Highly Integrated, Biostable, and Self-Powered DNA Motor Enabling

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Highly integrated, biostable and self-powered DNA motor enabling autonomous operation in living bodies Jing Wang, Dong-Xia Wang, An-Na Tang, and De-Ming Kong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00007 • Publication Date (Web): 18 Mar 2019 Downloaded from http://pubs.acs.org on March 18, 2019

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Analytical Chemistry

Highly Integrated, Biostable and Self-Powered DNA Motor Enabling Autonomous Operation in Living Bodies Jing Wang,1 Dong-Xia Wang,1 An-Na Tang,1 De-Ming Kong1,2* 1

State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular

Recognition, Research Centre for Analytical Sciences, College of Chemistry, Nankai University, Tianjin, 300071, P R China 2

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, 300071, P R China.

ABSTRACT: An ultimate goal of synthetic DNA motor studies is to mimic natural protein motors in biological systems. Here, we rationally designed a highly integrated and biostable DNA motor system with high potential for living body operation, through simple assembly of a Mn2+-dependent DNAzyme-powered DNA motor with a degradable MnO2 nanosheet. The motor system shows outstanding high integration and improved biostability. High integration confers the motor system with the ability to deliver all the core components to the target sites as a whole, thus enabling precise control of the spatiotemporal distribution of these components and achieving high local concentrations. At the target sites, reduction of the MnO2 nanosheet by intracellular glutathione (GSH) not only releases the DNA motor, which can then be initiated by the intracellular target, but also produces Mn2+ in situ to power the autonomous and progressive operation of the DNA motor. Interestingly, the resultant consumption of GSH in turn protects the DNA motor from destruction by physiological GSH, thus conferring our motor system with improved biostability, reduced false-positive outputs and consequently an increased potential to be applied in a living body. As a proof of concept, the highly integrated DNA motor system was demonstrated to work well for amplified imaging detection of survivin messenger RNA (mRNA), an important tumor biomarker, in both living cancer cells and living tumor-bearing mice. This work reveals concepts and strategies promoting synthetic DNA motor applications in biological systems.

Highly complicated and hierarchical molecular motors are ubiquitous in living systems. Many important biological processes, including cellular cargo transport, synthesis of peptides and proteins, and transfer of genetic information, rely on the cooperation of multiple molecular motors with different functions.1 Inspired by these biological phenomena, researchers have attempted to fabricate various artificial molecular motors that can be precisely controlled and exploited at both the molecular and macroscopic levels.2 Owing to its remarkable versatility and robustness, as well as welldefined Watson−Crick base pairing rules, DNA is a promising building block for artificial molecular motors.3 DNA motor devices, including tweezers,4 gears,5 cranes,6 robots,7-9 springs10 and walkers,3,11-18 are increasingly being reported. These DNA motors conduct designated mechanical movements along pre-designed one-dimensional (1D), two-dimensional (2D) or threedimensional (3D) tracks.17,19-24 To achieve the ultimate goal of mimicking natural functional proteins, artificial molecular motors should autonomously perform assigned tasks in living cells and living bodies. To date, most reported DNA motors have focused on in vitro principle verification. In the past two years, a breakthrough was realized in achieving the

operation of DNA motors in living cells;25-29 however, application in living bodies has not yet been reported. The DNA motors that fulfill living body applications should have the characteristics of suitable driving force, high integration and long-term biostability. To date, the reported DNA motors have commonly used protein enzymes, DNA strands, pH variation, metal ions and light to power autonomous motor operation in an effective manner.25,30-33 Because protein enzymes and DNA strands must often be externally supplemented, they are not highly suitable for applications in living cells and living bodies.34-36 By using a Mn2+-dependent DNAzyme as the driving force, the Le group constructed the first self-powered DNA motor and accomplished its operation in living cells.25 Owing to the need for external addition of the cofactor Mn2+, however, this DNA motor is still not desirable for application in living bodies. At nearly the same time, the Wang group also successfully achieved the use of DNA motors in living cells by using a Mg2+-dependent DNAzyme as the driving force.26,28,29 They have demonstrated that cellular endogenous Mg2+ can power the operation of a DNA motor. However, because the endogenous Mg2+ concentration is lower than the optimal concentration required by the DNA motor, the kinetics is relatively slow if no exogenous

