Biosensors and Bioelectronics 73 (2015) 19–25

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Cascade DNA nanomachine and exponential amplification biosensing Jianguo Xu a, Zai-Sheng Wu a,b,n,1, Weiyu Shen a, Huo Xu a, Hongling Li a, Lee Jia a,n a

Cancer Metastasis Alert and Prevention Center and Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350002, China State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China

b

art ic l e i nf o

a b s t r a c t

Article history: Received 10 April 2015 Received in revised form 15 May 2015 Accepted 21 May 2015 Available online 23 May 2015

DNA is a versatile scaffold for the assembly of multifunctional nanostructures, and potential applications of various DNA nanodevices have been recently demonstrated for disease diagnosis and treatment. In the current study, a powerful cascade DNA nanomachine was developed that can execute the exponential amplification of p53 tumor suppressor gene. During the operation of the newly-proposed DNA nanomachine, dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) was ingeniously introduced, where the target trigger is repeatedly used as the fuel molecule and the nicked fragments are dramatically accumulated. Moreover, each displaced nicked fragment is able to activate the another type of cyclical strand-displacement amplification, increasing exponentially the value of fluorescence intensity. Essentially, one target binding event can induce considerable number of subsequent reactions, and the nanodevice was called cascade DNA nanomachine. It can implement several functions, including recognition element, signaling probe, polymerization primer and template. Using the developed autonomous operation of DNA nanomachine, the p53 gene can be quantified in the wide concentration range from 0.05 to 150 nM with the detection limit of 50 pM. If taking into account the final volume of mixture, the detection limit is calculated as lower as 6.2 pM, achieving an desirable assay ability. More strikingly, the mutant gene can be easily distinguished from the wild-type one. The proof-of-concept demonstrations reported herein is expected to promote the development and application of DNA nanomachine, showing great potential value in basic biology and medical diagnosis. & 2015 Elsevier B.V. All rights reserved.

Keywords: Cascade DNA nanomachine Dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) p53 gene

1. Introduction Due to the obvious advantages such as specific base-pairing, predictable assembly, and single-stranded flexibility, DNA is commonly employed as a smart building block in the design of DNA based nanomachines (Dittmer et al., 2005; Modi et al., 2009; Seeman, 2003; Simmel and Dittmer, 2005; Song et al., 2013; Wang et al., 2010), some of which have been applied as the useful tools for bioassays with highly amplified efficiency (Chen et al., 2015; Wen et al., 2012). The efficient construction of DNA nanomachine has become a more and more active research area in medical research and clinical diagnosis. Generally, DNA nanomachine often consisted of one or more sequence-designed oligonucletide probes that can automatically produce a signal for readout through specific binding to the “fuel” n

Corresponding authors. E-mail addresses: [email protected] (Z.-S. Wu), [email protected] (L. Jia). 1 Present address: CMAPC, Fuzhou University, Fuzhou, Fujian 350002, China. http://dx.doi.org/10.1016/j.bios.2015.05.045 0956-5663/& 2015 Elsevier B.V. All rights reserved.

(namely trigger molecule). In order to screen disease-related genotypes, mutations, phenotypes or karyotypes for clinical purposes as well as the improvement of life quality, a few of DNA nanomachines with a variety of functions have been developed and are playing an increasingly important role in biological research over the past decades (Bi et al., 2014; Chen et al., 2004, 2015; Zhao et al., 2009). However, although some molecular machines can imitate biological phenomena or perform certain functions, their practical use still remains a great challenge due to the less desirable structure-determined functions. It is necessary and urgent to design powerful DNA nanomachines. Cancer is considered a worldwide mortal sickness and accounts for several millions of deaths every year, becoming a major public concern. Nevertheless, if accurate early diagnosis could be accomplished, there is a great chance of cure. For example, the 5-year survival rate is more than 90% when lung cancer is screened at its early stage. Thus, early diagnosis and prompt operation are essential for the successful treatment of cancers. In the clinical and basic biological research on the cancer-related diseases, the p53, also known as TP53, has been often highlighted as

