, enzyme cascade, fluorescence resonance energy transfer, gold nanoparticles, switch.

ABSTRACT: DNA nanostructures with controllable motions and functions have been used as flexible scaffolds to precisely and spatially organize molecules reactions at the nanoscale. The construction of dynamic DNA nanostructure with site-specifically incorporated functional elements is a critical step towards building nanomachines. Artificial self-assembled DNA nanostructure have also been developed to mimic key biological process, like various small biomolecule and protein-based functional biochemistry pathways. Here, we report a selfassembled dynamic trident-shaped DNA (TS DNA) nanoactuator which biomolecules can be tethered to the three “arms” of the TS DNA nanoactuator. The TS DNA nanoactuator is

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implemented as the mechanical scaffold for the reconfiguration of fluorescent/quenching molecules and the assembly of gold nanoparticles (AuNPs) which exhibiting controlled spatial separation. Furthermore, two enzymes (glucose oxidase, (GOx)/horseradish peroxidase, (HRP)) are attached to the two outer arms of the TS DNA nanoactuator, which show an enhanced cascade reaction efficiency compared with the free enzymes. The efficiency of the two-enzyme cascade reaction can be spatially regulated by switching the TS DNA nanoactuator between opened, semi-opened and closed states through adding the “thermodynamic drivers” (fuels or antifuels). This is the first report to precisely modulate the relative position of couple enzyme with multiple states and only based on one dynamic DNA scaffold. The present TS DNA nanoactuator with multistage conformational transitions functionality could be applied as a potential platform to precisely and dynamically control the multi-enzyme pathways and would broaden the scope of DNA nanostructures in single-molecule biology application.

In living systems, metabolism, a whole range of life-sustaining chemical transformations occurring within the cells, is mediated by myriads of complex and dynamic biomolecules reactions (e.g., enzyme reactions) which present specific and extraordinary catalytic efficiency.1,2 These biomolecules reactions could maintain organism’s structures and adjust their response to surrounding environments. With the continuous evolution of nature, the living systems become increasingly complex and multi-step than before. Therefore, the engineering of rationally designed artificial nanodevices,3-5 which precisely mimic those biomolecules reactions will not only facilitate us to understand the molecular mechanisms of the living system, but also help us to expand the application of these artificial nanodevices in many fields, such as in synthetic biology,6 biocatalysis,7 and biodiagnostics.8


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DNA nanotechnology,9-16 taking advantage of the molecular recognition properties of DNA to create artificial structures, is one of the most promising route towards building such artificial nanodevices. In the past few decades, DNA, with sequence-specific base pairing and wellestablished double-helix structure, has been extensively used for the fabrication of one-, two- and three-dimensional nanostructures with controllable sizes and shapes.17-24 Besides, based on toehold-mediated isothermal DNA strand displacement, a number of dynamic DNA devices, including DNA walkers,25-30 molecular gears,31 molecular spiders,32 interlocked DNA nanostructure,33,34 rolling motors35 and DNA tweezers36,37 have been constructed in solution and on surfaces. Such molecular nanostructures have been used for many applications including biological probes,38-40 cargo delivery,41-43 controlled synthesis,44-47 phase transfer48 and nanobioreactors.49 Furthermore, these DNA nanostructures have been used as molecular scaffolds with nanometre resolution for the assembly of heteroelements such as viruses,50 proteins51-55 and nanoparticles.56-63 For example, Xin et al. and Liu et al. reported that DNA tweezers could dynamically regulate the reaction between enzyme/enzyme pairs or enzyme/cofactor pairs by controlling the distance between two arms of the tweezers.64,65 Kuzuya et al. demonstrated a DNA origami pliers, which can be used to visually detect single-molecule target by a shape transition.66 Ke et al. constructed a rhombus-shaped DNA origami nanoactuator to tune the fluorescent behaviors of green fluorescent protein via long-range allosteric regulation.67 Kuzyk et al. demonstrated selective control of reconfigurable chiral plasmonic molecules assembled by pH and light-sensitive DNA triplex formation.68,69 These devices all showed good switchable ability to regulate biomolecular reaction with two-step (open/close) process. There are also many complex biomolecular reactions which consists of variety of multistep process (three or more) in living systems, which can’t be mimicked by existing DNA nanodevices, thus the new types of


