A dual-enzyme-assisted 3D DNA walking machine using T4

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A dual-enzyme-assisted 3D DNA walking machine using T4 polynucleotide kinase as activators and application in polynucleotide kinase assays Chang Feng, Zihan Wang, Tianshu Chen, Xiaoxia Chen, Dongsheng Mao, Jing Zhao, and Genxi Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04924 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on February 3, 2018

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Analytical Chemistry

A dual-enzyme-assisted 3D DNA walking machine using T4 polynucleotide kinase as activators and application in polynucleotide kinase assays Chang Feng,†, # Zihan Wang,‡, # Tianshu Chen, ‡ Xiaoxia Chen, ‡ Dongsheng Mao,‡ Jing Zhao,*,‡ and Genxi Li*,†,‡ †

State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China. Tel.: +86-25-83593596, Fax: +86-2583592510, E-mail: [email protected] ‡ Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China. Tel.: +86-21- 66138133, E-mail: [email protected] ABSTRACT: T4 polynucleotide kinase (T4 PNK) - an intracellular kinase catalyzes the phosphorylation of 5’-hydroxyl termini in nucleic acids plays a crucial role in DNA-related physiological activities. Malfunction of PNK is associated with the deregulation of many cellular activities and eventually induces a variety of human diseases. Herein, we report a smart three-dimensional (3D) DNA walking machine using PNK as an effective activator when coupled with duplex DNA nuclease-assisted cleavage reaction. The 3D DNA tracks benefit from high DNA loading capacity of gold nanoparticles, while high efficiency of duplex nucleases-mediated cyclic cleavage facilitates the movement of DNA machine in response to T4 PNK. The DNA machine is also applied for PNK assay based on the signal amplification from point to area during DNA walking process. The method achieves an excellent detection limit of 0.0067 U/mL with a linear range from 0.01 to 0.3 U/mL and a favorable specificity even in complex serum samples. Therefore, 3D DNA machine shows great potential in biochemical and molecular biology studies, drug discovery and clinic diagnostics.

In the past decades, nucleic acids have become the most popular molecules for the fabrication of diverse nanostructures. Benefiting from the precise Watson-Crick base pairing, the thermodynamics implicated in DNA hybridization effectively drive the construction of DNA machines. Subsequently, different DNA machines, such as DNA tweezers, motors, walkers, robots and switches, have been reported based on programmable and accurate DNA self-assembly, using nucleic acids as building blocks.1-8 These DNA machines can be activated by external input, and are usually accompanied by conformational changes of the functional DNA upon molecular interactions. In 2004, Seeman et al. firstly reported a precisely controlled molecular nanomachine using biped walking along a well-defined track.9 Later, Pierce et al. demonstrated a synthetic DNA walker to move along the linear DNA track using the external input of DNA stimuli, inspired by the intracellular movement of kinesin.10 Afterward, extensive studies of DNA walker have been conducted using different tracks. Seeman et al. designed a DNA walker that could move along a DNA origami-based track;11 Tan et al. reported a new light energypowered DNA walker with regulated autonomous movement along a specific single stranded DNA track;12 Reif et al. constructed a unidirectional DNA walker that moves along a selfassembled DNA track.13 While most of DNA walkers are only able to move along a one-dimensional (1-D) or a twodimensional (2-D) track, Elington et al. proposed a threedimensional (3-D) DNA machine using a colloidal nanoparticle as substrate, which opens a new way to better amplification performance as well as DNA enrichment capacity ascrib-

ing to large surface-to-volume ratio of nanoparticles.14 Meanwhile, different activators have been introduced to drive the movement of DNA machines besides DNA stimuli. Willner et al. fabricated a bipedal DNA walker using H+/OH- and Hg2+/cysteine as triggers;15 Mao et al. developed a new walking mechanism using both DNAzyme and strand displacement to drive the movement;16 Qu et al. designed a pH responsive DNA walker that can reversibly transport specific molecules under environmental stimuli.17 The use of non-nucleic acid activators to induce conformation changes of the functional DNA have the benefits of precise controllability and optimized freedom while preserving autonomous and progressive motion of DNA walkers. Phosphorylation of DNA with 5’- hydroxyl termini is an important regulatory process involved in a majority of normal cellular events, including DNA recombination, DNA replication, and DNA repair during strand interruption.18-21 Various exogenous and endogenous agents (e.g. ionizing radiation, chemicals, and nucleases) may induce the hydroxylation of 5’termini in nucleic acids, resulting in DNA damage and genomic instability.22-24 In this case, polynucleotide kinase (PNK)- a kinase catalyzes the transfer of the γ-phosphate group from adenosine triphosphate (ATP) to 5’- hydroxyl group of oligonucleotides or nucleic acids- is critical for repairing DNA strand breaks induced by endogenous or exogenous agents.25-29 Malfunction of PNK is also found to associate with the deregulation of many cellular activities and eventually leads to a variety of human diseases, such as loom’s syndrome, Werner syndrome, and Rothmund-Thomson syn-