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an ideal candidate maker due to its aberrant expression during the onset and progression of a variety of cancers (Nigro et al., 1989). Based on the above considerations, in the current contribution, a cascade DNA nanomachine was developed to implement the exponential amplification of stimulus and used for the highly sensitive detection of p53 tumor suppressor gene. A rational designed recognition probe (RP) tagged with fluorophore and quencher acts as the “track” for the operating of machine, which was constructed to be capable of recognizing target DNA, serving as enzymatic replication template, polymerization primer and signaling probe in the presence of the target DNA “fuel”. During the process for the hybridization event amplification, besides the target DNA acts as the trigger species to induce one cyclical stranddisplacement polymerization (RT-CNDP) reaction and is repeatedly used, nicked strands are dramatically generated, each of which can in turn activate other cyclical strand-displacement polymerization (RN-CNDP)-based amplifications, resulting in the cascade operation of DNA nanomachines and exponential amplification effect. Simply stated, via coupling with dual-cyclical nucleic acid strand-displacement polymerization (RT-CNDP and RNCNDP), a new-type of DNA nanomachine is for the first time designed as a proof-of-concept for the target binding exponential amplification. Moreover, this DNA nanomachine exhibits the desirable capability to discriminate the mutation point existing in p53 gene. Additionally, the hybridization event can be in situ signaled, circumventing the requirement for any additional reporting probe. In the text, the design of RP, molecular mechanism of signal conversion, characterization of cascade DNA nanomachine and assay capability are detailedly represented.

2. Experimental section 2.1. Materials and chemicals All oligonucleotide sequences designed in the study are listed in Table 1. The RP labeled with FAM and DABCYL was obtained via commercial synthesis by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd. (Shanghai, China) with HPLC purification, and its two italic fragments are complementary to each other. Other oligonucleotides were all supplied by Invitrogen Bio Inc. (Shanghai, China). All oligonucleotide stock solutions were prepared by dissolving DNA strands in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6) and stored at 4 °C refrigerator before usage. The Nt. BbvCI nicking endonuclease, Klenow Fragment (3′–5′ exo-) polymerase and low DNA ladder were purchased from New England Biolabs (USA) Ltd. The deoxynucleotide triphosphates (dNTPs) and Sybr Green I were obtained from Dingguo Changsheng Biotechnology Co., Ltd (Beijing, China). The 25-mM tris-buffer (pH 8.2, 100 mM NaCl, 50 mM KAc, 10 mM MgAc2 and 1 mM DTT) was used as the reaction solution (Xie et al., 2013). All other chemical reagents were of analytical grade and used without further purification. Ultrapure water used to prepare all aqueous solutions throughout the experiments was obtained through a Kerton lab MINI water purification system (UK) (resistance418 MΩ/cm). 2.2. Operation of DNA machine for amplification detection of target DNA RP was heated at 90 °C for 5 min, followed by slowly cooling down to room temperature. Subsequently, 3 μL of 5 μM RP and

Table 1 Oligonucleotide sequences designed in the current study.

Note RP (recognition probe)

Sequence (5’-3’) 5'-(DABCYL)gTCGCAGCACAAACACGCACCTCAAAGCCTGCGACt(FAM)C-3'

PT1 (polymerization template 1)

5'-GTCGCAGCACAAACACGCACCTCAgctgaggAGTC-3'

PT2 (polymerization template 2)

5'-GTCGCAGCACAAACACGCACCTCAgctgaggAGTCG-3'

PT3 (polymerization template 3)

5'-GTCGCAGCACAAACACGCACCTCAgctgaggAGTCGC-3'

PT4 (polymerization template 4)

5'-GTCGCAGCACAAACACGCACCTCAgctgaggAGTCTT-3'

Nicked fragment Target DNA (p53 gene)

5'-TCAGCTGAGGTGCGTGTTTGTGCTGCGAC-3' 5'-CAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTG-3'

MT1 (mutant target DNA1)

5'-CAGCTTTGAGGTGCaTGTTTGTGCCTGTCCTG-3'

MT2 (mutant target DNA 2)

5'-CAGCTTTtAGGTGCaTGTTTGTGCCTGTCCTG-3'

MT3 (mutant target DNA 3)

