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A Synthetic Light-Driven Substrate Channeling System for Precise Regulation of Enzyme Cascade Activity Based on DNA Origami Yahong Chen, Guoliang Ke, Yanli Ma, Zhi Zhu, Minghui Liu, Yan Liu, Hao Yan, and Chaoyong James Yang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b05429 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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Journal of the American Chemical Society

A Synthetic Light-Driven Substrate Channeling System for Precise Regulation of Enzyme Cascade Activity Based on DNA Origami Yahong Chen,† Guoliang Ke,*‡ Yanli Ma,† Zhi Zhu,† Minghui Liu,ǁ Yan Liu,ǁ Hao Yan*ǁ and Chaoyong James Yang*†§ †

Collaborative Innovation Center of Chemistry for Energy Materials, The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China § Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, 200127, China ‡ Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China ‖ Center for Molecular Design and Biomimetics, Biodesign Institute and School of Molecular Sciences at Arizona State University, Tempe, Arizona, 85287, United States Supporting Information Placeholder

ABSTRACT: Substrate channeling, in which metabolic intermediate is directly passed from one enzyme to the next enzyme in an enzyme cascade, accelerates the processing of metabolites and improves substrate selectivity. Synthetic design and precise control of channeling outside the cellular environment are of significance in areas such as synthetic biology, synthetic chemistry, and biomedicine. In particular, the precise control of synthetic substrate channeling in response to light is highly important, but remains a major challenge. Herein, we develop a photo-responsive molecule-based synthetic substrate channeling system on DNA origami to regulate enzyme cascade activity. The photoresponsive azobenzene molecules introduced into DNA strands enables reversible switch the position of substrate channeling to selectively activate or inhibit the enzyme cascade activity. Moreover, DNA origami allows precise control of inter-enzyme distance and swinging range of the swing arm to optimize the regulation efficiency. By combining the accurate and addressable assembly ability of DNA origami and the clean, rapid and reversible regulation of photo-responsive molecules, this lightdriven substrate channeling system is expected to find important applications in synthetic biology and biomedicine.

INTRODUCTION Substrate channeling is a fundamental natural process involving direct transfer of a metabolic intermediate from one enzyme to the next enzyme without the release of free intermediates to the bulk solution.1-3 One important mechanism of substrate channeling is mediated by swing arm, which typically uses a covalent linker for transferring intermediates between different active sites.3,4 By controlling metabolic intermediates, substrate channeling significantly improves the efficiency of natural biochemical reactions via accelerating the reaction and avoiding unnecessary byproducts. Inspired by this interesting and significant natural approach, the synthetic design and precise control of substrate channeling outside the cellular environment are of paramount importance in the areas such as synthetic biology, synthetic

chemistry and biomedicine.3,5 In particular, light-responsive enzyme activity is a fundamental means of signal transduction in nature. For example, as a light-sensitive receptor, rhodopsin is regulated by the photo-isomerization of the cis/trans forms of its cofactor retinal, which is an important G-protein-coupled receptor in phototransduction.6 Besides, light is a clean, waste-free fuel, and can be switched on and off rapidly. More importantly, light enables noninvasive manipulation with the features of high spatial and temporal resolution for in vivo application, such as optogenetics.7,8 Therefore, the design of a smart light-driven synthetic substrate channeling that directionally regulates the enzyme cascade activity in response to different wavelengths of light would be highly beneficial. To design light-driven substrate channeling in an enzyme cascade, two factors are of paramount importance. First, to regulate enzyme cascade by light, a rapid and reversible photoresponse element is basically required. Second, the accurate assembly and control of each component such as enzymes and cofactors are the fundamental basis. Although there have been several reports of light-regulation of enzyme activity based on photo-responsive molecule,9-12 their probability for light controllable substrate channeling has not been explored. In addition, the control of reaction stoichiometry, spatial position and orientation of cascade components remains poor, and presents obstacles for efficient activity regulation. Recently, the development of DNA origami13-23 has provided platforms for accurate arrangement of components (such as nanoparticles,24-30 proteins31-36 and small molecules37,38) with the advantages of addressable assembly, nanometer precision, and dynamic control. Taking advantage of these features,35,39-42 it is efficient to control the distance and stoichiometry of enzyme cascade via DNA origami.31,32,34,43-45 Despite these successes, the construction of a smart light-driven substrate channeling for photo-regulating the enzyme cascade activity still remains challenging and has not been reported. As a popular photoisomerizable molecule, azobenzene plays a critical role in the design of light driven artificial molecular machines.46-48 In particular, azobenzene can be incorporated into

