All-in-One Synchronized DNA Nanodevices Facilitating Multiplexed

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All-in-one Synchronized DNA Nanodevices Facilitating Multiplexed Cell Imaging Jing Xue, Feng Chen, Min Bai, Xiaowen Cao, Ping Huang, and Yongxi Zhao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00089 • Publication Date (Web): 12 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019

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Analytical Chemistry

All-in-one Synchronized DNA Nanodevices Facilitating Multiplexed Cell Imaging Jing Xue, †,§,‡ Feng Chen, §,‡ Min Bai, § Xiaowen Cao, § Ping Huang, † and Yongxi Zhao §,* State Key Laboratory for Mechanical Behavior of Materials, School of Materials Science and Engineering, Xi’an Jiaotong University, Xianning West Road, Xi’an, Shaanxi 710049, P. R. China †

The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xianning West Road, Xi’an, Shaanxi 710049, P. R. China §

ABSTRACT: Multifunctional DNA nanodevices perform ever more tasks with applications ranging from in vitro biomarkers detection to in situ cell imaging. But most developed ones consist of a series of split building blocks, suffering from asynchronous behaviors in complicated cellular microenvironments (endocytosis pathway, diffusion-limited cytoplasm, etc.) and causing the loss of stoichiometric information and additional post-assembly processes. Herein we constructed all-in-one DNA nanodevices to achieve synchronous multiplexed imaging. All DNA components, including two sets of probe module (each containing target-specific walkers, hairpin tracks with chemically-damaged base), are modified on individual gold nanoparticles. This design not only enable their integrated internalization into cells, circumventing inhomogeneous distribution of different building blocks and increasing the local concentrations of the interaction modules, but also avoid the impact of stochastic diffusion in viscous cytoplasm. A couple of intracellular enzymes in situ actuates the synchronized motion of the modules all on-particle after specific recognition of intracellular targets (such as RNAs and proteins), thus facilitating synchronized multiplexed cell imaging. Finally, the proposed all-inone nanodevices were successfully applied to monitor intracellular microRNA-21 and telomerase expression levels. The flexible design can be extended to detect other cytoplasmic molecules and monitor related pathways by simply change the sequences.

exogenous fuels or supplementary components into cytoplasm complicates the reaction procedures and limit the detection efficiency. Furthermore, the signal responses of these nanodevices without reference modules may be influenced by heterogeneous environments of different cells.

Multiple components and corresponding interaction networks synergistically regulate the metabolic processes in living cells.1 Consequently, simultaneously monitoring of multiplex intracellular biomarkers is critical to acquire comprehensive information for biological assays, medical diagnostics and drug delivery. Deoxyribonucleic acids (DNAs) are outstanding building materials for complex system construction due to the predictable hybridization and programmable assembly.2-4 The DNA-based nanodevices with excellent structural flexibility have been applied in broad applications for multiplexed targets detection and imaging.5-8

MicroRNAs (miRNAs/miRs) are a group of singlestranded, small-sized RNAs that can regulate the expression of genes at the post-transcriptional level. Their abnormal expressions are associated with various diseases, thus have been widely used as biomarkers in medical diagnostics and prognostics.19, 20 Telomerase, a unique ribonucleoprotein that extends specific repeats (TTAGGG) to the ends of telomeres, is overexpressed in primary human tumor cells. Its activity can be an indicator of cancer progression.21, 22 Therefore, microRNA21 (miR-21) and telomerase are selected as dual-targets for cell imaging. However, previous methods for miR-21 and/or telomerase detection such as polymerase chain reaction (PCR),23 nanoflares,24, 25 molecular probes26, 27 and electrochemiluminescence techniques28 suffer from complicated procedures, intracellular stability and easily influenced by environmental interferences resulting in errors. We program endogenous enzyme-powered, all-inone synchronized DNA nanodevices with self-reference

Existing DNA nanodevices, such as molecular robots,1-3 walkers4, 9, 10 and networks,11-16 can in principle be used to simultaneously monitor the alternative expression levels of multiple biomarkers in living cells. However, most of these systems are consisting of separate building blocks. When applied in live cell imaging, the asynchronous cellular uptake of different building elements could reduce the control of their distribution and stoichiometric information, resulting in unreliable signal response. In addition, many researchers have proved that diffusivity of macromolecules and nanoscale structures varies over orders of magnitude in mammalian cell cytoplasm and other solutions.17, 18 Co-delivery of multiple modules,