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Mg2+ is supplemented. Collectively, the above DNA motors share the same drawback of insufficient integration. Exogenous supplementation with some core components not only increases the complexity of motor systems but also makes controlling the spatiotemporal distribution of core components in target sites difficult, thus greatly limiting potential applications in living bodies. The Ye group has constructed an integrated DNA motor powered by endogenous ATP molecules and has autonomously performed a bioanalytical task of specific microRNA imaging inside living cells without any auxiliary additives.27 Nevertheless, complicated motor design and elaborate DNA sequence screening are needed to achieve satisfactory motor operation. High biostability is also a basic requirement for DNA motors working in living bodies. The above-mentioned intramolecular operating DNA motors are all constructed on the basis of DNA-functionalized gold nanoparticles (AuNPs) via the formation of Au-S bonds. Thus, the presence of biothiols, especially glutathione (GSH), in physiological environments may be one of the most challenging obstacles to their application in living bodies, because these DNA-functionalized AuNPs might undergo competitive ligand exchange with biothiols and thus lead to false-positive outputs.37,38 Therefore, preparation of DNA motors with long-term biostability and improved anti-interference ability is urgently required. In this work, we fabricated a highly integrated DNA motor system via simple assembly of a DNAzymefunctionalized AuNP-based DNA motor and MnO2 nanosheet. When the integrated motor system was delivered to the target sites as a whole, its motor operation was initiated by a specific biological target. Mn2+, a DNAzyme cofactor, can be produced in situ to power the autonomous motor operation via MnO2 reduction by intramolecular GSH.39 Consumption of GSH in turn weakens the interference from GSH, thereby conferring the DNA motor with improved biostability. The proposed highly integrated, self-powered DNA motor was demonstrated to work well in survivin messenger RNA (mRNA) imaging in living cells and living mice with tumors. RESULTS AND DISCUSSION Design of a highly integrated DNA motor The highly integrated DNA motor was constructed according to the procedures shown in Scheme 1 and Scheme S1. AuNPs, which can enter cells and possess distance-dependent optical features, acted as a cellular transporter and fluorescence quencher. The substrate strand and enzyme strand (Table S1) of a Mn2+dependent RNA-cleaving DNAzyme were conjugated on the AuNP surface via the formation of Au-S bonds. The substrate strand was fluorescently labelled with carboxyfluorescein (FAM), whose fluorescence was quenched by AuNPs. An oligonucleotide termed the locking strand was used to partly hybridize with the enzyme strand to prevent the formation of the substrate/enzyme hybrid, thus silencing the DNAzyme. Then, a highly integrated DNA motor, termed the DNA motor/MnO2, was easily prepared through simple assembly of the obtained silenced DNAzyme-

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functionalized AuNPs (DNA motor) with a MnO2 nanosheet via the strong physisorption between ssDNA nucleobases and the MnO2 nanosheet.40 After entering cells as a whole, the MnO2 nanosheet is reduced by intracellular GSH,41 thus releasing the adsorbed DNA motors and providing high local concentrations of Mn2+. In the presence of an intracellular target (e.g. mRNA), the locking strand is disassociated from the enzyme strand via a toehold-mediated strand displacement reaction. The released enzyme strand then hybridizes to the adjacent substrate strand and forms an active enzyme/substrate hybrid. In the presence of the cofactor Mn2+ provided by MnO2 reduction, the DNA–RNA chimeric substrate strand is cleaved into two parts, thus resulting in the release of the FAM-labelled segment and the recovery of fluorescence. The residual part of the substrate strand is insufficient to form a stable hybrid with the enzyme strand. The enzyme strand is released and subsequently hybridized with the next substrate strand, thereby triggering hybridization-cleavage-release cycles. Through monitoring of the fluorescence change of FAM, the operation process of the DNA motor can be monitored in real-time. Because all core components (except the target mRNA) are highly integrated in a nanosystem, they can be delivered to the target sites as a whole, thus making the precise control of spatiotemporal distribution of these components possible. Because the cofactor Mn2+ is produced in situ at target sites, the DNA motor can be self-powered to yield autonomous and progressive operation after initiation by the target mRNA without a need for any other components to be supplied. This design not only substantially simplifies the triggering and driving operations of the DNA motor but also makes the use of DNA motors in a living body possible.