5'-CAGCTTTtAGGTGCaTGTTTGTtCCTGTCCTG-3'

MT4 (mutant target DNA 4)

5'-CAGCTTTtAGtTGCaTGTTTGTtCCTGTCCTG-3'

For RP, both the FAM and DABCYL were attached onto the lowercase bases ‘t’ and ‘g’, respectively, and the bold fragment can hybridize with the bold one indicated in target DNA. The self-hybridization of two italicized fragments helps RP fold into a hairpin structure, while the gray segment may serve as a primer when binding to polymerization template. PT1, PT2, and PT3 were projected individually with 5, 6, and 7-base fragment with gray background complementary to the primer at the 3′ terminus of RP. Except for two ‘T’ bases at the 3′ terminus, PT4 has the same sequence as PT1. Nicked fragment was designed to have the same base sequence as the nicked/displaced oligonucleotide strand resulting from RN-CNDP (seen in Scheme 1 and in the text), in which the boxed portion can hybridize to the boxed fragment of RP. Mutant target DNAs have the same sequence as the target DNA but with one or more point mutations indicated in bold lowercase in the middle region.

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2 μL of target DNA at certain concentration were successively added into an eppendorf tube containing 15 μL of tris-buffer and mixed sufficiently, resulting in the RP/target DNA duplex. After incubation for 2 h at room temperature, 0.5 μL of 5 U/μL klenow fragment (3′–5′ exo-), 0.5 μL of 10 U/μL Nt. BbvCI nicking endonuclease, 1 μL of 10 mM dNTPs, and 3 μL of 5 μM PT1 were injected and incubated at 37 °C for 1 h. The volume of resulting reaction solution is 25 μL. Finally, the enzymatic reaction was terminated at 0 °C for 10 min (Xie et al., 2013). After adding 175 μL of tris-buffer, the fluorescence spectrum was collected. The target DNA concentration mentioned in the text means the value calculated from the 25-μL reaction solution. When characterizing the proposed DNA nanomachine and investigating the influencing factors, only one issue was changed and the experiments were carried out according to the same procedure. For example, other PTs were used instead of PT1 to evaluate the dependence of the signal intensity on the polymerization template, while the hybridization, polymerization and nicking were performed under identical conditions. 2.3. Apparatus The fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrometer with a Xenon lamp as the excitation source (Hitachi, Ltd., Japan) controlled by FL Solution software. The excitation wavelength of 492 nm was used to excite the sample, and the emission spectrum was collected from 500 to 600 nm with the integration time of 0.5 s at the scan rate of 240 nm/min. Both of the excitation and emission slits were set at 5.0 nm with a PMT Voltage at 600 V. The assay capability of the nanomachine was evaluated by recording the fluorescence peak at 518 nm.

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The 12% native polyacrylamide gel was freshly prepared at laboratory, and native-PAGE (polyacrylamide gel electrophoresis) was carried out at a constant voltage at 80 V for 80 min at a gel electrophoresis instrument (BIO-RAD, USA). The fluorescence images were taken to using a ChemiDoc XRSþ imaging system with Image Lab image acquisition and analysis software (BIO-RAD, USA).The Sybr Green I was only used for the fluorescent staining of ladders.

3. Results and discussion 3.1. The cascade operation of DNA nanomachines and dual-CNDPbased exponential amplification of target stimulus As a generic molecular system, design and fabrication of a DNA device for fast and convenient DNA analysis is clearly needed. To enable efficient amplification of the cargo on a molecular track, the development of autonomous devices has therefore become interesting. To our knowledge, CNDP was a very impressive amplification strategy frequently adopted for sensitive bioassays (Guo et al., 2009; Qiu et al., 2011; Xu et al., 2015), which has been proved to possess exceptional capability to enhance the target-induced signal and attractive multiplexing properties suitable for the development of versatile DNA nanomachines. However its a great challenge to simplify the amplification steps via changing conventional CNDP signaling fashion without sacrificing the assay capabilities. In the current contribution, a turn-on fluorescent cascade DNA nanomachine has been prepared, where RP severing as the nanomachine track was designed to execute different functions. Specially, the developed RP can recognize target DNA, signal hybridization event, switch its molecular structure to activate the machine operation