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modules for facilitated imaging of intracellular miR-21 and telomerase （Scheme 1A）. A couple of damage base repair enzymes in situ drive the motion of the modules all on-particle

EXPERIMENTAL SECTION Materials and Reagents. All reagents were used without further purification and are of analytical grade. Milli-Q water with resistance >18.2 MΩ was used for all reactions. Gold (III) chloride hydrate (HAuCl4•4H2O) 99%, trisodium citrate dehydrate (Na3C6H5O7•2H2O) 99%, were obtained from Sigma-Aldrich. And reactions here were RNase-free. CutSmart (50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(Ac)2, 100 μg/ml BSA, pH 7.9), Human 8oxoguanine DNA glycosylase (hOGG1), human apurinic/apyrimidinic endonuclease 1 (APE1) were obtained from New England Biolabs Inc. All of the oligonucleotides are synthesized and purified by Sangon Biotech Co., Ltd. The information of DNA sequences are listed in Table S1.

Scheme 1. (A) Schematic illustration of all-in-one synchronized nanodevices and split-module assemblies in cytoplasmic environments. a (B) Working principle of all-in-one synchronized DNA nanodevices.

All-in-one DNA Nanodevices Construction. Gold nanoparticles (AuNPs) were synthesized as follows. Trisodium citrate solution (1%, w/v) was fast added into a continuous boiling and stirred solution of HAuCl4. The solution was kept boiling and stirred for another 20 min, then gradually cooled to 20°C. The prepared AuNPs were kept at 4 °C. R-DW was blocked by partial complementary strand at a molar ratio of 1:2. The mixture was heated to 65 °C and gradually cooled to 4 °C. Thiolated oligonucleotides Prior to functionalization onto AuNPs, the thiol groups of DWs and DTs were activated by tris(2-carboxyethyl)phosphine (TCEP). AuNPs were mixed with DW and DT at the ratio of 1:30:270 (molar ratio). After incubated overnight, the mixed solution was successively added with NaCl solution (2.0 M, 10 mM PB) until the total NaCl concentration achieved 0.3 M. Additional 12-h incubation was needed. The resulted AuNP solution was centrifuged and the supernatant removed leaving a pellet of AuNPs, which were resuspended in 10 mM Tris-HCl buffer (pH 7.4) and stored at 4 °C until further use.

The endogenous enzymes hOGG1 and hAAG (light grey shapes) are inert and diffuse fast in cytoplasm. The proposed all-in-one DNA nanodevices perform in situ synchronized interaction processes. Whereas in split-module assembly, the process need exogenous assistance (green sphere) and can only be driven via three steps: asynchronous internalization of the split modules, diffusion/post-assembly and stochastic reaction. a

Examination of DNA Density Per AuNP. A competition strategy was applied to examine the DNA density per AuNP. 2-mercaptoethanol was used to release modified DNA strands. Then the supernatant containing released DT strands was acquired through centrifugation and the fluorescence was measured. Specifically, 2mercaptoethanol mixing with modified nanoparticles at a molar ratio of 1:2000 was incubated in dark overnight. After centrifuged at 12,000 g for 25 min, AuNPs were precipitated and 90% of the supernatant was collected for fluorescence analysis. Next, the concentration of DT strand was determined from a standard calibration curve of the track strand with fluorophore label. Finally based on the initial ratio of AuNP and DT, its average number per AuNP was obtained. The results exhibit that about 60 DT molecules were funtionalized onto each AuNP. Then approximately 7 DW strands were modified on a AuNP based on the molar ratio. The corresponding densities of DT and DW were circa 1.0×10-1 and 1.0×10-2 nm-2, respectively.

after specific recognition of intracellular targets, enabling synchronized imaging of distinct biomarkers. Their small molecular weights below 40 kDa enable them to diffuse freely as Brownian particles with negligible effects of molecular crowding.17, 29 Two sets of probe modules and one reference module, each containing single-foot DNA walkers (DWs) and densely-packed DNA tracks (DTs), are modified on individual gold nanoparticles (AuNPs).30, 31 As a result, the compact designed DNA devices enable their integrated internalization into cells, thereby circumventing inhomogeneous distribution of different building blocks, also avoid the impact of stochastic diffusion in viscous cytoplasm. In other words, the proposed all-in-one design bypasses the freely-diffusing split-module system (one case of AuNP-DTs/free DWs as a representative) by bringing the reactants closer together, reducing the effective reaction diameters and increasing the local concentrations of the interaction modules. Moreover, the integrated RMs can indicate heterogeneous environments of different cells for more reliable detection results.