Scheme 1. Schematic illustration of (a) preparation of highly integrated DNA motor/MnO2 nanoassembly and (B) its operation in living cells and living bodies.


Successful preparation of the DNA motor/MnO2 assembly was further verified through UV-Vis absorption spectroscopy, dynamic light scattering (DLS) and zeta potential analysis (Figure 2a). AuNPs and MnO2 nanosheets gave absorption peaks at 525 and 340 nm, respectively. The emergence of the characteristic DNA absorption peak at 260 nm in the DNA motor absorption spectrum confirmed that the DNA strands were successfully loaded on the AuNP surface, and the loading intensity was calculated to be ~80 substrate strands per AuNP. The coexistence of 340 and 525 nm absorption peaks in the nanoassembly absorption spectrum demonstrated the successful assembly of the DNA motor and MnO2 nanosheet. DLS analysis showed that the DNA motor/MnO2 assembly had an average hydrodynamic diameter of ∼200 nm, which was larger than those of the DNA motor (∼32 nm) and MnO2 nanosheet (~135 nm) (Figure 2b). Zeta potential analysis showed a large decrease in the zeta potential of the DNA motor/MnO2 assembly compared with the DNA motor and MnO2 nanosheet (Figure 2c), thus revealing remarkable alterations in the surface charges as a result of the assembly process. Collectively, all the above characterizations demonstrated the successful formation of an integrated DNA motor/MnO2 assembly. The prepared DNA motor/MnO2 assembly was stable in aqueous solutions. No visible precipitate was observed (Figure 2d), and its hydrodynamic size showed almost no change after incubation for 72 h in 25 mM Tris-HAc buffer (pH 7.4) containing 200 mM NaCl.

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Synthesis and characterization of the DNA motor/MnO2 nanoassembly A highly integrated DNA motor/MnO2 nanoassembly was prepared through two steps (Figure 1a): separate preparation of the DNA motor and MnO2 nanosheet, and their subsequent assembly. The DNA motor was obtained by functionalization of 13 nm AuNP by a thiolterminated substrate strand and enzyme strand (which was previously silenced by a locking strand) via Au-S bond formation. The MnO2 nanosheet was prepared at high yield through a rapid and facile chemical oxidation route of Mn2+ ions in the presence of tetramethylammonium cations in an aqueous solution.42 Then, via Van der Waals forces between DNA nucleobases and the MnO2 nanosheet,43 the DNA motor was easily adsorbed on the basal plane of the MnO2 nanosheet to form an integrated DNA motor/MnO2 assembly. The spherical shape of the DNAzymefunctionalized AuNP, the ultrathin structure of the MnO2 nanosheet and the corresponding integrated DNA motor/MnO2 assembly could be clearly seen through transmission electron microscopy (TEM) (Figure 1b, c, d), thus demonstrating the successful preparation of the highly integrated DNA motor/MnO2 nanoassembly step by step. High-angle annular dark-field scanning TEM (HAADF-STEM)-based elemental mapping (Figure 1e) and energy dispersive X-ray spectroscopy (EDS) characterization (Figure S1) confirmed that all representative elements (e.g., Au, Mn, N, O, C) were included in the nanoassembly, thus further demonstrating the successful assembly of the DNA motor and MnO2 nanosheet.

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Figure 2. (a) UV–Vis absorption spectral analysis, (b) DLS and (c) zeta potential characterization of DNA motor, MnO2 nanosheet, and DNA motor/MnO2 nanoassembly. (d) DLS analysis of the nanoassembly after storage for different time at room temperature. The insert shows the digital photos of corresponding aqueous solutions at 72 h. Optimization of DNA motor construction The proposed DNA motor was constructed on the basis of a Mn2+-dependent DNAzyme. According to the proposed working mechanism shown in Scheme 1, the prepared DNA motor should remain silent in the absence of target mRNA but be rapidly triggered by target mRNA. Thus, the length of the locking strand was carefully selected. Three locking strands with the same toehold