Scheme 1. Schematic illustration of cascade operation of DNA nanomachine and fluorescent exponential amplification detection of p53 gene based on dual-CNDP (cyclical nucleic acid strand-displacement polymerization) mechanism. ① Hybridization to target DNA; ② Binding to PT (polymerization template); ③ First polymerization and nicking by exposure to polymerase and nickase to execute the RT-CNDP and to generate the trigger of RN-CNTP; ④ Hybridization of displaced polymerization products to RPs; ⑤ Binding to PT; ⑥ Second polymerization and nicking by exposure to polymerase and nickase to accomplish the RN-CNDP and leading to more triggers. ⑦ Similar to the fourth stage ④. More details are expounded in the corresponding section of the text.

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and serve as polymerization primer, generating the exponential amplification effect. The sequence of RP and functional fragments are described in Table 1, while the cascade operation of DNA nanomachines and the exponential amplification detection of target p53 gene is illustrated in Scheme 1. The RP was designed to fold into a loop-stem structure, and the recognition element for target p53 gene was contained in the loop portion. Besides, the chemically attached fluorophore and quencher were kept in close proximity by the hairpin structure, and two wobble bases were engineered at the 3′ end. Due to the effect of fluorescence resonance energy transfer (FRET) (Marras et al., 2002; Tyagi and Kramer, 1996), no obvious fluorescence peak was observed in this case. In the absence of target DNA, the primer is locked by RP stem and cannot hybridize with PT (polymerization template), inhibiting the subsequent isothermal polymerization and other related reactions. On the contrary, the addition of p53 gene into the bioassay system can induce successive enzymatic reactions and hybridization reactions, resulting in cascade operation of molecular machines and the exponential amplification of target hybridization event. Briefly, the target hybridization amplification consists of seven sequential steps. ① Hybridization of RP to target DNA. The target DNA hybridization not only induces the formation of rigid target DNA/RP helix that can force the stem open and restore the fluorescence of FAM, but also unlocks the locked polymerization primer. ② Binding to PT. Note that, once the target DNA/RP binds to PT, two types of polymerization primers are simultaneously activated, making the extension of the 3′ termini of RP and PT possible. ③ First polymerization and nicking. With the help of polymerase, PT is extended from its 3′ terminus using the RP as polymerization template, during which the pre-hybridized target DNA is peeled out from the RP. The displaced target DNA can hybridize to another RP and trigger the next round reaction of stranddisplacement polymerization. The reaction cycle is called RP/target DNA-mediated cyclical nucleic acid strand-displacement polymerization (RT-CNDP) in view of the target DNA displacement. Meanwhile, the extension of RP occurs on the PT, producing the nicking site. Thus, polymerization products can be nicked by nickase and generate the new primers that activate the next polymerization. The extension of newly-nicked polymerization product in the upstream region along the PT is able to displace the nicked polymerization product in the downstream region, leading to the continuous reaction of polymerization/nicking/displacement responsible for primary accumulation of nicked fragments.④ Hybridization of displaced polymerization products resulting from the third stage with other RPs, releasing more primers pre-locked by RP