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dynamic DNA nanostructures which can regulate the multistage conformational transitions are needed. In the current work, we have designed a self-assembled multistage dynamic and reconfigurable trident-shaped DNA (TS DNA) nanoactuator system that can adopt four distinct structural states, compared with previous two-state systems. The TS DNA nanoactuator contains three “arms” which biomolecules can be tethered. The spatial distances between the three arms can be reversibly tuned by strand displacement reaction (Figure 1a), and the TS DNA nanoactuator was implemented as a mechanical scaffold for the reconfiguration of fluorescent/quenching molecules and gold nanoparticles (AuNPs). Furthermore, glucose oxidase (GOx)/horseradish peroxidase (HRP) are attached to the two outer arms of the TS DNA nanoactuator, and show an enhanced cascade reaction efficiency compared with the free enzymes. The efficiency of the two-enzyme cascade reaction can be spatially regulated by switching the nanoactuator between opened, semi-opened and closed states. Therefore, the present TS DNA nanoactuator would offer an exciting inspiration for studying other types of complicated enzyme cascade systems or as regulatory biological circuits for diagnostic and therapeutic applications. EXPERIMENTAL SECTION Materials. All oligonucleotides were synthesized and purified using polyacrylamide gel electrophoresis (PAGE) by Sangon Biotechnology (Tables S1-S3). GOx, HRP, Acrylamide/bisacrylamide (30%), Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and Bis (psulfonatophenyl) phenylphosphine dihydrate dipotassium salt (BSPP) were purchased from Sigma-Aldrich. PBS, TBS, TAE, TBE buffer, 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) and Dialysis tube 10 kDa MWCO were purchased from Sangon


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Biotechnology.

N-[ε-maleimidocaproyloxy]

sulfosuccinimide

ester

(Sulfo-EMCS)

was

purchased from Thermo Fisher Scientific. Gold colloids 5 nm and 10 nm were purchased from British Biocell. Metaphore agarose was purchased from Lonza. Amicon ultra 30 kDa, 100 kDa MWCO were purchased from Millipore. Deionized water from Millipore water purification system (18.2 MΩ•cm) was used throughout this study. Instruments. Transmission electron microscopy (TEM) images were obtained by using a HITACHI HT7700 Transmission Electron Microscope (Japan) operated at an accelerating voltage of 100 kV. All UV/Vis results were acquired on a SH-1000 UV/Vis spectrophotometer and processed with the Origin Lab software. The fluorescence data was acquired on a Cary Eclipse Fluorimeter (Varian Inc). Atomic force microscopy (AFM) images were taken using a Mulitmode 8 microscope (Scanasyst in fluid mode, Bruker, USA). Gel images were captured using a ChemiDocTM Touch Imaging System from Bio-Rad Laboratories. Preparation and Characterization of TS DNA Nanoactuator. The assembly of the TS DNA nanoactuator was performed by mixing equimolar amounts of short DNA staple strands (DNA-1 to DNA-13, H1, H2, more details in Table S1) at a concentration of 0.5 µM in 1×TAE/Mg buffer (40 mM Tris, 20 mM acetic acid, 2 mM EDTA, 12.5 mM MgCl2, pH = 7.5), and then annealing from 95 °C to 20 °C at a rate of −0.2 °C•min−1. The TS DNA was purified from the mixture by using agarose gel electrophoresis. Target band was excised from an agarose gel and crushed in Freeze ‘N Squeeze DNA Gel Extraction Spin Columns. The cup plus gel pieces was put in a 20 °C freezer for 5 minutes, then removed and immediately centrifuged at 13,000 x g for 3 minutes at room temperature. Agarose debris is retained within the filter cup; the liquid at the