DNA motor/MnO2 assembly preparation and the feasibility of DNA motor/MnO2 assembly applications in living cells and living bodies, given that uptake of 500 μM Mn2+ has been reported not to affect the viability of cells.44 After being silenced by locking strand, the DNA motor showed both Mn2+ and target mRNA dependence (Figure 3b). Neither Mn2+ nor target mRNA was able to initiate the DNA motor alone. After addition of both Mn2+ and target mRNA, however, the DNA motor was activated to cleave the substrate strand, thereby initiating autonomous operation of the motor, which was accompanied by a rapid increase in FAM fluorescence. In addition, the dependence on Mn2+ was highly specific. None of the various relevant metal ions, including Co2+, Cu2+, Ba2+, Pb2+, Fe2+, Ni2+, Mg2+ and Ca2+, could substitute for Mn2+ and yield an obvious fluorescence signal enhancement (Figure S5).

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sequence (8 nucleotides) and different lengths of enzyme strand-binding sequence (21, 22, and 23 nucleotides for Lock-21, Lock-22 and Lock-23, respectively) were compared. In these three locking strands, Lock-22 gave the best performance (Figure S2a, 2b), not only completely silencing the DNA motor, as reflected by the negligible changes of FAM fluorescence in the absence of target mRNA, but also being rapidly released by target mRNA to trigger the operation of DNA motor, thus resulting in a significant increase in FAM fluorescence. Polyacrylamide gel electrophoresis (PAGE) analysis (Figure S3) also demonstrated that Lock-22 hybridized with the enzyme strand to form a stable duplex and then was released from the enzyme strand by forming a more stable duplex with target mRNA. After being initiated by target mRNA, the DNA motor was able to automatically and rapidly operate. Correspondingly, the fluorescence signal increased and reached a plateau in 30 min. This rapid operation rate was due to the curvature design of DNA motor tracks and the local high concentration of DNA strands. Then, the molar ratio of enzyme strand to substrate strand on the AuNP surface was optimized. As shown in Figure S2c, when the molar ratio increased from 25:500 to 500:500, the signal-to-noise ratio F/F0 (where F and F0 are the fluorescence signal with and without target mRNA, respectively) increased first and then declined. The largest F/F0 was observed at the ratio of 100:500. Therefore, this optimal molar ratio was used in the preparation of the DNA motor. Dependence of the DNA motor on Mn2+ The cofactor Mn2+ plays an important role in ensuring the rapid and progressive operation of the DNA motor. The dependence of the DNA motor on Mn2+ was investigated. First, the effects of Mn2+ concentration on the performance of free DNAzyme, which was not immobilized on the AuNPs, were tested. PAGE analysis showed that the cleavage of substrate strands was highly Mn2+ concentration-dependent (Figure S4). After incubation at room temperature for 60 min, more than 90% of the substrate strands were cleaved when the Mn2+ concentration was equal or greater than 100 μM. When the Mn2+ concentration was increased to 500 μM, the substrate strands were nearly completely cleaved. These results suggested that the DNAzyme can be well activated in a broad of Mn2+ concentration range. After immobilization of the substrate and enzyme strands (without a locking strand) on the AuNP surface, the obtained unlocked DNA motor was also highly Mn2+ concentration-dependent (Figure 3a). Without Mn2+, the DNA motor could not operate even it was not silenced by the locking strand. After the addition of Mn2+, the DNA motor was activated, as reflected by the time-dependent fluorescence increase, and the fluorescence change was Mn2+ concentration-dependent. A rapid fluorescence increase was observed when more than 300 μM Mn2+ was added, and the fluorescence change in the presence of 500 μM Mn2+ was comparable to that in that presence of 1000 μM Mn2+. These results indicated that the DNA motor could also work in a broad Mn2+ concentration range. Such a broad Mn2+ concentration dependence increases both the flexibility of subsequent