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Figure 1. Schematic illustration of synthetic light-driven substrate channeling for precise regulation of enzyme cascade activity on DNA origami with different wavelength of light. (a) Trans-cis photoisomerization of azobenzene by UV/Vis light. (b) Light responsive hybridization (under Vis) and dehybridization (under UV) of HJ-arm-AZO and anchor-AZO. (c) G6pDH-LDH enzyme cascade and NAD+-HJ swing arm assembly on DNA origami scaffold. Due to the hybridization of HJ-arm-AZO and anchor-AZO under Vis, the swing arm is fixed to be far away from the two enzymes, turning off enzyme cascade activity. When UV light is applied, the swing arm is released to freely swing between both enzymes to switch on the enzyme cascade activity. DNA strands to photo-regulate the hybridization/dehybridization of double-stranded DNA.11,49-53 Herein, combining photoresponsive azobenzene molecules and DNA origami scaffold, for the first time, we demonstrated a concept of light-driven substrate channeling for photo-regulation of enzyme cascade activity. DNA origami was employed for accurate and addressable assembly of the enzyme cascade, swing arm and anchor, while photoresponsive azobenzene was utilized to control the position of swing arm for enzyme cascade activity regulation. As shown in Figure 1, our model system consists of glucose-6phosphate dehydrogenase (G6pDH) and lactate dehydrogenase (LDH) as the enzyme cascade, and nicotinamide adenine dinucleotide (NAD+) as the cofactor. The cascade reaction starts with the oxidation of glucose-6-phosphate by G6pDH, with the reduction of NAD+ to generate NADH. NADH is subsequently used by LDH to reduce pyruvate to lactate, regenerating NAD+ to continue the cascade reaction (Figure S1).5 Due to its critical role, NAD+ is conjugated to a Holliday junction (HJ, Figure S2), which serves as a swing arm between G6pDH and LDH on DNA origami. Similar to natural substrate channeling, the presence of synthetic channeling has a significant influence on the G6pDHLDH cascade activity. To achieve light-controllable swing arm movement, photo-responsive azobenzene molecules are introduced into the binding DNA strands on HJ and the anchor strand on DNA origami to control their mutual hybridization (Figure 1b). The azobenzene-modified arm of HJ (called HJ-armAZO) hybridizes with the azobenzene-modified anchor strand (called anchor-AZO) under visible light (Vis) irradiation (Figure 1b), leading the swing arm far away from the enzyme cascade, which switched off its activity (left panel in Figure 1c). When ultraviolet light (UV) is applied, the isomerization of azobenzene results in the dehybridization of HJ-arm-AZO and anchor-AZO (Figure 1b), and the swing arm is released to swing between the

two enzymes, thus strongly enhancing the cascade activity (right panel in Figure 1c).

RESULTS AND DISCUSSION Photo-regulation of azobenzene-modified DNA behavior on DNA origami. In a proof-of-concept experiment to show the photoregulation of azobenzene-modified DNA behavior on DNA origami, we first explored the photo-switching property of the azobenzene-modified single-stranded oligonucleotide. Here we used sequence-complementary A3X/B4X (A3X: CGTXTAXGTXTCA; B4X: TGXAAXCTXAAXCG; X stands for the azobenzene) as the basic photo-responsive modules.50 To experimentally investigate the isomerization rate of azobenzenemodified oligonucleotide, the UV-Vis spectrum of B4X was measured at various irradiation time. The absorbance of azobenzene at 340 nm sharply decreased within 2 min under UV treatment (365 nm) (Figure 2b), and subsequently, recovered within 10 min under Vis irradiation (450 nm) (Figure 2c). These results confirmed that the rapid photo-response of azobenzenemodified oligonucleotide. We then investigated the photoresponsive DNA hybridization property of azobenzene-modified dsDNA in bulk solution using native polyacrylamide gel electrophoresis (PAGE). As shown in Figure 2d, the DNA duplex formed when the solution was irradiated at 450 nm. In contrast, the intensity of the DNA duplex band significantly decreased with simultaneous increase of intensity of single strand band after the solution was irradiated at 365 nm. More importantly, three cycles of Vis/UV irradiation led to three cycles of hybridization and dehybridization, suggesting the excellent photo-responsive reversibility of azobenzene-modified dsDNA.