stems. This is followed by binding to other PTs (namely, ⑤ stage). ⑥ Second polymerization and nicking. In this stage the extension of PT and polymerization/nicking/displacement can continuously proceed as did in the third stage, generating a large amount of displaced nicked fragments and achieving RP/nicked fragment-mediated cyclical nucleic acid strand-displacement polymerization (RNCNDP). ⑦ Each nicked fragment from the sixth stage can trigger another cyclical amplification similar to the fourth stage, leading to the cascade operation of DNA nanomachines and exponential accumulation of nicked fragments. In the current nanomachine, both RT-CNDP and RN-CNDP can be continuously repeated, and the number of primers and templates taking part in the nucleic acid strand-displacement polymerization would increase exponentially. As a result, more and more DNA machines operate, and large numbers of RPs are supposed to be opened even though few trigger DNA are involved. Thus, the fluorescence signal is dramatically enhanced, achieving a powerful dual-CNDP-based cascade molecular nanomachine suitable for the exponential amplification of target recognition events. 3.2. Exponential amplification capability of DNA nanomachine and gel eletrophoresis analyses For the current DNA machine, an impressive feature is the dualCNDP-mediated exponential amplification of target stimulus, during which RT-CNDP and RN-CNDP can simultaneously operate, but only two easily-designed oligonucleotide probes are involved. Seemingly, although DNA machine operation is very simple and convenient, it would be not easy to accomplish such an powerful target amplification. Thus, in the initial stage, comparative experiments were carried out to confirm the dual-CNDP-based exponential amplification of target DNA resulting from DNA nanomachine operation. The experimental results are presented in Fig. 1A. As demonstrated by the direct comparison of lines a and b, target DNA injection only leads to a weak fluorescent change (line b) for the only RP-contained sensing system, in which one target DNA hybridization event makes only one RP open responsible for a slight fluorescence restoration. Upon addition of polymerase, as illustrated in the difference between line d and line c, an obvious increase in fluorescence response is observed after addition of target DNA. However, the fluorescence peak in line d is only slightly higher than the value of line b, seemingly implying no substantial polymerization because of the week interaction

Fig. 1. The DNA nanomachine operation and dual-CNDP-based exponential amplification for p53 gene detection: (A) Fluorescence emission spectra under different conditions: (a) RP; (b) RP þtarget DNA; (c) RP þPT1 þpolymerase; (d) RP þ PT1þ target DNA þ polymerase; (e) RP þPT1 þ polymerase þnickase; (f) RP þ PT1þ target DNA þ polymerase þ nickase. Inset: comparative data on the fluorescence response of different assay systems: (i) RP, (ii) RP þ PT1þ polymerase, and (iii) RP þ PT1þ polymerase þ nickase. F and F0 represent the fluorescence intensity at 518 nm in the presence and absence of target DNA, respectively. (B) The 12% native PAGE analyses of the same samples. The concentrations of RP, PT1, and target DNA are 600 nM, 600 nM, and 100 nM, respectively.

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between RP and PT in the absence of nickase. A particularly exciting achievement is that, as demonstrated in line f, a dramatic increase in the fluorescence intensity can be induced by target DNA at the same concentration when the polymerase and nickase coexist in the machine system. Moreover, no obvious increase in the background fluorescence is detected as shown in line e, suggesting the hairpin structure of RP could not be opened in the absence of target DNA and the polymerization primer was tightly locked. Obviously, in this section, three different systems, including non-amplification system (system i), polymerization amplification system (system ii) and polymerization/nicking amplification system (system iii, DNA nanomachine system), are involved. To obtain the accurate comparative data, the difference in fluorescence peak (fluorescence difference) between target sample and blank, as well as the corresponding fluorescence peak ratio (called fluorescence ratio), is shown in the Inset. Clearly, the fluorescence ratio of systems i, ii and iii are about 3, 4 and 13, respectively. These results are consistent with the trend implied by the data of fluorescence differences, indicating the quite powerful target reporting capability of system iii and validating the operation of DNA machines. To directly confirm the DNA nanomachine operation and dualCNDP amplification process, the native PAGE analyses were performed to visualize the involved DNA elements. As shown in Fig. 1B, an easily detectable band with a relatively lower mobility was observed in lane b compared with lane a. This should be attributed to the formation of RP/target DNA duplex (69 bases). Similarly, lanes c and d display analogical bands at the identical positions compared with lanes a and b, respectively. Moreover, a band in the lower part of lane d can be still clearly seen, indicating the residual hairpin-type RP in the closed state. These experimental results are consistent with the observations from fluorescence spectra shown in Fig. 1A. On the contrary, as shown in lane f, when polymerase and nickase coexist in the RP sensing system (namely, DNA nanomachine system), the RP band completely disappears in the presence of target DNA, suggesting the opening of almost all RP loop-stem structures and sufficient restoration of FAM fluorescence. More strikingly, three new bright bands (i, ii and iii) are detected, and the RP/target DNA duplex band becomes vague and dim. The new bands demonstrate the formation of new