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Figure 3. Mn2+ dependence of (a) unsilenced DNA motor, (b) silenced DNA motor and (c) DNA motor/MnO2 assembly. (a1) Working mechanism of unsilenced DNA motor. (a2) Fluorescence responses of the unsilenced DNA motor to different concentrations of Mn2+. (b1) Working mechanism of silenced DNA motor. (b2) Dependence of the silenced DNA motor operation on Mn2+ and target mRNA. (c1) Working mechanism of DNA motor/MnO2 assembly. (c2) UV-Vis absorption spectra of MnO2 nanosheet (30 μg/mL) with or without 1 mM GSH. The insert shows the absorbance at 340 nm of different concentrations of MnO2 nanosheet in the presence of 1 mM GSH. (c3) Real-time fluorescence monitoring of DNA motor/MnO2 nanoassembly in the presence of different concentrations of GSH. [target mRNA] = 2.5 μM. According to the proposed working mechanism, no exogenous Mn2+ is needed by the DNA motor/MnO2 assembly, and the cofactor Mn2+ is provided by the


reduction of the MnO2 nanosheet by intracellular GSH. As shown in Figure 3c, after the addition of GSH, the characteristic absorption band of MnO2 nearly disappeared, thus demonstrating the destruction of the nanosheet by GSH. This destruction reaction was very fast and could be completed in 3 min. A 30 µg/mL MnO2 nanosheet was completely destroyed by 1 mM GSH. Because the intramolecular GSH concentration (1–10 mM) was higher than 1 mM,41,45 as long as the MnO2 nanosheet concentration was lower than 30 µg/mL, complete destruction of MnO2 nanosheet and thus efficient release of the DNA motor from the assembly was ensured. To demonstrate that MnO2 nanosheet destruction could produce enough Mn2+ to fulfill the requirement of the DNA motor, we monitored the fluorescence response of 1 nM DNA motor/MnO2 assembly (AuNP concentration, corresponding to 30 µg/mL MnO2) to 2.5 µM target mRNA in the presence of different concentrations of GSH in real-time. As shown in Figure 3c, without GSH, almost no fluorescence signal change was observed, thus indicating that no Mn2+ was produced, and the DNA motor/MnO2 assembly remained intact. With an increase in GSH concentration, a rapid fluorescence increase resulted from the DNA motor/MnO2 assembly. The effect of GSH nearly reached saturation when the concentration was above 250 µM. These results suggested that the intramolecular GSH concentration is sufficient to produce adequate Mn2+ to support the rapid operation of the DNA motor. In vitro response of the DNA motor/MnO2 nanoassembly to target mRNA Having demonstrated the proposed working mechanism, we investigated the in vitro response of the DNA motor/MnO2 assembly to target mRNA by recording corresponding fluorescence~time curves. As a proof of concept, synthetic survivin mRNA fragment (termed survivin mRNA, Table S1) was used as a target mRNA model. Survivin mRNA is a tumor biomarker. It is significantly associated with cell division and is overexpressed in most cancer cells.46-48 The results in Figure 4a showed that the assembly had a rapid response to survivin mRNA in the presence of 1 mM GSH. With the addition of survivin mRNA, the fluorescence signal increased rapidly and then levelled off to a saturation value in a short time of 30 min. In addition, both the signal rate of increase and the signal intensity were survivin mRNA concentration-dependent. By recording the fluorescence signal at 30 min, we observed a linear relationship between fluorescence intensity and survivin mRNA concentration in the range of 2.5 to 20 nM (Figure 4b). These results suggested that the proposed DNA motor/MnO2 assembly can be used for target mRNA quantification. The detection limit was calculated to be 1.2 nM on the basis of the rule of 3σ method. This detection limit was comparable to the above-mentioned exogenous Mn2+-assisted DNA motor and obviously lower than those of the survivin mRNAsensing without signal amplification steps (Figure S6), thus suggesting that the DNA motor/MnO2 assembly can work with a similar efficiency to free DNA motor, but eliminating the need for additional Mn2+. By comparing the slopes of the standard curves given by the DNA motor and the sensing system without amplification

steps, it can be calculated that each target mRNAbinding event can result the cleavage of ~147 FAMlabelled substrate strands in our motor system. In addition, our DNA motor/MnO2 assembly was able to easily differentiate the perfectly matched target mRNA from mismatched variants (Figure S7). The introduction of only a single-base mismatch greatly decreased the initiating efficiency toward the DNA motor, and nontargeted TK1 mRNA could not initiate the DNA motor at all.