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bottom of the tube contains the recovered DNA. Finally, AFM was used to characterize the TS DNA nanoactuator. Real-time Fluorophore/quencher Experiment. The preparation of fluorescent/quenching molecule-modified TS DNA nanoactuator was similar to that of the construction of TS DNA nanoactuator, except that the DNA-11, 12, 13 strands were substituted by Cy3-11, BHQ2-12, Cy5-13 respectively. Fluorophore/quencher experiments were carried out in a Cary Eclipse Fluorescence Spectrophotometer at 25 °C. The experimental concentration of DNA nanoactuator was 50 nM. The fluorophores Cy3 and Cy5 were excited at 545 nm and 630 nm, and emission wavelengths were recorded at 566 nm and 670 nm, respectively. The concentrations of the respective fuels and antifuels were added at a 1:1 ratio, and were in excess compared to the concentration of the TS DNA nanoactuator. Synthesis and Characterization of the TS DNA nanoactuator Modified with DifferentSized AuNPs. The commercial AuNPs (5 or 10 nm) were mixed with an excess of BSPP (0.4 mg/mL) and stirred overnight. Then, the AuNPs were concentrated by 100 kDa MWCO Amicon filtering tubes and stored at +4 °C for further use. The thiol-modified DNA were diluted to a final concentration of 15 µM with 15 mM TCEP and stored at -20 °C for further use. 5 nm AuNPs (3 µM) and the thiol-modified SH-11 (4 µM) were first prepared in a solution that contained 0.5×TBE, 0.4 mg/mL BSPP, 100 mM NaCl. Then, the auxiliary strand (Fu, 10 µM, which could partially hybridize with SH-11) was added to improve subsequent separation efficiency by gel electrophoresis.


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10 nm AuNPs (0.5 µM) and the thiol-modified SH-13 (1.5 µM) were first prepared in a solution containing 0.5×TBE, 0.4 mg/mL BSPP and 100 mM NaCl. 10 µM auxiliary strand (Fu, which could partially hybridize with SH-13) was added to improve subsequent separation efficiency by gel electrophoresis. 3% w/v agarose gel was used to separate the single-strand modified AuNPs from the mixtures. The mixtures were firstly mixed with glycerol (final concentration is 12% v/v), and added in the wells of the gel. The gels were run at a constant voltage of 8 V/cm. When visually satisfying separation was observed, the corresponding bands were cut, and subjected to a voltage of 100 V inside a closed dialysis membrane filled with 0.5×TBE buffer. The single-strand modified AuNPs which trapped in the 100 kDa dialysis membrane was collected and filtered with a 0.22 µm syringe filter, followed by concentration with 100 kDa MWCO Amicon filtering tube. The single-strand modified AuNPs were not very stable with time, thus the stabilization of the AuNPs is needed. In detail, we incubated the single strand-modified AuNPs with 100 mM NaCl, 0.5×TBE, 0.4 mg/mL BSPP and 100-fold excess of 5T-DNA. A fully complementary strand (Fu*) was added to the solution to hybridize with the auxiliary strand Fu and Fu was released from the AuNPs. The mixture was incubated at 25 °C for 24 h and then washed for four times by 100 kDa MWCO Amicon filtering tubes to remove strand Fu, Fu* and excess 5T-DNA. At the end, the concentration of the AuNPs was determined by UV spectrum and the sample was stored at +4 °C for further use. The single SH-11 functionalized 5 nm AuNPs and single SH-13 functionalized 10 nm AuNPs were co-incubated with the semi-finished TS DNA nanoactuator (without DNA-11 and DNA-13) at a molar ratio of 1:1:1 in all-opened state, at 25 °C for 10 hours. Then, the products of all-


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opened AuNPs-functionalized TS DNA nanoactuator were assembled. The other states (semiopened and all closed states) of AuNPs-functionalized TS DNA nanoactuator were transformed from all-opened state by adding the corresponding fuels and antifuels. TEM Experiments. The AuNPs-functionalized DNA nanoactuator with different states were diluted to a final concentration of 0.25 nM in a 200 mM NaCl solution. For TEM analysis, 3 µL of different samples were deposited on the copper grids and dried at 30 °C for three hours, then the images were obtained with a HITACHI HT7700 Transmission Electron Microscope. Importantly, the sample were analyzed by TEM at least three independent preparations, and the images were acquired from different regions of the copper grids. AFM Imaging. For AFM imaging, 3 µL of NiCl2 (10 mM) was deposited onto a freshly cleaved mica and left to adsorb for 5 min, then, washed three times with a 1xTAE/Mg buffer (40 mM Tris, 20 mM acetic acid, 2 mM EDTA, 12.5 mM MgCl2, pH = 7.5). 10 µL of the sample was dropped on the mica and left to adsorb for another 3 min at ambient temperature. Finally, 200 µL of deionized water was added to the liquid cell and AFM was performed in Scanasyst in fluid mode. Enzyme-DNA Conjugation. DNA-enzyme conjugates were prepared using Sulfo-EMCS as a bifunctional crosslinker. 1×10-5 M GOx or HRP were incubated with 1×10-3 M Sulfo-EMCS in PBS buffer (12.5 mM, pH=8-8.5) for 4 hours at room temperature, allowing amine-reactive Nhydroxysuccinimide (NHS) esters to react with the lysine residues on the protein surface. The excess Sulfo-EMCS was removed by 30 kDa MWCO Amicon filtering tubes. Then the SulfoEMCS-modified GOx or HRP was further incubated with 10-fold excess of SH-13 or SH-11 for another 5 h, respectively. Finally, the resulting products (GOx-SH-13 and HRP-SH-11) were