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drome.30 Moreover, PNK is a promising therapeutic target in the radio therapy of somatic cancers because of the positive effect of PNK inhibitors.31, 32 To date, some methods have been reported for PNK assay for its considerable importance in the fundamental biochemical and molecular biology studies. However, traditional methods for evaluating PNK activities, such as polyacrylamide gel electrophoresis (PAGE), autoradiography and radical isotope 32P-labeling techniques, always suffer from intrinsic drawbacks of radioactive hazards, timeconsuming, complicated, inefficient and costly operations.33-38 Therefore, both the extensive application and effective determination are definitely worth exploring on PNK. In this paper, a dual nuclease-assisted 3D DNA walker is developed using T4 PNK as a trigger, which could also be successfully applied for detection of polynucleotide kinase activity. In this DNA machine, the integration of λ exonuclease and nicking enzyme facilitated the movement of swing DNA arm and thus released hundreds of fluorophore-labelled oligonucleotides in response to a PNK recognition event on the surface of gold nanoparticle (AuNP). Accordingly, the sensitive and specific detection of T4 PNK activity could achieve in term of the smart DNA walker, exhibiting the advantages of convenient manipulation, high efficiency and improved sensitivity.

EXPERIMENTAL SECTION Chemicals and Materials. All the oligonucleotides are synthesized and purified by Sangon Biotechnology Co., Ltd. (Shanghai, China), and their sequences are listed in Table S1. T4 polynucleotide kinase (10 units/µL), λ exonuclease (5 units/µL), Nb. BbvCI (10 units/µL) and phi 29 DNA polymerase were obtained from New England Biolabs Inc. Tris (hydroxymethyl) aminomethane (Tris), adenosine triphosphate (ATP), dithiothreitol (DTT), adenosine diphosphate (ADP), protein kinase A (PKA), ammonium sulfate (NH4)2SO4), Thrombin, bovine serum albumin (BSA), IgG, ovalbumin (OVA) and Transferritin were bought from Sigma-Aldrich. All chemicals were of analytical grade and used without further purification. All solutions were prepared with Milli-Q water (18.2 MΩ · cm−1) from a Milli-Q purification system (Millipore Corp, Milford, MA). Preparation of 13 nm AuNPs. AuNPs were prepared following literature methods.39 Specially, 100 mL of 1 mM of HAuCl4 (Sigma Aldrich) was taken in round bottomed flask and brought to boiling with vigorous stirring, followed by the quick addition of 10 mL of 38.8 mM sodium citrate (Sigma Aldrich) to the above solution. Then, boiling was continued for additional 20 minutes. After the removal of heating, the solution was brought to room temperature with continuous strong stirring. The gold nanoparticles were thus prepared for further experiments. Preparation of DNA-functionalized AuNPs. The swing arm was mixed with report probe in 20 mM Tris-HCl (20 mM MgCl2, pH 8.0) at different molar ratio, the total concentration of which was 500 nM. A measured aliquot of this mixture was added into 1 mL of 13 nm AuNP solution to make the molar ratio of total oligonucleotide to AuNP 500:1. This solution was then incubated at room temperature overnight. Then, 0.1 M NaCl was added in the solution consisting of sonication (20 s) and followed incubation (1 h) at room temperature for eight times. After incubation at room temperature for another 8 h, the solution was centrifuged at 13,000 g for 20 min to separate the AuNPs from the unconjugated