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Figure 4. In vitro response of the DNA motor/MnO2 assembly to target mRNA. (a) Time-dependent fluorescence changes of 1 nM assembly upon addition of different concentrations of survivin mRNA. [GSH] = 1 mM. (b) Survivin mRNA concentration-dependent fluorescence change at 30 min. The inset shows the linear relationship between the fluorescence intensity and survivin mRNA concentration in the range of 0~20 nM. Stability and cytotoxicity of the DNA motor/MnO2 assembly To achieve applications in living cells and living bodies, the prepared DNA motor/MnO2 assembly should have high biostability and low cytotoxicity. Nucleic acids are susceptive to digestion by nucleases that are ubiquitous in physiological environments. The stability of nucleic acids greatly increases after being assembled on the surface of nanomaterials (e.g., AuNPs, graphene oxide and so on), because the increased steric hindrance prevents the access of nucleases.49-51 In the prepared DNA motor/MnO2 nanoassembly, nucleic acid strands are simultaneously assembled on the surface of AuNP and the MnO2 nanosheet and thus might have enhanced biostability. As expected, after incubation with 5 U/mL deoxyribonuclease I (a concentration much higher than those in living cells) or cell culture medium for 1 h, almost no obvious fluorescence change was observed (Figure S8), thereby indicating that the prepared DNA motor/MnO2 nanoassembly was highly resistant to enzymatic degradation and highly stable in biological fluids. Our highly integrated DNA motor/MnO2 nanoassembly utilized GSH to reduce MnO2 to yield a Mn2+. In fact, intramolecular GSH, especially when overexpressed GSH in tumor cells, is usually the most challenging obstacle to the application of DNA-functionalized Au nanoparticles in living cells and living bodies, because GSH can destroy Au-S bonds,37,38 thus yielding falsepositive results. The biostabilities of the DNA motor and DNA motor/MnO2 assembly in the presence of GSH were compared by following their time-dependent fluorescence changes (Figure 5a). The fluorescence intensity scarcely changed even after incubation of the DNA motor/MnO2 assembly with 1mM GSH for 12 h.


Analytical Chemistry Even when the GSH concentration was increased to 10 mM, only a little background fluoresce increase could be observed for the assembly after incubation for 6 h (Figure S9). In contrast, a distinctly enhanced fluorescence signal was observed for the free DNA motor (8.2-fold at 12 h) in the presence of 1mM GSH, owing to the breaking of Au-S bonds by GSH. Of note, the DNA motor/MnO2 assembly showed a much higher level of resistance to GSH than the simple DNA motor. The reason for this result was attributed to the redox reaction between MnO2 and GSH. This reaction converts active GSH to inactive glutathione disulfide (GSSG), thereby resulting in a greatly decreased local GSH concentration around the released DNA motor. Because the GSH concentrations (about 2–20 μM) in the circulation and in extracellular fluids are approximately 100- to 1000-fold lower than those in cells,52,53 the prepared DNA motor/MnO2 assembly should remain intact before entering cells. Herein, degradable MnO2 nanosheets were selected to construct a highly integrated DNA motor. One reason for this selection was that manganese is a necessary nontoxic element involved in physiological metabolism.54 As expected, more than 80% of the cells were alive when as much as 40 μg/mL MnO2 nanosheet was incubated with cells for 24 h (Figure S10). After assembly with the DNA motor, the obtained DNA motor/MnO2 assembly also exhibited good biocompatibility (Figure 5b), thus revealing that it could be used in living cells.

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liberation of the Cy5-labelled locking strand from the DNA motor, thus recovering the Cy5 fluorescence. At the same time, the released enzyme strand hybridized with adjacent substrate strands to initiate the intramolecular operation of the DNA motor, thus resulting in the recovery of FAM fluorescence. That is, both Cy5 and FAM fluorescence responded to survivin mRNA. By carefully comparing the fluorescence signals emitted by these two fluorophores (Figure S11), we found that (i) the FAM fluorescence was much stronger than that of Cy5, thus demonstrating the signal amplification due to autonomous and progressive operation of the DNA motor; (ii) the fluorescence signal changes of FAM and Cy5 were nearly synchronous, a result suggesting that the hybridization between survivin mRNA and the locking strand might be the rate-determining step. After the locking strand left from the DNA motor, the released enzyme strand “walked” rapidly along the tracks on the AuNP surface, in agreement with the results mentioned above; (iii) the fluorescence images of FAM perfectly overlapped with those of Cy5, indicating that the DNA motor/MnO2 assembly might be used for in situ amplified imaging of the intracellular survivin mRNA target. The integrated DNA motor/MnO2 nanoassembly also showed high specificity in living cells, as demonstrated by the negative performance of the mutant DNA motor/MnO2 assembly (Figure 6). In this mutant assembly, three-base mismatches were introduced in locking strand/survivin mRNA duplex. The survivin mRNA was not able to release the locking strand from the enzyme strand and thus could not trigger the DNA motor. As expected, after incubation of HeLa cells with mutant assembly for 4 h, neither Cy5 nor FAM fluorescence was observed.