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Journal of the American Chemical Society

Figure 2. Photo-regulation of azobenzene-modified DNA behavior: (a-c). Kinetics study of photoisomerization of azobenzene-modified ssDNA under UV (b) or Vis (c). (d) 12% native PAGE characterization of the reversible hybridization of A3X/B4X by alternating Vis/UV for three cycles. Lane 1: A3X; lane 2: B4X; lane 3, 5, 7, 9: A3X/B4X under Vis; lane 4, 6, 8: A3X/B4X under UV. (e) Schematic illustration (upper) and gel characterization (lower) of light driven HJ-FAM anchoring or leaving from DNA origami (left: fluorescein imaging for HJ-FAM detection; right: GelRed staining for DNA origami detection). Lane 1: HJ-FAM; lane 2: Ori-AZO-FAM under Vis; lane 3: Ori-AZO-FAM under UV; lane 4: Ori-FAM under Vis; lane 5: Ori-FAM under UV. Furthermore, using agarose electrophoresis, we studied the photo-regulation of HJ binding behavior on the DNA origami (Figure 2e). The top arm of HJ was labeled with a fluorescent dye (HJ-FAM) in place of NAD+ to track the behavior of HJ. The HJ was assembled on DNA origami through the sequence-specific hybridization of HJ-arm-AZO and anchor-AZO on DNA origami (named Ori-AZO-FAM). The band for HJ-FAM and band for DNA origami were overlaid under Vis irradiation (lane 2 in Figure 2e), while were separated under UV irradiation (lane 3 in Figure 2e). Thus, we concluded that HJ-FAM was successfully anchored on or released from DNA origami in response to different wavelengths of light. In order to further confirm the photoisomerization-dependent hybridization regulation on DNA origami, we designed a control group in which the hybridization sequences between HJ-FAM and DNA origami were native DNA strands (named Ori-FAM) (Figure S4). The result showed the HJFAM co-localized with DNA origami regardless of the wavelengths used for light irradiation (lane 4 and lane 5 in Figure 2e). These results strongly confirmed that the photo-regulation of DNA hybridization and dehybridization worked well on DNA origami. Light-switching enzyme cascade activity by regulating the position of substrate channeling. After confirming the photoresponsive DNA hybridization behavior on DNA origami, we then assembled the G6pDH-LDH enzyme cascade on a rectangular DNA origami (ca. 60 × 90 nm2). G6pDH and LDH were successfully coupled with respective DNA sequences through bi-functional succinimidyl 3-(2-pyridyldithio) propionate (SPDP) linker (Figure S5, S6 and S7).54 Through the sequencespecific hybridization of the DNA strands labeled on the enzymes and the extended DNA capture probes on DNA origami, the two enzymes were expected to achieve addressable locations on the specific binding sites on the DNA origami scaffold. This was fully confirmed by gel electrophoresis (Figure 3a) and the direct visualization using atomic force microcopy (AFM) imaging (Figure 3b). More importantly, the assembly yield of enzymes on DNA origami was similar under Vis and UV light (lane 3 and 4 in Figure 3a), indicating that irradiation has no effect on the enzyme assembly. We then verified the photo-regulation enzyme cascade activity of G6pDH-LDH on DNA origami. To study the effect of light irradiation on enzyme activity, we explored the enzyme catalytic

activity kinetics after different periods of light irradiation. The activities of both enzymes had little change after lengthy Vis or UV irradiation (at least 16 min) (Figure S8 and Figure S9), indicating the negligible effect of light irradiation on the catalytic activity of the two enzymes individually. According to our previous report,44 the presence of substrate channeling is critical to enzyme cascade activity. Thus we utilized the light-driven presence and absence of substrate channeling to regulate enzyme cascade activity. That is, the azobenzene-modified anchor strand (anchor-AZO) hybridizes with the arm of HJ to fix the substrate channeling to be far away from enzyme cascade, thus the activity was turned off under Vis (named AZO-Ori-Vis in Figure 3c). When UV light is applied, the swing arm is released from anchorAZO to freely swing between the enzyme cascade, thus switching on the enzyme cascade activity (named AZO-Ori-UV in Figure 3c). To further assess the real state of cascade activity under the two wavelengths, we designed two control groups with known opposite activity states. In the negative control group (named negative control in Figure 3c), there was no azobenzene modified on DNA, allowing the anchor to be hybridized with HJ-arm regardless of light irradiation. In this case, the swing arm stayed away from G6pDH-LDH, leading to enzyme cascade in the inactive state. The positive control group (named positive control in Figure 3c) had no anchor strand modified on DNA origami. Thus, the swing arm swung freely, promoting substrate channeling occurrence between G6pDH and LDH, leading to the active of enzyme cascade. The negative control and positive control indicated the real activity off and on states, and the result showed their activities were about 5 fold different (Figure 3d and S11). As expected, the activity of AZO-Ori-Vis had same activity as negative control, indicating that the activity of enzyme cascade was switched off under Vis irradiation. In contrast, the activity of AZO-Ori-UV behaved similarly to positive control, which was 5 fold higher than inactive state activity. Therefore, efficient photoregulation of enzyme cascade activity was successfully achieved by photo-switching the position of swing arm on DNA origami. Effects of inter-enzyme distance and swinging range of the swing arm. The accurate and addressable DNA origami enables us to further investigate the effects of inter-enzyme distance and the swinging range of swing arm on the enzyme cascade. The distance between G6pDH and LDH was the first parameter to be