DNA. The AuNPs were re-suspended in 20 mM Tris-HCl (pH 8.0) solution to a final concentration of 3.5 nM, and stored at 4 °C prior to use. Performance studies of PNK-activated DNA walker. All oligonucleotides were dissolved in 20 mM Tris buffer. The swing arm was diluted to 10 µM using annealing buffer (20 mM Tris, pH 8.0, 20 mM MgCl2) and stayed at 95 °C for 5 min. Then, the solution was slowly cooled to room temperature. The phosphorylation reaction system (30 µL) consisting of 1 µM Swing arm, 0.1 mM ATP, 3 U T4 PNK and 3 µL of 10 × T4 polynucleotide kinase reaction buffer was incubated at 37 °C for 1 h and terminated by deactivating the enzymes at 95 °C for 10 min. After phosphorylation reaction, 3 U lambda exonuclease and 3 µL of 10 × lambda exonuclease reaction buffer were added into 30 µL of reaction system and incubated for 1 h at 37 °C, which was also terminated by deactivating the enzymes at 95 °C for 10 min. Then, 1 µM report probe and 2 µL of Nb. BbvCI (~6 units) were added to the above solution and the mixture was incubated at 37 °C for 1 h to perform the nicking cleavage reaction. Fluorescence measurements and electrophoresis analysis. For the detection of the reaction products, fluorescence analysis was adopted by a HitachiF-7000 fluorescence spectrophotometer (Tokyo, Japan). The emission spectra were recorded at a scan rate of 2 nm/s with an excitation wavelength of 494 nm, and the fluorescence intensity at 522 nm was recorded for data analysis. 12% nondenaturating polyacrylamide gel electrophoresis was performed for the characterization of the reaction products. The electrophoresis experiments were carried out in 1 × Tris boric acid EDTA (TBE) at 120 V for 90 min. Then, the gel was stained with SYBR Green I for 30 min. The imaging of the gel was performed using the Gel Doc XR Imaging System (Bio-Rad, USA). Different ratio of swing arm and report probes loading on the AuNPs were separately monitored by 1% agarose electrophoresis in 1 × TBE for 30 min at 100 V. DNA walker-based T4 PNK detection. The DNA walker-based reaction system containing 3.5 nM DNAfunctional AuNPs, 0.1 mM ATP, 0.01 - 10 U of T4 PNK, 1 × T4 polynucleotide kinase reaction buffer, 10 U lambda exonuclease, 10 × lambda exonuclease reaction buffer was incubated at 37 °C for 1 h and terminated by deactivating the enzymes at 95 °C for 10 min. Then, 8 U Nb. BbvCI was added to the above solution and the mixture was incubated at 37 °C for 1 h to perform nicking enzyme-mediated cleavage reaction on the surface of AuNPs. Inhibition assay. In the inhibition experiment, to evaluate the effects of inhibitors on the PNK-catalyzed phosphorylation process, several kinds of inhibitors, including different concentrations of adenosine diphosphate (0 - 6 mM) or (NH4)2SO4 (0 - 50 mM), were contained in the reaction buffer. The following procedures were similar as above using 3 U T4 PNK.

RESULTS AND DISCUSSION Principle of T4 PNK-activated 3D DNA walker. The smart T4 PNK-activated DNA walker on AuNP surface is described in Figure 1. Both the swing arms and the report probes were immobilized on the AuNP surface through Au-S self-assembly. The swing arm was a hairpin with a stem-loop structure, in which the loop contained recognition site of the nicking enzyme (NEase) - Nb.BbvCI, and 5’ end of the stem contained a hydroxyl group for PNK-actuated phosphorylation reaction. The report probe contained a fluorophore (FAM) at


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Analytical Chemistry 5’ end and a sulfhydryl group (SH) at 3’ end. In the presence of T4 PNK, γ-phosphate residue of adenosine triphosphate (ATP) was transferred to 5’ - hydroxyl terminus of the swing arm by the catalysis of PNK. Then, the lambda exonuclease recognized the phosphorylated swing arm and specifically degraded dsDNA duplexes through the removal of 5’- mononucleotides one by one. The degradation destroyed the stem structure of the hairpin and thus activated the DNA machine on AuNP surface. Then, the swing arm moved along the AuNP surface and formed a stable hybridization with the report probe, introducing a short recognition duplex of the nicking enzyme. The NEase-catalyzed cleavage of report probe liberated the swing arm, making it available to hybridize with another report probe on the same AuNP surface. In this sense, the dual enzymatic cleavage drove the swing arm to move autonomously along AuNP surface, and the machine stopped until all cleavages are completed. Because of high DNA loading efficiency and quenching capability of AuNPs, the coupled cleavage realized high efficient manipulation of DNA machine by the combination of lambda exonuclease and nicking enzyme. Since the release of fluorophore-labelled probe DNA is corresponding to PNK-initiated DNA phosphorylation, 3D DNA machine could be used to sensitively measure T4 PNK activity by tracing the fluorescent signals.