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Figure 5. Stability and cytotoxicity of the DNA motor/MnO2 assembly. (a) Background fluorescence changes of DNA motor and DNA motor/MnO2 assembly in the presence of 1 mM GSH. (b) Cell viability of HeLa cells after treatment with 1 nM DNA motor, 30 μg/mL MnO2 nanosheet or 1 nM DNA motor/MnO2 assembly for different time. Imaging detection of survivin mRNA in living cells Having demonstrated that the integrated DNA motor/MnO2 nanoassembly could be used for in vitro quantitation of synthetic survivin mRNA fragments, we next sought to demonstrate that the assembly could also be used for visualizing intracellular natural long-stranded survivin mRNA. Herein, a human cervical cancer cell line (HeLa) was selected as the model cancer cell, in which survivin mRNA is over-expressed. To obtain comprehensive information about the assembly operation in living cells, we labelled the locking strand and substrate strand with the fluorophores Cy5 and FAM, respectively. Their fluorescence signals were both quenched by AuNPs. After entering living cells, the MnO2 nanosheets were destroyed rapidly and released the DNA motor, survivin mRNA and then triggered the

Figure 6. Confocal fluorescence images of living HeLa cells incubated with 1 nM DNA motor/MnO2 nanoassembly or mutant DNA motor/MnO2 nanoassembly for 4 h. The scale bar indicates 10 μm. We sought to further confirm the ability of our integrated DNA motor/MnO2 assembly to be used for the analysis of target mRNA in living cells. The intracellular survivin mRNA expression level was down-regulated by the imidazolium-based small-molecule compound YM155,55 a potent repressor of survivin mRNA expression. After treatment with increasing concentrations of YM155, significantly decreased fluorescence was observed in HeLa cells (Figure S12), thereby demonstrating the successful inhibition of survivin mRNA expression by YM155 and providing robust evidence of the practicality of our integrated DNA


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motor/MnO2 assembly for target mRNA imaging in living cells. Improved intracellular biostability In living cells, the cofactor Mn2+ was provided by the redox reaction between the MnO2 nanosheet and GSH. To investigate whether sufficient Mn2+ could be provided in living cells, we used inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis to determine the intramolecular concentration of the Mn element. After incubation with 1 nM DNA motor/MnO2 assembly for 4 h, the intramolecular Mn concentration was estimated to be around 300 μM, whereas the intramolecular GSH concentration is much higher than this concentration. Therefore, most of the Mn element may be present as Mn2+ ions. Because Mn2+ is produced in situ, the local Mn2+ concentration around DNA motor might be higher than 300 μM, a level sufficient to support rapid and autonomous operation of DNA motor in living cells. As mentioned above, the reaction between MnO2 and GSH not only provides the cofactor Mn2+ but also decreases the interference from GSH. To further verify the increased anti-interference ability of the integrated DNA motor/MnO2 assembly, we functionalized AuNPs by substrate strands only, then further assembled them with MnO2 nanosheets. The obtained substrate-functionalized AuNP/MnO2 assembly was unable to function as a DNA motor because of the lack of the enzyme strand. However, if the FAM-labelled substrate strands were separated from AuNPs because of the breaking of Au-S bonds by GSH, nonspecific background fluorescence might be observed. After incubation with HeLa cells for identical times, especially for 12 h, the substratefunctionalized AuNP/MnO2 assembly yielded obviously weaker fluorescence images than did simple substratefunctionalized AuNPs (Figure 7), thus suggesting that assembly with MnO2 nanosheets might also confer the DNA motor with enhanced biostability and improved resistance against GSH in living cells. Substrate-functionalized AuNP/MnO 2