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purified by 30 kDa MWCO Amicon filtering tubes and washing two time with 1×PBS buffer (pH 7.4) containing 1 M NaCl, and six times with 1×PBS buffer. The resulting products characterized with 8% PAGE which stained by Gelred and Coomassie Blue. GOx/HRP Co-assembly on TS DNA Nanoactuator. GOx-SH-13 and HRP-SH-11 were incubated with all-opened semi-TS DNA nanoactuator (without DNA-11 and DNA-13) at a molar ratio of 1:1 within 1×TAE/Mg buffer (pH 7.5) at 25 °C for 5 h, resulting in the products of all-opened enzyme-functionalized TS DNA nanoactuator. The other states of enzymefunctionalized TS DNA nanoactuator were obtained from all-opened states by adding corresponding fuels and antifuels. 3% agarose gel was used to characterize the assemblies. Enzyme Assay. GOx/HRP co-assembled TS DNA nanoactuator was diluted to 0.25 nM for enzyme cascade reaction assays, which were performed on a 96 well plate at 25 °C. GOx/HRP cascade activity of different states was determined by monitoring the increase signal of absorbance at 410 nm, in 1×TBS/Mg buffer (tris buffered saline with 1 mM MgCl2, pH 7.5) in presence of 1 mM Glucose and 2 mM ABTS2-. At least, three replicates of each sample were measured. RESULTS AND DISCUSSION Synthesis of the TS DNA Nanoactuator. The TS DNA nanoactuator with two DNA triplecrossover (TX) motifs20 immobilized by Holliday junctions at all-closed state was prepared through a fast and versatile annealing procedure. Compared with duplex, the TX motif with rigidity and variability in the nanoactuator could provide flexible and adjustable internal binding sites for the two hairpin structures without decreasing the rigidity of the TS DNA nanoactuator. The TS DNA nanoactuator was constituted of thirteen single strand DNA and two hairpin DNA,


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and the two hairpin DNA can circulate between stem-loop structure and double helix structures through a strand-displacement reaction (Figure 1a and Figure S1).

Figure 1. (a) Schematic illustration of the TS DNA nanoactuator with three arms that can be switched to open or close by the strand displacement reaction. (b) Demonstration of the switchable properties of the upper half-completed TS DNA nanoactuator and completed TS DNA nanoactuator by native PAGE. (Lane M, DNA Marker; Lane 1, upper half-completed TS DNA. Lane 2, upper half-completed TS DNA+F1; Lane 3, upper half-completed TS DNA+F1+ aF1; Lane 4, completed TS DNA; Lane 5, completed TS DNA+F1+F2; Lane 6, completed TS DNA+F1+F2+aF1+aF2;) (c) AFM characterization of TS DNA nanoactuator.