Figure 1. Schematic presentation of smart 3D DNA walking machine for T4 polynucleotide kinase assay based on coupled exonuclease and nicking enzyme cleavage reaction. Validation of DNA walker with PAGE. We conducted a preliminary non-denaturing PAGE experiment to investigate the initiation of DNA walker – T4 PNK-catalyzed phosphorylation of swing arm, followed by lambda exonuclease digestion. Figure 2A illustrates non-denaturing PAGE studies of the oligonucleotide products relating to different conditions. A 76nt band was observed in the absence of either lambda exonuclease (lane 2) or T4 PNK (lane 3), which is the same as that with the swing arm I alone (lane 1). In the presence of T4 PNK and lambda exonuclease, new bands of 70-nt and 57-nt appeared (lane 4-5) and the band of 57-nt (lane 5) become more distinct as the concentration of T4 PNK increased. The result indicates that PNK enables the addition of γ-phosphate groups from ATP to 5’- hydroxyl termini of swing arm. Accordingly, a 57-nt product is generated from lambda exonuclease-catalyzed cleavage, while a 70-nt band stands for incompletely cleavage by lambda exonuclease. We further verified NEase-catalyzed cleavage of report probe after T4 PNKcatalyzed phosphorylation of swing arm in the solution. As

shown in Figure 2B, the band of swing arm I or report probe was obviously observed in the absence of either T4 PNK or NEase, suggesting that no report probe was cleaved by NEasecatalyzed digestion of swing arm I (lane 3-5). As expected, no obvious band of report probe emerged in the presence of both T4 PNK and NEase (lane 6). The result is quite reasonable. PNK is able to successfully transfer the γ-phosphate residue from ATP to the 5’-hydroxyl termini of swing arm and thus initiates dual enzymes-mediated cleavage of report probe using swing arm I as template. To demonstrate the importance of swing arm I in dual enzyme-mediated cleavage reaction, a control swing arm II with a 10-bp length of the stem was designed as shown in Figure 2C. The band of report probe disappeared even without T4 PNK (lane 4). The result suggests that the stability of hairpin structure within swing arm is critical to prevent the unwanted NEase-catalyzed cleavage, as the shortening of the stem (9-bp length) could decrease the stability of rigid structure of hairpin DNA compared to swing arm I.

Figure 2. (A) T4 PNK-catalyzed phosphorylation of swing arm I, followed by lambda exonuclease digestion at the 5’terminus. (B) NEase-catalyzed cleavage of report probe after T4 PNK-catalyzed phosphorylation of swing arm I, (C) and swing arm II. Determination of DNA loading on AuNPs. To demonstrate the movement of DNA walker on the colloid surface, we studied the effect of DNA loading on AuNP surface by fluorescent monitoring DNA walker. AuNP for the loading of DNA walker was successfully prepared and characterized (Figure S1). TEM images confirmed the good disperse of AuNP before and after DNA conjugation (Figure S1A), and the dynamic light scattering data showed a slightly increased hydrodynamic diameter of AuNP (26.3 nm vs. 17.7 nm) after the surface coating with DNA (Figure S1B). UV-Vis spectrum of AuNP-DNA also presented the successful conjugation of DNA sequences to gold surface by a characteristic DNA peak at 260 nm (Figure S1C). The interaction of gold nanoparticles and the loading DNA was then studied via fluorescent method (Figure 3A). As shown in Figure 3B, a high fluorescence value