Figure 3. (a) Characterization of the G6pDH-LDH assembled on DNA origami with 1.2% agarose gel. Lane 1: M13mp18 ssDNA; lane 2: rectangular origami; lane 3: G6pDH-LDH assembled on DNA origami under Vis; lane 4: G6pDH-LDH assembled on DNA origami under UV. (b) AFM imaging of the DNA origami with G6pDH-LDH, scale bar: 50 nm. (c-d) Schematic illustration (c) and relative activities (d) of the four state of enzyme cascade/DNA origami assembly. studied with the length of swing arm held constant. By fixing the location of G6pDH on origami, the location of LDH was changed to different binding sites to adjust the horizontal distances between the two enzymes to 10 nm, 25 nm, 35 nm and 40 nm (Figure 4a, see Figure S10 for design detail). As shown in Figure 4b and S12, the activity of G6pDH-LDH under UV irradiation was higher than that under Vis irradiation, regardless of the interenzyme distance. In contrast, with increasing inter-enzyme distance, the activity at inactive state (after Vis) decreased significantly due to the decrease of interaction between the swing arm and the two enzymes. To quantify the photo-regulation efficiency of the enzyme activity, the enhancement of the enzyme cascade activity in the active (after UV) state compared to the inactive states (after Vis) was chosen as the criterion. As shown in Figure 4c, the activity improved when the distance increased from 10 nm to 35 nm, then declined when the distance continued to increase, suggesting that the swing arm serves as a bridge to connect the enzyme cascade in a certain swinging range. When the distance became longer, swing arm could not interact efficiently between the two enzyme. These results also powerfully showed the benefit of the DNA origami platform, which enables precise control of the inter-enzyme distance at the nanometer scale. Subsequently, we investigated the influence of swinging range of the swing arm by fixing the inter-enzyme distance of G6pDH-LDH at 35 nm. The swinging range was determined by the linker length between cofactor NAD+ and the HJ top arm. Different poly T linkers (T0, T10, T20) were designed (Figure 4d). As shown in Figure 4e and S13, with increasing poly T length, the enzyme cascade activity showed little increase under UV irradiation, while dramatically improving under Vis irradiation,

Figure 4. The effects of inter-enzyme distance (a-c) and the length of the swing arm (d-f) on the photo-regulation efficiency of enzyme cascade activity. The schematic representations (a, d), relative enzyme cascade activity under different light (b, e), and activity enhancement fold (c, f) are shown.

Figure 5. Rapid and reversible photo-regulation of enzyme cascade activity on DNA origami. (a-b) Enzyme cascade activity under the different exposure time of UV (a) and Vis (b). (c) Regulatory cycling of the enzyme cascade activity under Vis or UV irradiation. largely due to the increasing swinging range of NAD+. Based on the active/inactive ratio (Figure 4f), the activity enhancement of T0 was about 7 fold, then sharply decreased to about 3.5 fold when the linker was T10, and finally decreased to about 2.6 fold when the linker was T20. As shown in these results, both the inter-enzyme distance and swing range of swing arm significantly influence the enzyme cascade activity regulation, since they determine the interact efficiency between cofactor and enzymes, as well as the activity difference of enzyme cascade between active and inactive state.5,44 These results demonstrated that the swing arm is an important functional component to regulate enzyme cascade activity. Rapid and reversible photo-regulation of enzyme cascade activity. Finally, we studied the kinetics and reversibility of photo-switching enzyme cascade activity. We first tested the ki-