Fluorescence Measurements. All fluorescence analysis of DNA Nanodevices in vitro were performed at


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Analytical Chemistry walker-track duplexes. Each R-DW is silenced by a blocking strand (BS, its length was optimized in Figure S1) and the target miR will hybridizes with it via a stranddisplacement reaction, releasing the blocking strand and unlocking R-DW. Then free R-DW can hybridize with RDT on AuNP surface to form a duplex. On the other hand, T-DWs ended with telomerase primers, can be elongated in the presence of telomerase in the intracellular environment to generate telomeric repeats. The repeated sequences are complementary to T-DTs, which can produce oG-containing walker-track duplexes. Afterwards for both of the resulting duplexes, freely diffused Human 8-oxoguanine glycosylase 1(hOGG1) could specifically bind to oG sites and excise these damaged bases to generate AP sites. Then AP sites will be hydrolyzed by downstream enzyme apurinic/apyrimidinic endonuclease 1 (APE1) of base-excision repair (BER) pathway, inducing break/dissociation of DTs simultaneous with releasing of DWs. Then, free DWs and other DTs could generate new DNA duplexes. This process cycles and enables DWs moving fast along on-particle DTs, meanwhile outputs fluorescence signals for monitoring the motion. Moreover, in the all-in-one design, RMs represent similar structure comprising of DWs and DTs directly complementary to each other with an oG-base in the loop. The corresponding RM fluorophore will be unloaded respond to hOGG1 and APE1, which can function as self-reference for the variation of enzyme activities in different cells.

37 °C for 60 min using LightCycler 96 (Roche Applied Science, Mannheim, Germany). hOGG1 and APE1 used in the reactions were 4 U/mL and 2000 U/mL respectively. Ficoll 400 (1, 2.5, 5, 6 wt%) were added into the buffer to mimic cytoplasm-like conditions. Corresponding viscosity was measured by a commercial viscometer (SNB-1, Fangrui, China). Intracellular Imaging. HeLa cells were cultured in a humidified incubator containing CO2 (5%) at 37 °C. Dulbecco’s modified Eagle medium (DMEM) supplemented with 1% antibiotics penicillin/streptomycin (100 U/mL) and 10% fetal bovine serum (FBS) was used. 8well chambered cover glass (Cellvis, USA) was seeded with 105 HeLa cells each well at 37 °C for 12 h. Then cells were incubated in 200 μL DMEM (serum-free) with 10% (v/v) all-in-one DNA nanodevices for 4 h. Before imaging, three times of wash were performed with 1×PBS. Nikon A1 laser scanning confocal microscope (lasers of 488, 561 and 633nm as excitation sources) was used for fluorescence images acquiring. Flow Cytometry Analysis. HeLa cells were seeded in a 12-well plate and incubated overnight to 80-90% confluency. Then the cells were incubated in 500 μL DMEM (serum-free) with 10% (v/v) all-in-one DNA nanodevices for at 37 °C for 4 h. After washing with 1×PBS for three times, the cells in each well were detached from the plate by 0.25% trypsin and kept in 1×PBS on ice and in dark for flow cytometry measurement. Flow cytometry analysis was carried out using a FACS Canto system (BD Biosciences, USA). Totally 105 cells were analyzed for each sample. Particle Fluorescence Analysis. The fluorescence imaging of single-particle was performed on the cover slides. The cover slides were pre-treated as follows. First, they were immersed in a mixture of 30% H2O2 and concentrated H2SO4 (1:3 v/v) for 15 min. Then the cover slides were washed with deionized water and ethanol, and immersed in 10% (v/v) APTES solution for 2 h to complete surface aminosilanization. Then all-in-one DNA nanoparticles and particles with DTs only (for the splitmodule control) were deposited onto the aminofunctionalized cover slides and incubated for 10 min at room temperature in dark. After washing with 1×Cutsmart buffer, fluorescent images were acquired by a high resolution laser confocal microscope (TCS SP8 STED 3X, Leica). 488-nm laser and 100× oil-immersion objective were selected. Imaging was started immediately after target miR-21, hOGG1 and APE1 successively adding to allin-one designed sample. Free DWs were added together with miR-21 and two enzymes for split-module control.