was obtained when there was only report probe in the solution (black curve). When adding AuNPs, low fluorescence signals were obtained in both supernate (pink curve) and re-dispersed precipitate (green curve) after centrifugal operation. The results not only reconfirm DNA loading on AuNP surface through Au-S interaction, but also reveal that AuNPs could efficiently quench the fluorescence of surface-immobilized probe DNA as a super-quencher. When the conjugated DNA was released from AuNP surface using 20 mM dithiothreitol (DTT), AuNP precipitate and the supernatant were separated after centrifugal operation. Fluorescence intensity of both the report probe solution and supernate were evaluated, respectively (red curve and blue curve). The results show that the supernate remained 70% of original fluorescence intensity from DTT-released report DNA, indicating ~70% report DNA was successfully modified on AuNP surface. We also investigated the effect of DNA loading on AuNP surface by varying the composition ratio of the loading DNA. The fluorescence intensity of DTT-released report probe from AuNP surface enhanced with the increasing proportion of report probe in the total loading DNA. The fluorescence intensity also exhibited a linear correlation with the proportion of report probe from 0% - 100% (Figure 3C). where Y is the fluorescence intensity and X is the proportion of report probe. The agarose electrophoresis results in the inset of Figure 3C also confirmed the loading proportion of report DNA on AuNP surface, as the mobility of AuNPs would increase with the increasing ratio of report probe due to the reduced size of DNA coating membrane.

DNA until it was 96% (red curve), which was be approximate to the fitting curve from Figure 3C (black curve). However, the fluorescence intensity decreased sharply after the proportional of report probe reached 96%, suggesting that the report DNA reached the maximum amount for ensuring enough swing arms to drive DNA machine. In contrast, almost no fluorescence signal was detected in the control group without T4 PNK, owing to the lack of driving force of DNA walker (blue curve). We also demonstrated the performance of the DNA walkers using another control swing arms III as shown in Figure 4B. Unlike swing arm II that may initiate unwanted NEase-catalyzed cleavage of report probe, swing arm III with only 3-nt conjugating within the stem greatly impedes the enzyme access of T4 PNK for reduced distance between 5’hydroxyl termini of swing arm and the surface of AuNPs. Therefore, nearly no fluorescence signal changed with the increasing of report probe on the AuNPs, no matter whether T4 PNK was present or not (red curve and blue curve).

Figure 4. (A) The fluorescence measurement of DNA walker induced by PNK (red curve) and the control without PNK (blue curve). (B) The fluorescence measurement of DNA walker with the swing arm III induced by PNK (red curve) and the control without PNK (blue curve).

Figure 3. (A) The schematic studies of DNA-functionalized AuNPs and (B) fluorescence measurement corresponding to the modification process. I: the total fluorescence value of only report probes. II and III: the fluorescence value of report probes in supernate and re-dispersed precipitate after centrifugal operation. IV: the fluorescence value of conjugated report probes in supernate that was released using 20 mM dithiothreitol (DTT). V: the fluorescence value of re-dispersed precipitate after treatment of DTT. (C) Fluorescence measurements of different ratio of report probes in total of report probes and swing arm (insert: mobility of AuNPs with the increasing ratio of report probe in total DNA amount by agarose electrophoresis). The fluorescent characterization of DNA walker. To demonstrate the availability of the movement of the DNA machine, the fluorescence signal of NEase-released report probe was monitored in response to the activation of T4 PNK. As shown in Figure 4, an improved fluorescence signal was observed for the activation of DNA walker in the presence of T4 PNK. The fluorescence intensity was proportional to the percentage of report probes in the total amount of loading

Figure 5. The verification of the activator for DNA walker using different proteins. The verification of the activator of DNA motor. To verify that DNA motors was specifically activated by T4 PNK, we further examined the response of DNA machine in the presence of seven other proteins including thrombin, bovine serum albumin (BSA), protein kinase A (PKA), IgG, ovalbumin (OVA), transferritin and phi29 DNA polymerase. As


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Analytical Chemistry shown in Figure 5, no fluorescence signal was detected in all the control groups, while a significant signal was detected in the presence of PNK, indicating that our proposed DNA motor is highly specific against non-targeted proteins or enzymes. Given that adenosine diphosphate (ADP) and ammonium sulfate (NH4)2SO4) are the effective inhibitors of T4 PNK, the inhibitory effect on DNA machine has also been investigated as shown in Figure S2-3. The fluorescence intensity decreased with the increasing concentrations of ADP, owing to the reversible PNK-catalyzed phosphorylation reaction in the presence of both ADP and 5’-phosphoryl nucleic acids (Figure S2). Similarly, the increasing concentrations of ammonium sulfate also resulted in the decrease of fluorescence intensity due to the inhibitory effect of (NH4)2SO4 on PNK activity (Figure S3). The half maximal inhibition values (IC50) of ADP and (NH4)2SO4 were calculated to be 0.83 mM and 7.42 mM, respectively, which are comparable to previous studies.40, 41 Besides reconfirming high selectivity of the activator on DNA walker, the results also suggest that the potential use of DNA walker for inhibitor screening of T4 PNK in the drug development. Overall, both the control experiments and inhibitory studies have demonstrated the importance of PNK for activation of the movement of DNA walker.