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As shown in Figure 1a, by adding the corresponding fuel(s) (F1, F2) and antifuel(s) (aF1, aF2) to control the open and close of the H1 and/or H2, the TS DNA nanoactuator could be reconfigured between four states (state I to state IV). The stepwise synthesis of the TS DNA nanoactuator was characterized by the native PAGE (Figure S2). With the increasing number of the DNA strands to assemble the TS DNA nanoactuator, the bands migrated slower as the molecular weights of the products increase, implying that the relative molecular weights of each complex are consistent with the design. For demonstrating the open and close of the TS DNA nanoactuator by gel electrophoresis (Figure 1b and Figure S3), the completed TS DNA nanoactuator and the half-completed TS DNA nanoactuator were first synthesized and purified from agarose gel. As mentioned above, the hairpin DNA in the TS DNA nanoactuator could hybridize with the fuel, forming the duplex, thus leading to the opening of the TS DNA nanoactuator. The hybridization of the fuels would increase the molecular weight of the opened TS DNA nanoactuator, and the opened conformation will also make the TS-DNA nanoactuator run slower than the more compact closed conformation. In Figure 1b, lane 1-3 show the band of the half TS DNA nanoactuator. By adding the fuel F1 to open the hairpin structure H1, the relatively higher molecular weights and opened conformation of the opened-half TS DNA nanoactuator reduce the movement of the bands lane 2. The fuel strand could hybridize with the antifuel and release from the half-TS DNA nanoactuator structure, thus leading to switch the opened-half TS DNA nanoactuator back to the initial closed-half TS DNA nanoactuator state (lane 3). Furthermore, the gel electrophoresis results of the completed TS DNA nanoactuator also follow the same trend as using fuels or antifuels to open or close the TS DNA nanoactuator. As shown in Figure 1b, lanes 4-6, due to the relative higher molecular weight and the larger spatial size, the opened TS DNA nanoactuator moves slightly slower than that of the closed TS


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DNA nanoactuator. The successful synthesis of the TS DNA nanoactuator was also verified by AFM (Figure 1c and Figure S5), which showed well-defined assemblies with distinct three arms. The PAGE results, in conjunction with the AFM images provide evidence that the complexes assembled as intended. Real-time Fluorophore/quencher Experiment. As our designed, the TS DNA nanoactuator exhibits four states (state I to state IV) with significant difference of the distances between each arm. The gel electrophoresis results have preliminarily proved the open and close of the TS DNA nanoactuator. The dynamic regulation of the TS DNA nanoactuator was further investigated by the Fluorophore/quencher experiment. As shown in Figure 2a, the fluorophores Cy5 and Cy3 were modified on the arm 1 and arm 3 respectively, while the black hole quencher BHQ2 was modified on arm 2. The quenching of the fluorophores by the quencher provides the readout signals for the switchable reconfiguration of the TS DNA nanoactuator, which is powered by the fuel(s)/antifuel(s) stimuli. The TS DNA nanoactuator is initially set at state I (all-opened state), in which the fuels F1 and F2 were hybridized with H1 and H2 respectively. Due to the larger distance from the BHQ2 unit, the fluorophores Cy5 and Cy3 are less efficiently quenched. Addition of aF2 to state I yields state II in which the hairpin H2 is reconstructed by the stranddisplacement reaction (F2 is hybridized with aF2 and released from the TS DNA nanoactuator, thus leading to the close of arm 2/arm 3). The proximity between Cy3 and BHQ2 leads to the effective quenching of the fluorophore, while the fluorescence intensity of Cy5 is not affected. Similarly, adding aF1 to form the F1/aF1 duplex would transfer the state I to state III, by releasing F1 from the TS DNA nanoactuator, returning H1 back to hairpin structure to close arm 1/arm 2. Therefore, the fluorescence of Cy5 is quenched and the Cy3 is not affected. Addition of the aF1 and aF2 at the same time, the TS DNA nanoactuator would transit from opened state I to


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closed state IV, leading to the effective quench of both Cy3 and Cy5. Treated with appropriate stimuli, the TS DNA nanoactuator would transit to the as-demand state, and the fluorescence changes of both fluorophores would follow the corresponding trend as the state transition. The time-dependent fluorescence intensity changes of Cy3 and Cy5 upon the transformation among the four states are shown in Figure 2b. At initial state I, fluorophores Cy3 and Cy5 were spatially separated from the quencher BHQ2 by the two-opened hairpin structure (F1 and F2 were hybridized with H1 and H2 respectively), leading to inefficient quenching of both fluorophores. When aF2 strand was added to state I, the lower half of TS DNA nanoactuator was closed (state II) by the formation of hairpin H2, shortening the distance between fluorophore Cy3 and

Figure 2. Monitoring of the switchable reconfiguration of the TS DNA nanoactuator in different states by fluorescence intensity changes. (a) Schematic illustration of fluorophore/quencher experiment of the TS DNA nanoactuator. (b) Time-dependent fluorescence changes of the fluorophore probes Cy3 and Cy5 upon the reversible transition of the device between the states I →II→IV→III→I→IV→I. The experimental concentration of TS DNA nanoactuator is 50 nM.