Figure 1. The operation of all-in-one and split-module DNA nanodevices in diffusion-limited environments. (A) Walking performance in cytoplasm-like viscous and crowded solutions. (B) Single-particle fluorescence analysis using STED microscopy, scale bar=5 μm.

To illustrate how all-in-one design accelerates DNA motion in cytoplasm-like environment, we practically compared the performances of our compact DNA nanodevice and a freely-diffusing reaction system based on assembling of split modules (DT-modified AuNPs and free DWs) in different crowded and viscous environments (miR-21 selected as representative target). As shown in Figure 1A and S2, all initial reaction velocities for the two systems gradually decreased with the increase of solution

RESULTS AND DISCUSSION Scheme 1B depicts the overall operation of the all-in-one DNA nanodevices. Two individual stem-loop structured DTs (R-DTs and T-DTs) both containing one chemically damaged 8-oxoguanine (oG) site are labelled with a terminal fluorophore. The fluorescence is quenched by AuNP before activation. DWs (R-DWs and T-DWs) are designed to hybridize with corresponding DTs to form



parameters fixed (Figure 2B and S4). With the increase of DW density, the reaction velocity first rise and then declined notably. The decline of the velocity value illustrated high DW density would result in inhibition of the signal output. This phenomenon might result from the increasing electrostatic and steric hindrance among adjacent DWs. Furthermore, optimizing other reaction conditions such as enzyme concentrations could enhance the walking kinetics. Then we examined the operation of the DNA nanodevices in response to various concentrations of target miR-21. As expected, higher concentrations of miR-21 led to larger fluorescence signal (Figure 2C), consistent with more walkers being initiated by higher concentrations of target. Then the specificity of our all-in-one nanodevices was examined by testing two variants with one and two bases mismatch. All the variants were unable to drive specialized DNA walking system (Figure 2D and S5), indicating its excellent resistance to non-specific targets.

viscosity, indicating experimental results are consistent with the mentioned principle that diffusion of nanoscale objects is limited in mammalian cell cytoplasm. And in each crowded solution, all-in-one nanodevices showed much higher initial velocity and signal output, achieving acceleration of DNA walking procedure. Then we also monitored the corresponding processes of the two reaction systems in a viscous environment by stimulated emission depletion (STED) microscopy (Figure 1B and S3). The particles were immobilized on chemically treated cover slides via electrostatic adsorption. The walking of DWs induced enzymatic digestion of DTs, releasing fluorophore into reaction solution and leading to fluorescence loss of the single particles at a particular focusing plane under STED. For all the reactions before activation, the fluorescent spots in the imaging area were obviously bright. After sequentially adding of target miR21 and the couple of enzymes, the images of spots taken by the same time interval showed much darker fluorescence for our system. Instead, the freely-diffusing split-module strategy complicated the whole process and the fluorescence of the spots in the viscous solution present no clear change observed by naked eyes.

Figure 2. The reaction kinetics of the proposed all-in-one DNA nanodevices. (A) Verification of on-particle rather than interparticle DNA mobility of the device. Au-DT, Au-DW and Au-DW-DT indicate AuNPs modified with DT, DW and DW/DT, respectively. (B) The effect of on-particle DW density on initial reaction velocity of single-target nanodevices. (C) Detection range of all-in-one DNA nanodevices. (D) Specificity of all-in-one DNA nanodevices.

Figure 3. Monitoring intracellular miR-21 using corresponding all-in-one DNA nanodevices. (A) The cell images of three walker systems. Flow cytometry analysis is consistent with cell imaging. (B) Fluorescence images of miR-21 specific DNA nanodevices with RMs in different cell samples. Cell sample 1: untreated HeLa cells. Cell sample 2: HeLa cells treated with 2 μM NCA. Scale bar: 20 μm.