Figure 6. (A) Real-time response of the DNA machine to various concentrations of T4 PNK from 0.01 U/mL to 10 U/mL. (B) Change of fluorescence intensity at the plateaus with the T4 PNK concentration. Error bars represent the standard deviation of three experiments. The sensitivity of PNK assay. Having observed that active machine operates with a similar progress profile, we reasoned that the release of report probe in solution is proportional to the T4 PNK concentration. To test this, we measured progress curves resulting from various concentrations of T4 PNK (Figure 6A). As expected, the overall fluorescence signals gradually increased as the concentrations of PNK varied from 0.01 to 10 U /mL, which was proportional to the concentration of T4 PNK. The operation of machine completes and the fluorescence achieves plateaus when the reaction time reaches 60 min, so we use the fluorescent value at 60 min for PNK assay. The fluorescence value at 60 min was linear with the PNK concentration in the range from 0.01 to 0.3 U/mL. The linear relationship was described as F = 2241.073C + 255.186 with the correlation coefficient of R2 = 0.992, where F is the fluorescence intensity and C is the PNK concentration (Figure 6B). The detection limit (3σ/slope) estimated to be 0.0067 U/mL (the detailed calculation is shown in Table S2).42 Therefore, the machine is able to differentiate as low as 0.0067 U/mL T4 PNK from the blank, which is almost 10 times higher than those of chemiluminescent and fluorescent assays (0.05 U/mL).43-44 A comparison study for better highlighting the advantages of the developed detection strategy was also conducted by making use of a molecular beacon. As shown in Figure S4, the fluorescence signals also gradually increased

with the PNK concentration, but fluorescence value was much lower than that using DNA walker. The detection limit estimated to be 0.084 U/mL, clearly demonstrating enhanced sensitivity of our method. Since the above-mentioned studies of the activator have proven the high selectivity of our method, we have further demonstrated the applicability of this assay in clinical diagnosis. The proposed DNA walker for T4 PNK detection was conducted in the complex serum samples, which were compared with the results in the above-mentioned buffer. As shown in Figure S5, the fluorescence signal gradually increases with the PNK concentration in the same linear range as that in the buffer, and detection limit of 0.0092 U/mL is comparable to that in the buffer. Therefore, the result suggests the smart 3D DNA walking machine is applicable in the complex environment.

CONCLUSION In summary, we have proposed a smart 3D DNA walking machine on AuNP surface using T4 PNK as trigger, which could also be used as an effective biosensor for sensitive detection of T4 PNK. The operation of DNA walker, powered by enzymatic cleavage of conjugated oligonucleotides, could contribute to degradation of hundreds of oligonucleotides in response to T4 PNK catalytic event. Control experiments using different control proteins and PNK inhibitors demonstrated the selectivity of T4 PNK-activated performance of DNA walker. In addition, our DNA walker is proven to enhance the sensitivity of PNK sensitivity with a limit of detection of 0.0067 U/mL, exhibiting a wide dynamic range from 0.01 to 0.3 U/mL. Therefore, our DNA walker is not only promising in quantitatively monitoring the activity of T4 PNK, but also can be used for inhibitor screening of T4 PNK in the drug development. Furthermore, the proposed DNA walker may be used to simultaneously monitor multiple nucleotide kinases through the design of different DNA self-assembly within DNA machine. Importantly, given the crucial roles of kinases in some biological processes, the sensitive and efficient DNA machine-based sensing platform is promising in developing on-chip, highthroughput assays for drug discovery and clinical diagnostics. Such a 3D DNA controllable molecular machine is an excellent construction in dynamic DNA nanotechnology which may bring a breakthrough to DNA nanodevices and perform multiple and complex function.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Table S1. Sequences of oligonucleotides. (PDF) Figure S1. (A) Transmission electron microscope (TEM) images of AuNPs. (B) Dynamic light scattering (DLS) characterization of AuNPs. (C) UV-Vis absorption spectra of the AuNPs (red curve) and DNA - modified AuNPs (black curve). (PDF) Figure S2. Change of the relative activity of PNK with the concentrations of ADP. The T4 PNK concentration is 10 U/mL. Error bars show the standard deviations of three experiments. (PDF) Figure S3. Change of the relative activity of PNK with the concentrations of (NH4)2SO4. The T4 PNK concentration is 10 U/mL.