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quencher BHQ2, thus leading to strong quenching of the fluorophore Cy3. Treating the state II with aF1, the newly formed hairpin H1 led to an effective quenching of the Cy5 (state IV). Further addition of F2 to separate the fluorophore Cy3 away from the BHQ2 (state IV to state III) by new formed F2/H2, restored the fluorescence of Cy3. Similarly, addition of F1 to state III result in the transformation of TS DNA nanoactuator to state I where the fluorescence of Cy5 was regenerated. The formation of state IV from state I was accompanied by the decrease in the fluorescence of both Cy3 and Cy5. The reconfiguration of state IV into state I was accompanied by the increase in the fluorescence of Cy3 and Cy5. The slow hybridization of a fuel strand to a self-folded hairpin structure (H1 or H2) resulted in the sluggish process of TS DNA opening. That's why the process of quenching is faster than de-quenching. The reduced yield of Cy3 fluorescence observed over time could be attributed to photo bleaching. Evidently, the fluorescence features of the two fluorophores provided the effective optical labels to probe the states of the TS DNA nanoactuator. Organization of AuNPs-modified TS DNA Nanoactuator. The fueled transition of the TS DNA nanoactuator have been demonstrated by the fluorophore/quencher experiment. In order to simulate the distances of the three arms in different states, the TS DNA nanoactuator was further implemented as a molecular device for programming switchable organization of different sized AuNPs (Figure 3). AuNPs with diameter of 5 nm and 10 nm were firstly functionalized with single thiol-modified SH-11 and SH-13, respectively (Figure S6), and then hybridized with the semi-TS DNA nanoactuator (without DNA-11 and DNA-13). Figure 3a, left showed the initial state I of the assembly of AuNPs on the TS DNA nanoactuator and the TEM images (Figure 3b, left) showed that the two AuNPs were spatially separated with a statistic characteristic distance of ∼25.5 nm (center to center, Figure 3c, left and Figure S7). Treating the AuNPs functionalized


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TS DNA nanoactuator of state I with antifuel strand (aF2 or aF1) yielded a semi-opened (state II and III) AuNPs functionalized TS DNA nanoactuator (Figure 3a, middle). Figure 3b, middle shows representative TEM images of the resulting dimers in state II and III with a calculated separation distance of ∼17.5 nm (Figure 3c, middle and Figure S8). Similarly, when state II or state III were exposed to aF1 or aF2, a compact assembly of all-closed AuNPs functionalizes TS DNA nanoactuator configuration (state IV) was obtained (Figure 3a, right). It looked like that the

Figure 3. Programmed assembly of AuNPs by the TS DNA nanoactuator. (a) Modification of the TS DNA nanoactuator at state I (all opened state) with 10 nm and 5 nm AuNPs at arm 1 and arm 3, respectively, and spatial regulation of the AuNPs assembly in states I-IV. (b) Representative TEM images of the AuNPs associated with states I-IV (scale bars: 20 nm). (c) Gaussian fittings of interparticle distances of the AuNPs dimers in different states.