Next, we investigated the reaction kinetics of all-in-one DNA nanodevices. As verified above that our design operated much faster than split-module diffusing assemblies, we further explored the interparticle influence of the reaction process. As shown in Figure 2A, the anchored DW preferred to walk along DTs on individual particle rather than interact with other particles. This proved again all-in-one architecture plays a pivital role in accelerating the reaction kinetics. Longer DW possibly lead to its jumping to DTs on adjacent particles and even cause unwanted nanoparticle aggregation. Moreover, the impact of on-particle DW density was studied with other

Finally, we monitored the operation of the all-in-one DNA nanodevices in cytoplasmic milieus for individual target miR-21 or telomerase (Figure 3 and S6). For singletargeted devices, non-specific reaction systems (no DWs and random DWs) are designed as negative controls. Each of these three types of nanodevices are synchronously entering into living cells, avoiding asynchronous uptake of different modules. It can be seen in Figure 3A and S6A that two negative devices induce no obvious signals in HeLa cells, while specific DW anchored


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Analytical Chemistry DNA nanodevices show high and bright fluorescence responses. For single-targeted all-in-one nanodevices with/without RMs, the walking processes are studied in different cell samples with altered enzyme activities (Figure 3B, S6B and S7). HeLa cells are treated by an APE1 inhibitor CRT0044876 (7-nitroindole-2-carboxylic acid, NCA). The reference signal RM only depends on the activities of hOGG1 and APE1, clearly indicating the signal fluctuation from the change of intracellular enzyme levels, but not the levels of targets.

Ptelomerase and Rsignal, are recorded in different cell samples. MATLAB was used to extract the intensity distribution information of each pixel in these cell images and related scatter plots are shown on the right. It can be seen that inhibition of telomerase lead to decrease of Ptelomerase whereas the signal PmiR stays unaffected. For NCA treated cell sample 3, both Ptelomerase and PmiR fluorescence turn down as APE1 level declines, while Rsignal can reflect the activities of hOGG1 and APE1. Thus, all the above reliable results verify the successfully design of our all-in-one DNA nanodevices. The design can also be combined with various techniques (such as microfluidics) to participate in other live cell analysis and/or cargo release applications.

In addition, dual-targets induced intracellular imaging has also been evaluated. For the DNA nanodevices without RMs, the change of APE1 activity lead to the decline of both miR-21 and telomerase specific fluorescence responses (Figure S8). This result further confirm that lack of RMs can cause unreliable signal output. Then as shown in Figure 4 and S9, all-in-one nanodevices with RMs are operated in three distinct HeLa cell samples (untreated cells, cells treated with telomerase inhibitor EGCG and cells treated with NCA). We simplify the reaction as PmiR = ƒ(XmiR, XhOGG1, XAPE1) and Ptelomerase = ƒ(Xtelomerase, XhOGG1, XAPE1) considering the signals are affected by three componets. P represents the performances of the all-in-one

It is well known that intracellular damage base repair enzymes hOGG1 and APE1 are overexpressed in various human tumor cells. We testify their sufficiency for our imaging systems in HeLa cells incubated with a series of NCA concentrations (Figure S10). At first when NCA concentration rises from zero in a narrow range, the reference fluorescence decreases while the signal fluorescence remains at a steady level. Yet as NCA concentration increases substantially, the intensity of signal and reference both decline. The results demonstrate enzymes are excessive for our all-in-one DNA nanodevices in HeLa cells. Cells use spatial constraints to control and accelerate the flow of information in enzyme cascades and signaling networks.32-35 Such spatial constraints accelerate the interactions between modules by two efficient paths: 1) increasing the effective concentrations of the modular reactants; 2) turning freely diffusing components into fixed integrity. Such principle has been involved in synthetic DNA-based nanodevices.1-4, 9, 10, 36-40 Although these DNA nanodevices perform well in water solutions or other cell-free settings owing to the low viscosity, reactants diffusing and multistep assembly still exist. Thus far, intracellular operating of DNA nanodevices with split reaction modules41-43 suffer from diffusional limitation and low reaction efficiencies. All-in-one synchronized DNA nanodevices can simultaneously solve these tough problems fundamentally.44 In addition, exogenous fuels or supplementary components limit the reaction procedures as well. Thus on the basis of our work that developing MnO2 nanozymes combined with endogenous proteins for monitoring the intracellular DNA base-excision repair pathways,45 all-in-one DNA nanodevices initiated by intracellular enzymes have been successfully constructed.