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Journal of the American Chemical Society netics of the switching-on process under UV irradiation. The assembly taken directly from the thermal cycler system was defined as the initial state (named UV-0 min ). In this state, the enzyme cascade activity was switched off. Equal amount of samples after different time of UV irradiation (2 min, 4 min, 8 min and 10 min, respectively) were tested. For testing the kinetics of the switching-off process, the sampled pretreated with UV for 10 min was defined as initial state (named Vis-0 min). Similarly, equal amount of samples was tested after 10 min, 15 min and 20 min of Vis irradiation. As shown in Figure 5a and Figure S14, the enzyme cascade activity was enhanced largely after 2 min UV irradiation, and switched on completely after 4 min. The activity switched off completely after 10 min Vis irradiation and longer Vis treatment times had little effect on the activity of the off state assembly (Figure 5b and S14). These experiments clearly demonstrated that the light-driven substrate channeling responded rapidly to light irradiation. To further investigate the reversibility of our system, we tested the enzyme cascade activity after several cycles of UV or Vis irradiation. As shown in Figure 5c and S15, the activity remained steady after different cycles of UV light irradiation. Although the activity after Vis was slightly increased, it still kept inactive after three cycles. These results indicated that the enzyme cascade activity on DNA origami could be reversibly regulated through different wavelengths of light irradiation, which is of great importance in many areas.55-57

CONCLUSIONS In summary, we have for the first time demonstrated the concept of light-driven substrate channeling to regulate the enzyme cascade activity using DNA origami and photoresponsive molecules. In our model system, the presence of substrate channeling was achieved by photo-responsive cofactor covalently linked to the swinging arm, whose location could be reversibly controlled by light to selectively activate or inhibit the enzyme cascade reaction in response to different wavelengths of light. Moreover, taking advantage of the addressable assembly of DNA origami, precise control of the inter-enzyme distance and swing arm successfully improved the regulation efficiency. Therefore, a clean, rapid and reversible light-regulation of enzyme cascade activity on DNA origami was finally achieved. Considering the critical role of substrate channeling, this lightdriven substrate channeling strategy is expected to be widely applied in areas such as synthetic biology and biomedicine,58 e.g. optogenetics.

EXPERIMENTAL SECTION Materials and Reagents. Glucose-6-phosphate dehydrogenase (G6pDH, from Leuconostoc mesenteroides), Lactate dehydrogenase (LDH, from rabbit muscle), lactate oxidase (LOX, from Pediococcus sp.), horseradish perpxidase (HPR), glucose-6phosphate (G6p), sodium pyruvate, N-Succinimidyl 3-(2pyridyldithio)-propionate (SPDP), β-Nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH), βNicotinamide adenine dinucleotide hydrate (NAD+), AmplifluTM Red and Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Sigma (Ontario, CA, USA). Phosphatebuffered saline (PBS), sodium N-(2-hydroxyethyl) piperazine-N'(2-ethanesulfonate) (HEPES) were purchased from Sangon Biotech (Shanghai, China). All aqueous solutions were prepared using ultrapure water (18 MΩ, Milli-Q, Millipore). Preparation of azobenzene-modified DNA and DNA origami relative oligonuleostrands. All DNA oligonucleotides except azobenzene-modified DNA were purchased from Sangon Biotech (Shanghai, China). The azobenzene-modified DNA sequences (Table S1) were synthesized on a 12-Column DNA