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two dimers of AuNPs surfaces become close to each other at state IV (Figure 3b, right and Figure S9). The corresponding separation distance of the AuNPs was ∼9.5 nm in state IV (Figure 3c, right). Furthermore, the theoretical relative distance between the two outer arms estimated by the trigonometric function (cosine law and double angle formulas) was ∼29.9 nm (Figure S4). All the distances estimated by AuNPs were in the reasonable range. Therefore, we think that the assembly of AuNPs was a feasible way to calibrate the structures, especially, to simulate the relative distances of the different states. Spatial Regulation of Enzyme Cascade Reaction. Recently, many efforts proved that the efficiency of the enzyme cascade reaction could be regulated by DNA nanostructures.64, 70-77 For our TS DNA nanoactuator, the experiments of fluorophore/quencher experiment and reversibly switchable AuNPs assembly suggested it would be a precise scaffold for spatially regulating the efficiency of the enzyme cascade reaction. Herein, the GOx (160 kDa)-HRP (44 kDa) cascade reaction system, which is the most frequently used enzyme pair, was chose as a model for the study of the multi-enzyme system on the TS DNA nanoactuator (Figure 4a). Firstly, GOx and HRP were conjugated with thiol-modified SH-13 and SH-11, respectively, by bifunctional crosslinker Sulfo-EMCS. The resulting products (GOx-SH-13 and HRP-SH-11) were characterized by PAGE (Figure S10) and UV-Vis spectroscopy (Figure S11), which proved that the enzyme was successfully attached to the thiol-modified DNA. After that, the GOx-SH-13 and HRP-SH-11 were co-incubated with semi-TS DNA nanoactuator (without DNA-11 and 13) to form the GOx/HRP co-assembled TS DNA nanoactuator. In order to achieve high co-assembly yields of the GOx/HRP pairs, we started with the semi-finished DNA nanoactuator at all-opened


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Figure 4. Design of GOx/HRP-assembled TS DNA nanoactuator. (a) Schematic of spatial regulation the efficiency of enzyme cascade reaction by TS DNA nanoactuator with four states. (b) Time-dependent absorbance changes of different conditions of the enzyme catalytic systems. Error bars represent the standard deviation from three experiments. state to give enough space for two enzymes. The individual GOx and individual HRP functionalized TS DNA nanoactuator were synthesized by the same method, respectively. The successful assembly of GOx/HRP with TS DNA nanoactuator was characterized by agarose gel (Figure S12). As we can see, the bands of individual GOx (lane 2) and individual HRP (lane 3) functionalized TS DNA nanoactuator were slower than the band of TS DNA nanoactuator without enzyme (lane 1), suggesting that the individual GOx-SH-13 and HRP-SH11 were immobilized on the TS DNA nanoactuator, respectively. Furthermore, the band of the GOx and HRP co-functionalized TS DNA nanoactuator sample (lane 4) migrated slightly slower than that of the single enzyme-functionalized TS DNA nanoactuator (lane 2, 3), indicating that


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the relatively higher molecular weight product was form. Therefore, we could conclude that the GOx/HRP co-assembled TS DNA nanoactuator was successfully assembled. The GOx/HRP enzyme cascade reaction beginning with the GOx catalytic oxidation of glucose to gluconic acid, with the concomitant formation of H2O2, which is subsequently used by HRP to oxidize ABTS2- to ABTS-, with the increment of absorbance at 410 nm. The spatial regulation of the enzyme cascade reaction was achieved by strand displacement reaction and verified by real time absorbance measurements. In Figure 4b, shorting the distance between GOx and HRP by strand displacement reaction to close the two hairpins one by one resulted in an increase in the efficiency of the enzyme cascade reaction (S1 > S2 > S3). The all-closed state (S1) showed the highest enzyme cascade reaction efficiency, which was 1.5 times higher than the efficiency of the free enzymes (C1). In S1, S2 and S3, the samples containing co-assembled GOx/HRP on TS DNA nanoactuator exhibited obvious higher efficiency than C1. At the same time, the control solutions C2 (with free enzymes and free TS DNA nanoactuator) had similar efficiency as free enzymes (C1), and the only free TS-DNA (C3) showed no changed in time-dependent absorbance. Evidently, TS DNA nanostructure didn’t affect enzyme cascade reaction efficiency in bulk solution and the enhanced efficiency maybe caused by closer distance between two enzymes on TS DNA scaffold. It should be noted that there were also some unloaded enzymes (i.e., enzymes without TS-DNA) in the reaction systems, thus the maximum enhancement of the cascade reactions efficiency could be more than 1.5-fold in theory. For a GOx/HRP enzyme cascade reaction, the effective biocatalysed formation of ABTS- was attributed to the high local concentration of intermediate H2O2. As for the mechanism of the intermediate H2O2 transfer on DNA scaffold, Fu et al. have reported that it followed the dimensionally-limited diffusion mechanism for closely spaced enzymes (the inter-enzyme