Figure 4. Intracellular dual-targets analysis using specific allin-one DNA nanodevices. Cell sample 1 corresponds to untreated HeLa cells. Cell sample 2 refers to HeLa cells treated with telomerase inhibitor epigallocatechin gallate (EGCG, 60 μg/mL). Cell sample 3 represent the cells treated with APE1 inhibitor NCA (2 μM). MATLAB was used to extract the intensity information (0-256) for each pixel in the cell images (Figure S9). Right panel is the corresponding intensity distribution plots (vertical direction). High scatter density and intensity values indicate strong fluorescence response. The reference signals (red) can indicate the change of hOGG1 and APE1 activity in different cell samples. Scale bar: 20 μm.

CONCLUSION In summary, we have programmed all-in-one synchronized DNA nanodevices to facilitate intracellular multiplexed imaging. A couple of endogenous damage base repair enzymes in situ actuate the walking via damage base excision/hydrolyzation reactions. Two distinct types of intracellular molecules miR-21 and telomerase are selected as imaging targets. Single gold nanoparticles are set as spatially-localized templates for

nanodevices and X indicates the levels of the corresponding influence factors. Accordingly, Rsignal = ƒ(XhOGG1, XAPE1) denotes the reference of the signals. The fluorescence of green, purple and red, representing PmiR ,



(3) Omabegho, T.; Gurel, P.S.; Cheng, C.Y.; Kim, L.Y.; Ruijgrok, P.V.; Das, R.; Alushin, G.M.; Bryant, Z. Controllable Molecular Motors Engineered from Myosin and RNA. Nat. nanotechnol. 2018, 13, 34-40. (4) Jung, C.; Allen, P.B.; Ellington, A.D. A Simple, Cleated DNA Walker That Hangs on to Surfaces. ACS Nano 2017, 11, 8047-8054. (5) Chakraborty, K.; Veetil, A. T.; Jaffrey, S. R.; Krishnan, Y. Nucleic Acid-Based Nanodevices in Biological Imaging. Annu. Rev. Biochem. 2016, 85, 349-373. (6) Yang, Y.; Zhong, S.; Wang, K.; Huang, J. Gold Nanoparticle Based Fluorescent Oligonucleotide Probes for Imaging and Therapy in Living Systems. Analyst 2019, 144, 1052-1072. (7) He, X.; Zeng, T.; Li, Z.; Wang, G.; Ma, N. Catalytic Molecular Imaging of MicroRNA in Living Cells by DNA Programmed Nanoparticle Disassembly. Angew. Chem. Int. Ed. 2016, 55, 3073-3076. (8) Yue, R.; Li, Z.; Wang, G.; Li, J.; Ma, N. Logic Sensing of MicroRNA in Living Cells Using DNA-Programmed Nanoparticle Network with High Signal Gain. ACS Sens. 2019, 4, 250-256. (9)Wang, D.; Vietz, C.; Schröder, T.; Acuna, G.P.; Lalkens, B.; Tinnefeld, P. A DNA Walker as Fluorescence Signal Amplifier. Nano Lett. 2017, 17, 5368-5374. (10) Khara, D.C.; Schreck, J.S.; Tomov, T.E.; Berger, Y.; Ouldridge, T.E.; Doye, J.; Nir, E. DNA Bipedal Motor Walking Dynamics: An Experimental and Theoretical Study of the Dependency on Step Size. Nucleic Acids Res. 2017, 46, 1553-1561. (11) Hong, F.; Zhang, F.; Liu, Y.; Yan, H. DNA Origami: Scaffolds for Creating Higher Order Structures. Chem. Rev. 2017, 117, 12584-12640. (12) He, X.; Sha, R.; Zhuo, R.; Mi, Y.; Chaikin, P.M.; Seeman, N.C. Exponential Growth and Selection in Self-Replicating Materials from DNA Origami Rafts. Nat. Mater. 2017, 16, 993997. (13) Abendroth, J.M.; Bushuyev, O.S.; Weiss, P.S.; Barrett, C.J. Controlling Motion at the Nanoscale: Rise of the Molecular Machines. ACS Nano 2015, 9, 7746-7768. (14) Ko, S.H.; Chen, Y.; Shu, D.; Guo, P.; Mao, C. Reversible Switching of pRNA Activity on the DNA Packaging Motor of Bacteriophage Phi29. J. Am. Chem. Soc. 2008, 130, 17684-17687. (15) Hu, L.; Lu, C.-H.; Willner, I. Switchable Catalytic DNA Catenanes. Nano lett. 2015, 15, 2099-2103. (16) Li, H.; Wang, M.; Shi, T.; Yang, S.; Zhang, J.; Wang, H.H.;