Error bars show the standard deviations of three experiments. (PDF) Figure S4. T4 PNK detection using a hairpin-based molecular beacon (from 0 to 10 U/mL). Insert is the linearship between change of fluorescence intensity and the T4 PNK concentration from 0.1 to 3.0 U/mL. Error bars represent the standard deviation of three experiments. (PDF) Figure S5. T4 PNK detection in serum (black curve) and in buffer (red curve) using the smart 3D DNA walker assay. Insert shows linearship of fluorescence intensity with the T4 PNK concentration in both serum and buffer from 0.01 to 0.3 U/mL. Error bars represent the standard deviation of three experiments. (PDF) Table S2. Detection limit of T4 PNK using 3D DNA walking machine. (PDF)

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

Author Contributions #

C.F. and Z.W. contributed equally to this work.

Present Addresses † State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China. ‡ Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant Nos. 21575088, 81671781).

REFERENCES (1) Yang, Y.; Huang, J.; Yang, X.; He, X.; Quan, K.; Xie, N.; Ou, M.; Wang, K. Anal. Chem. 2017, 89, 5850-5856. (2) Yang, Y.; Huang, J.; Yang, X.; Quan, K.; Wang, H.; Ying, L.; Xie, N.; Ou, M.; Wang, K. Anal. Chem. 2016, 88, 5981-5987. (3) Bhattacharjee, M.; Sharma, R.; Goel, R.; Balakrishnan, L.; Renuse, S. Clin Proteomics. 2013, 10, 11. (4) Tian, Y.; He, Y.; Chen, Y.; Yin, P.; Mao, C. Angew. Chem. Int. Ed. 2005, 44, 4355– 4358. (5) Liu, M.; Fu, J.; Hejesen, C.; Yang, Y.; Woodbury, N. W.; Gothelf, K.; Liu, Y.; Yan, H. Nat. Commun. 2013, 4, 2127. (6) Cha, T. G.; Pan, J.; Chen, H.; Salgado, J.; Li, X.; Mao, C.; Choi, J. H. Nat. Nanotechnol. 2014, 9, 39– 43. (7) Amir, Y.; Ben-Ishay, E.; Levner, D.; Ittah, S.; Abu-Horowitz, A.; Bachelet, I. Nat. Nanotechnol. 2014, 9, 353– 357. (8) Wang, F.; Liu, X.; Willner, I. Angew. Chem. Int. Ed. 2015, 54, 1098– 1129. (9) Sherman, W. B.; Seeman, N. C. Nano Letters 2004, 4, 1203– 1207. (10) Shin, J. S.; Pierce, N. A. J. Am. Chem. Soc. 2004, 126, 10834–10835. (11) Gu, H.; Chao, J.; Xiao S. J.; Seeman, N. C. Nature 2010, 465, 202–205.