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DNA complexes with higher molecular weight. The band ii should correspond to the extended RP/extended PT complex (134 bases), while the band iii would be the RP/extended PT (104 bases) where hybridized target DNA is displaced during the extension of PT. In another angle to explain, because target DNA was peeled out from RP in band ii and band iii, the appearance of the two bands plausibly provides an evidence that RT-CNDP was successfully implemented during the polymerization amplification. When nicked strands are displaced by the latter polymerization, because of their longer fragment complementary to RP than target DNA, they can easily hybridize with other RPs or RP fragment-contained complexes (e.g., extended RP/PT or extended RP/target DNA/PT), generating the more complicate complexes with sticky ends. These DNA complexes could interact with each other via the sticky-end pairing effect (Li et al., 2014; Wu et al., 2010), producing advanced DNA assemblies responsible for band i with the highest molecular weight. More supporting data are seen in the following section (Fig. 2). The formation of new DNA products, corresponding to bands i, ii and iii, not only causes the opening of more RPs but also forces the amplification reactions sufficiently proceed, leading to the disappearance of RP band. Additionally, the blank band in lane e is almost identical to the bands in lane a and lane c, demonstrating that the RP did not open and the background fluorescence was efficiently suppressed. The PAGE images not only support the fluorescence responses to target DNA shown in Fig. 1A, but also verify the molecular mechanism of DNA nanomachine operation and dual-CNDP-based cascade amplification in Scheme 1 for the sensitive detection of p53 gene to some extent. 3.3. Characterization of nicked fragment-mediated amplification during DNA nanomachine operation In order to affirm that the nicked fragment exerts the function similar to target DNA and induces directly the formation of polymerization products with high molecular weight, more gel electrophoresis analyses were performed where commercially synthesized nicked fragment (same as the one generated from the polymerization/nicking process) were used instead of target DNA under identical conditions, and the experimental results are seen in Fig. 2. The slight difference is the appearance of a dim band in lane d with the gel mobility similar to band ii of lane f. This would result from the formation of extended RP/extended PT complex (134 bases) from RP/PT hybrid even without target DNA, suggesting that nicked fragment triggers more easily the polymerization compared with target DNA (see lane d of Fig. 1B). However, taking it by and large, the electrophoresis bands obtained in Fig. 2 are similar to those in Fig. 1B, suggesting the nicked fragment is capable of triggering the dual-CNDP in an analogous fashion. 3.4. Comparative study between single-CNDP and dual-CNDP involved during DNA machine operation

Fig. 2. The 12% native PAGE characterization of nicked fragment-mediated reactions. The experiments were conducted where Nicked fragment was used instead of target DNA according to the identical procedure. Lane a: RP; Lane b: RP þNicked fragment; Lane c: RP þ PT1 þpolymerase; Lane d: RP þPT1 þ Nicked fragmentþ polymerase; Lane e: RP þPT1 þpolymerase þ nickase; Lane f: RP þPT1 þNicked fragmentþpolymerase þ nickase. The concentration of RP, PT1, and Nicked fragment are 600 nM, 600 nM, and 100 nM, respectively.

In the current DNA nanomachine, the involved dual-CNDP amplification consists of RT-CNDP and RN-CNDP, cooperatively contributing to the exponential amplification. Implementing the comparative study between single- and dual-CNDP amplification capability and evaluating the synergistic amplification of polymerase and nickase are the primary aim of this section. As clearly shown in the histogram in Fig. 3, the amplification capability of three sensing systems increases in the order: RT-CNDP oRNCNDPo dual-CNDP. Such a trend seems to be reasonable when taking the following issues into account. For RT-CNDP, only one target DNA is involved during the amplification process though it is displaced and used repeatedly. In contrast, once the RN-CNDP is activated, not only are more and more nicked fragments generated

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Fig. 3. The comparative data on the assay performance between single-CNDP (RTCNDP or RN-CNDP) and dual-CNDP amplification systems. RT-CNDP: nickase was not involved, so that the nicking reaction did not occur, and RN-CNDP was incapable of proceeding. RN-CNDP: PT4 was used instead of PT1, which has two-base overhang (“TT”) at its 3′ end to inhibit the enzymatic extension and cannot sever as polymerization primer. Thus, the target DNA was not displaced and RT-CNDP was prevented. However, the enzymatic extension from the 3′ terminus of RP and nicking reaction can proceed. Dual-CNDP: the DNA nanomachine-based exponential amplification system where both PT1 and nickase were used. “Blank” and “Target” indicate the sensing systems in the absence or presence of target p53 gene, respectively. The concentration of RP, PT1, and PT4 were 600 nM, while target DNA was 100 nM.