synthesizer (PolyGen GmbH). The HJ sequences are listed in the Table S2. The M13mp18 ssDNA was purchased from Bayou Biolabs (Los Angeles, CA, USA). The DNA origami tiles sequences were listed in Supporting Information Table S3 and S4. Enzyme conjugated DNA. In order to conjugate the enzymes with DNA, succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was chosen as a bifunction coupling linker connected by amineto-sulfhydryl conjugation. The experimental details was similar to that described in the previous report.44 First, 100 µL of 40 µM LDH or G6pDH in 10 mM HEPES (pH 7.5) was mixed with SPDP (5-fold excess for LDH, 3-fold excess for G6pDH). After adjusting the pH to 8-8.5, the mixture was incubated for 1 hour at room temperature. Excess SPDP was removed using Amicon 50kD cutoff filters. The SPDP coupling efficiency was estimated to be 1.2-2.5 SPDP per enzyme, by monitoring the increase absorbance at 343 nm for the release of pyridine-2-thione (extinction coefficient 8080 M-1cm-1). Next, the SPDP modified LDH and G6pDH were assembled with oligonucleotide. LDH was coupled with WS2 DNA (5’-SH-TTTTTGGCTGGCTGG-3’), while G6pDH was coupled with WS3 DNA (5’-SHTTTTTGCGTGCGTGC-3’). Thiol modified DNA was fresh prepared by incubating with 10 fold TCEP for 1 hour at room temperature. Excess TCEP was removed using Amicon 3-kD cutoff filters. Then enzyme-SPDP was incubated with 10 fold excess SH-DNA in 10 mM HEPES (pH 8.0-8.5) for 1 hour at room temperature. The excess DNA was removed using Amicon 50-kD cutoff filters. The final concentration of enzyme and conjugation ratio are listed by Supporting Information Figure S7. NAD+-DNA conjugation. The experimental details was described in the previous report.44 Briefly, 100 µL 200 µM aminemodified oligonucleotide was treated with anion exchange DEAESepharose resin (Sigma) by charge adsorption. Then 200 µL of 150 mM disuccinimidyl suberate (DSS) linker was conjugated with the oligonucleotide in N,N-dimethylformamide (DMF) with 2% (v/v) N,N-diisopropylethylamine (DIPEA) for 1 hour at room temperature. Then 10 fold excess of amino-modified NAD+ analog was incubated with the oligonucleotide in 1 M HEPES (pH 8.0) for 1 hour. After incubating, 50 mM HEPES with 1.5 M NaCl (pH 8.0) was added to elute the DNA-NAD+ from the resin. The final eluate was purified by an Eclipse XDB-C18 column (Agilent) on HPLC (Agilent 1200). The mobile phase was 100 mM TEAA and acetonitrile, with an acetonitrile gradient from 21% to 37%. Preparation of enzyme assembled DNA origami. All the tiles were prepared in 1×TAE/Mg (40 mM Tris, 20 mM acetic acid, 2 mM EDTA and 12.5 mM magnesium acetate, pH 8.0). The mixtures of 30 nM M13mp18 ssDNA and 150 nM tiles and probes were annealed in a BioRad thermocycler with the following temperature gradient: the beginning temperature was set to 90 ℃, and was decreased to 78 ℃ (2 ℃/step, hold for 5 min at each temperature), then decreased from 76 ℃ to 24 ℃ (4 ℃/step, hold for 5 min at each temperature) and finally held at 24 ℃. After the assembly, excess ssDNA strands were removed by 100-kD cut-off filters. Then the concentration of DNA origami was quantified by the absorbance at 260 nm, based on the extinction coefficient of ~1.091 × 108 M-1cm-1. According to the concentration, one-fold of NAD+ conjugated HJ (pre-annealed) and 5-fold of LDH-WS2 and G6PDH-WS3 were mixed with DNA origami. The annealing protocol was listed as follow: the start temperature was 37 ℃, which was held for 5 min; then was decreased from 36 ℃ to 15 ℃ (1 ℃/step, hold for 2 min at each temperature) and finally held at 15 ℃. Enzyme cascade activity array. For G6pDH-LDH enzyme cascade activity measurement, a coupled assay of lactate oxidase/horseradish peroxidase was performed. Lactate (the



product of the G6pDH-LDH enzyme cascade) could be oxidized by LOX to generate hydrogen peroxide, which is used by HRP to oxidize Ampliflu Red, to give the final fluorescent product resorufin (ex 544 nm/em 590 nm). In brief, 20 nM DNA origami enzyme cascade, 1 mM glucose-6-phosphate, 1 mM pyruvate, 10 nM lactate oxidase, 10 nM horseradish peroxidase, and 200 µM Ampliflu Red were mixed in 100 mM HEPES (pH 8.0) buffer. The signal of the cascade was followed by the increasing fluorescence intensity (ex 544 nm/em 590 nm) using a multimode plate reader (EnSpire®, PerkinElmer, USA). Error bars are shown as the standard error of the mean from three independent measurements.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Supporting Figure S1-S15 and Tables S1-S4 as described in the text. (PDF)

AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected] * [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We are grateful for the National Science Foundation of China (Grants 21735004, 21435004, 21775128, 21705024, 21521004 and 21705038), Program for Changjiang Scholars and Innovative Research Team in University (Grant IRT13036) and a DODNavy-ONR MURI award (W911NF-12-1-0420) for their financial support. We gratefully acknowledge Professor Chuanliu Wu for providing multimode plate reader (EnSpire®, PerkinElmer, USA) and Huimin Zhang for helpful discussions.

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