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distance less than 10 nm), and three-dimensional diffusion model for the inter-enzyme distances large than 20 nm.71 For our TS DNA-enzyme system, the relative distances of two enzymes in different states (opened: ∼25.5 nm, semi-opened: ∼17.5 nm, closed: ∼9.5 nm) were simulated by the 10 nm and 5 nm AuNPs (Figure 3c), whose diameters were similar to the GOx (∼10 nm) and HRP (∼5 nm), respectively. Three-dimensional diffusion model is the distance-dependent diffusion of H2O2 in the bulk solution (the longer distance, the lower concentration of H2O2). In the all-opened state (S3), the average distance between couple enzyme was ∼25.5 nm (Figure 3c, left), where the H2O2 concentration was obvious higher than that of the free enzymes in solution (C1), which spacing is ∼1.84 µm (0.25 nM couple enzymes). Therefore, the 1.35-fold higher efficiency of S3 in Figure 4b could be explained by the theory of 3D diffusion. In other side, the dimensionally-limited diffusion mechanism is that H2O2 does not generally escape into the bulk solution but instead transfers from GOx to HRP along their mutual, connected protein surface (the hydration layers of GOx and HRP) when the GOx/HRP assembled at very close space. As shown in Figure 3b, right, the surfaces of the two AuNPs are adjacent to each other. Thus, we could speculate that the surfaces of two enzymes are essentially close to each other. Therefore, the dimensionally-limited diffusion mechanism was highly compatible with our results which the cascade efficiency of S1 was higher than that of S3. In the semi-closed states (S2) which statistics spacing became longer (∼17.5 nm), the 3D diffusion model occupied a dominant for the transporting H2O2. However, owing to the shapes of the enzymes were not standard sphere, there might still be a small fraction of couple enzymes close to each other, the hydration layers still control the H2O2 transfer between this part of couple enzymes. The synergistic effect of the two models lead to the little higher enzyme cascade reaction efficiency of S2 than S3. The different enzyme cascade reaction efficiencies explained by the 3D diffusion model and dimensionally-


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limited diffusion model proved that the TS-DNA would be an excellent nanodevice for spatial regulation of enzyme cascade reaction. CONCLUSIONS In conclusion, we have successfully designed and constructed an artificial TS DNA nanoactuator which can be applied to organize biomolecules (for instance, fluorescent/quenching molecules, different sized AuNPs dimer structures and enzyme cascade system) with multiple states. We use gel electrophoresis and AFM to characterize the successful construction of TS DNA nanoactuator, and the fluorophore/quencher experiment real time monitoring the reversible allosteric motion of the three arms. We have spatially modulated the efficiencies of enzyme cascade reaction on DNA scaffold by regulating three dynamic arms of the TS DNA nanoactuator to change the relative position of the couple enzymes. The three-dimensional diffusion model and emerging dimensionally-limited diffusion model were used to explain the enhanced enzyme cascade reaction efficiency of enzyme-assembled TS DNA nanostructures. This TS DNA nanoactuator for regulating the efficiencies of enzyme cascade reaction through changing enzyme distance between different states could be applied to study numerous of other life processes (such as three or more enzyme cascade reactions and inter-domain electron transfer), and offer a potential platform to precisely and dynamically control the multi-enzyme pathways (like the control of substrate channeling). Furthermore, the TS DNA nanostructures would broaden the scope of DNA nanostructures in single-molecule biology and serve as regulatory biological circuits for diagnostic and therapeutic applications. ASSOCIATED CONTENT


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Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Additional table and figures (PDF) AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected] ORCID Huang-Hao Yang: 0000-0001-5894-0909 Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (Nos. 21775025, U1705281, U1505221, 21475026, 21635002), Natural Science Foundation of Fujian Province of China (No. 2015H6011, 2018J01687), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT15R11). REFERENCES


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SYNOPSIS


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Using artificial nanodevices to precisely mimic dynamic biomolecules reactions is of great significance in biocatalysis and synthetic biology. A strategy for the spatial regulation the efficiencies of enzyme cascade reaction based on a switchable trident-shaped DNA nanoactuator in opened, semi-opened and closed states is presented.


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Spatial Regulation of Biomolecular Interactions with a Switchable

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