modification of all building blocks. Such all-in-one design circumvents asynchronous internalization as well as inefficient freely-diffusion/post-assembly procedures in various solutions including diffusion-limited ones. The integrated reference module can indicate alternative enzyme catalysis ability of distinct cells. And several types of DNA nanodevices have been successfully operated in different cell samples. Our flexible design can be extended to detect other cytoplasmic molecules and monitor related pathways by simply change the sequence design. In addition, these nanodevices may also be used to construct computing systems with external inputs and release molecular cargo as outputs, etc. All-in-one synchronized DNA nanodevices with spatially-localized modules may provide a new path towards sensing, monitoring and delivering molecules in cytoplasmic environments.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

Author Contributions ‡J.X. and F.C. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was financially supported by the National Science Foundation of China (No.21475102, No.21705124, No.21874105 and No.31671013), the China Postdoctoral Science Foundation (No.2017M613102 and No.2018T111032), the Fundamental Research Funds for the Central Universities and “Young Talent Support Plan” of Xi’an Jiaotong University. Funding for open access charge: the National Science Foundation of China (No.21874105).

ASSOCIATED CONTENT Supporting Information

Nie, Z. A DNA‐Mediated Chemically Induced Dimerization (D‐ CID) Nanodevice for Nongenetic Receptor Engineering To Control Cell Behavior. Angew. Chem. 2018, 130, 10383-10387. (17) Etoc, F.; Balloul, E.; Vicario, C.; Normanno, D.; Liße, D.; Sittner, A.; Piehler, J.; Dahan, M.; Coppey, M. Non-Specific Interactions Govern Cytosolic Diffusion of Nanosized Objects in Mammalian Cells. Nat. Mater. 2018, 17, 740-746. (18) Delarue, M.; Brittingham, G.; Pfeffer, S.; Surovtsev, I.; Pinglay, S.; Kennedy, K.; Schaffer, M.; Gutierrez, J.; Sang, D.; Poterewicz, G. mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding. Cell 2018, 174, 338-349. (19) Wu, Z.; Fan, H.; Satyavolu, N.S.R.; Wang, W.; Lake, R.; Jiang, J.H.; Lu, Y. Imaging Endogenous Metal Ions in Living Cells Using a DNAzyme–Catalytic Hairpin Assembly Probe. Angew. Chem. 2017, 129, 8847-8851. (20) Novina, C.D.; Sharp, P.A. The RNAi Revolution. Nature 2004, 430, 161-164. (21) Ma, W.; Fu, P.; Sun, M.; Xu, L.; Kuang, H.; Xu, C. Dual Quantification of MicroRNAs and Telomerase in Living Cells. J. Am. Chem. Soc. 2017, 139, 11752-11759. (22) Li, W.; Xiaogang, Q. Cancer Biomarker Detection: Recent

The Supporting Information is available free of charge on the ACS Publications website. (Table S1) Sequences of oligonucleotides; (Figure S1) Optimizing the length of block strand; (Figure S2) Comparison of two DNA nanodevices in different viscous solutions; (Figure S3) Time-lapse singleparticle STED images; (Figure S4) Influence of onparticle DW density; (Figure S5) Specificity of all-inone DNA nanodevices triggered by miR-21; (Figure S6S10) Intracellular performances of corresponding nanodevices(PDF)

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