Substrate-functionalized AuNP

------------------------------------------------4h 12 h 4h 12 h

Applications of DNA motors in living bodies have not previously been reported. Our highly integrated DNA motor/MnO2 assembly was able to control the spatiotemporal distribution of the core components of DNA motors in target sites through synchronous delivery of these components as a whole, thus making applications in living bodies possible. To verify this application, a near infrared fluorescence dye Cy5labelled substrate strand was used instead of a FAMlabelled one, and BALB/c nude mice bearing HeLa xenograft tumors were used as the living body model. The mice were divided into three groups and treated with DNA motor/MnO2 assembly, mutant DNA motor/MnO2 assembly or simple DNA motor. As shown in Figure 8, the mice treated with the DNA motor/MnO2 assembly showed a continuous and marked enhancement of fluorescence signals in the tumor region, thus indicating that the DNA motor was successfully activated by survivin mRNA over-expressed in tumor cells. In contrast, much weaker tumor images were observed for both mutant DNA motor/MnO2 assembly and simple DNA motor-treated mice, thus confirming the response specificity of the DNA motor and the importance of the cofactor Mn2+ for DNA motor. Of note, the simple DNA motor gave a slight stronger fluorescence signal than the mutant DNA motor/MnO2 assembly, a result consistent with the enhanced biostability of the DNA motor/MnO2 assembly. That is, the destruction of the simple DNA motor by GSH resulted in a nonspecific signal, but the protection of the DNA motor by MnO2 decreased the background fluorescence. 10 min 60 min 90 min 120 min

1000.0

3022.1

DNA motor/MnO 2 10 min 120 min 10 min 120 min

FAM

5044.2

7066.3

Bright field

Mutant DNA motor/MnO 2

Merge

Figure 7. Confocal fluorescence images of HeLa cells incubated with 1 nM substrate-functionalized AuNP/MnO2 assembly or 1 nM substrate-functionalized AuNP for different time. Application in the living body

9088.4 DNA motor

Figure 8. Fluorescence images of HeLa tumor-bearing mice after uptake of different DNA motor systems. The arrows indicate the tumor regions. To further demonstrate the response specificity of the DNA motor/MnO2 assembly, we simultaneously administered identical amounts of DNA motor/MnO2 assembly to the tumor and adjacent hypodermis regions of the same mouse (Figure S13). A strong fluorescence image was observed in the tumor region within 120 min after injection of the assembly, whereas only a very weak fluorescence signal was observed in the hypodermis region. These results are consistent with the



over-expression of survivin mRNA in tumor cells but not normal cells, thus suggesting that the integrated DNA motor/MnO2 assembly can be applied for amplified tumor imaging in living bodies. Conclusions In summary, applications of DNA motor in living bodies were realized by construction of the first reported highly integrated DNA motor/MnO2 nanoassembly. The nanoassembly was conveniently prepared via a simple one-step assembly of a cofactor Mn2+-dependent DNAzyme-powered DNA motor and a biodegradable MnO2 nanosheet. Highly integrated characteristics allowed the assembly to deliver the core components of the DNA motor as a whole, and thus increased the control of the spatiotemporal distribution of these components in target sites. After being taken up by cells as a whole, the reduction of the MnO2 nanosheet by intracellular GSH not only led to a release of the DNA motor and the production of cofactor Mn2+, thus enabling self-powered and autonomous operation of target mRNA-activated DNA motor without supplying any other components, but also increased the resistance of the DNA motor to GSH interference, thus greatly decreasing the possibility of false positive results. This assembly was demonstrated to work well in specific and amplified imaging of target mRNA in cancer cell and tumor-bearing nude mouse models. This work reveals new concepts and strategies in promoting synthetic DNA motor applications in biological systems.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional information as noted in text: full experimental details, optimization of experimental conditions, the selectivity, and stability of this nanoassembly, and biological experiments.

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

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 21874075, 21728801), the National Natural Science Foundation of Tianjin (No. 16JCYBJC19900) and the Fundamental Research Funds for the Central Universities.

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Highly integrated, biostable and self-powered DNA motor enabling autonomous operation in living bodies 117x91mm (300 x 300 DPI)


Highly Integrated, Biostable, and Self-Powered DNA Motor Enabling

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