(12) You, M.; Chen, Y.; Zhang, X.; Liu, H.; Wang, R.; Wang, K.; Williams, K. R.; Tan, W. Angew Chem Int Ed. 2012, 51, 24572460. (13) Yin, P.; Yan, H.; Daniell, X. G.; Turberfield, A. J.; Reif, J. H. Angew Chem Int Ed. 2004, 43, 4906-4911. (14) Jung, C.; Allen, P. B.; Ellington, A. D. Nat. Nanotechnol. 2016, 11, 157-163. (15) Wang, Z. G.; Elbaz, J.; Willner. I. Nano Lett. 2011, 11, 304– 309. (16) Tian, Y.; He, Y.; Chen, Y.; Yin, P.; Mao, C. Angew Chem Int Ed. 2005, 44, 4355-4358. (17) Wang, C.; Ren J.; Qu, X. Chem. Commun. 2011, 47, 1428– 1430. (18) Tang, Z. W.; Wang, K. M.; Tan, W. H.; Ma, C. B.; Li, J.; Liu, L. F.; Guo, Q. P.; Meng, X. X. Nucleic Acids Res. 2005, 33, e97. (19) Ma, C. B.; Yeung, E. S. Anal. Bioanal. Chem. 2010, 397, 2279–2284. (20) Wang, L. K.; Lima, C. D.; Shuman, S. EMBO J. 2002, 21, 3873–3880. (21) Whitehouse, C. J.; Taylor, R. M.; Thistlethwaite, A.; Zhang, H.; Karimi-Busheri, F.; Lasko, D. D.; Weinfeld, M.; Caldecott, K. W. Cell 2001, 104, 107–117. (22) Henner, W. D.; Rodriguez, L. O.; Hecht, S. M.; Haseltine, W. A. J. Biol. Chem. 1983, 258, 711–713. (23) Lown, J. W.; McLaughlin, L. W. Biochem. Pharmacol. 1979, 28, 1631–1638. (24) Torriglia, A.; Perani, P.; Brossas, J. Y.; Chaudun, E.; Treton, J.; Courtois, Y.; Counis, M. F. Cell Biochem. 1998, 18, 3612– 3619. (25) Lu, L.; Wang, M.; Liu, L. J.; Wong, C. Y.; Leung, C. H.; Ma, D. L. Chem. Commun. 2015, 51, 9953-9956. (26) Lin, S.; Kang, T. S.; Lu, L.; Wang, W.; Ma, D. L.; Leung, C. H. Biosens. Bioelectron. 2016, 86, 849-857. (27) Jiao, H.; Wang, B.; Chen, J.; Liao, D.; Li, W.; Yu, C. Chem. Commun. 2012, 48, 7862-7864. (28) He, H. Z.; Leung, K. H.; Wang, W.; Chan, D. S.; Leung, C. H.; Ma, D. L. Chem. Commun. 2014, 50, 5313-5315. (29) Novogrodsky, A.; Tal, M.; Traub, A.; Hurwitz, J. J. Biol. Chem. 1966, 241, 2933−2943. (30) Sharma, S.; Doherty; Kevin, M.; Brosh; Robert, M. Biochem. J. 2006, 398, 319−337. (31) Mereniuk, T. R.; Maranchuk, R. A.; Schindler, A.; PennerChea, J.; Freschauf, G. K.; Hegazy, S.; Lai, R.; Foley, E.; Weinfeld, M. Cancer Res. 2012, 72, 5934−5944. (32) Allinson, S. L. Future Oncol. 2010, 6, 1031−1042. (33) Wang, L. K.; Shuman, S. J. Biol. Chem. 2001, 276, 26868– 26874. (34) Bernstein, N. K.; Williams, R. S.; Rakovszky, M. L.; Cui, D.; Green, R.; Karimi-Busheri, F.; Mani, R. S.; Galicia, S.; Koch, C. A.; Cass, C. E.; Durocher, D.; Weinfeld, M.; Glover, J. N. M. Mol. Cell 2005, 17, 657–670. (35) Karimi-Busheri, F.; Daly, G.; Robins, P.; Canas, B.; Pappin, D. J. C.; Sgouros, J.; Miller, G. G.; Fakhrai, H.; Davis, E. M.; Le Beau, M. M.; Weinfeld, M. J. Biol. Chem. 1999, 274, 24187– 24194. (36) Meijer, M.; Karimi-Busher, F.; Huang, T. Y.; Weinfeld, M.; Young, D. J. Biol. Chem. 2002, 277, 4050–4055. (37) Chappell, C.; Hanakahi, L. A.; Karimi-Busheri, F.; Weinfeld, M.; West, S. C. EMBO J. 2002, 21, 2827–2832. (38) Karimi-Busheri, F.; Lee, J.; Tomkinson, A. E.; Weinfeld, M. Nucleic Acids Res. 1998, 26, 4395–4400. (39) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735-743. (40) Lin, L.; Liu, Y.; Yan, J.; Wang, X.; Li, J. Anal. Chem. 2013, 85, 334−340. (41) Wang, L. J.; Zhang, Q.; Tang, B.; Zhang, C. Y. Anal Chem. 2017, 89, 7255-7261. (42) Jiang, H. X.; Kong, D. M.; Shen, H. X. Biosens. Bioelectron. 2014, 55, 133−138.


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Analytical Chemistry (43) He, H.-Z.; Leung, K.-H.; Wang, W.; Chan, D. S.-H.; Leung, C.-H.; Ma, D.-L. Chem. Commun. 2014, 50, 5313−5315.

(44) Lin, L.; Liu, Y.; Zhao, X.; Li, J. Anal. Chem. 2011, 83, 8396−8402.

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