from one PT via the polymerization/nicking/displacement cycle, but also the displaced nicked fragments may hybridize with other RPs and help the RPs bind to more PTs, leading to the enzymatic extension of more RPs, as well as the continuous polymerization/ nicking/displacement reaction on more polymerization templates. More apparently, the coexistence of polymerization and nicking, via combining with the cyclical use of target DNA, could result in the much higher amplification capability of dual-CNDP. Note that the increase in fluorescence intensity upon target DNA hybridization for dual-CNDP is more than 2060 (a.u.), which is more than twice larger than the sum (about 940 a.u.) of fluorescence changes from both RT- and RN-CNDP. This suggests that the much higher amplification capability of dual-CNDP originates not from the superposition of RT- and RN-CNDP in a simple manner but from the synergistic effect of polymerization and nicking who together operate DNA machines. 3.5. Capability of DNA nanomachine to sense target stimulus The sensitivity of DNA nanomachine to sense its stimulus is of

central importance to various applications and is evaluated in this section. Additionally, as is known that sensitive identification and accurate quantification of p53 tumor suppressor gene is of fundamental significance, especially in early-stage diagnosis of cancers, a series of target DNA samples at the concentration ranging from 0 to 300 nM were quantitatively detected to verify the sensing sensitivity of proposed nanomachine. The fluorescence data are represented in Fig. 4. Fig. 4A depicts the fluorescence spectra in the absence and presence of target stimuli at different concentrations, and a monotonic increase in fluorescence intensity is observed when increasing the target DNA concentration. The Inset displays the fluorescence spectra in the low target DNA concentration range from 0 to 1 nM. Clearly, the 50 pM target DNA induces a detectable fluorescence increase compared with the fluorescence intensity of the blank, and this target concentration is defined as the detection limit, indicating the target DNA acted as the fuel of DNA nanomachine, which produced detectable signal for readout. Because the fluorescence intensity is determined by the concentration of RP in the final mixture, the detection limit is much lower (about 6.2 pM) when taking into account the final volume of detected mixture. Fig. 4B exhibits the linear relationship between target DNA concentration and the fluorescence peak at 518 nm in a relatively wide concentration range (0.05–150 nM). The regression equation is Sqrtpi (F)¼ 23.33þ0.573C with a correction coefficient of 0.9995, where F and C represent the fluorescence intensity and target concentration, respectively. No obvious fluorescence change is detected when increasing further the target DNA concentration to a higher concentration (for example, 200 nM), and the dose-response points are not within the linear response range. Additionally, the average relative standard deviation estimated from all datum points is approximately 5.0%, indicating a satisfactory reproducibility. 3.6. Initiation specificity of DNA nanomachines toward target stimuli Gene mutation is a very common phenomenon, which is closely related to some genetic diseases. DNA nanomachines are required to be specifically initiated by the stimulus especially employed for mutation point detection of target DNA. As a tumor suppressor gene, the wild-type gene of p53 can inhibit tumor growth and progression in vivo. Inactivation of the p53 tumor suppressor is a frequent event in tumorigenesis, and the mutant type of p53 gene was screened in at least half of all human tumors (Bykov et al., 2002). Thus, reliable detection of p53 gene mutation is of great importance for early diagnosis, targeted therapy, prognostic evaluation and monitoring of tumors. In the present study, a single-base-mutation DNA (MT1) closely related to cancers and

Fig. 4. The dynamic relationship between fluorescence response and target DNA concentration. (A) Fluorescence emission spectra of RP in the presence of various concentrations of target DNA or blank. Inset magnifies the fluorescence spectra in the low target concentration range. (B) the linear relationship between target concentration and fluorescence response. Sqrtpi (F) means the Fπ , while F and C indicate the fluorescence peak and target DNA concentration, respectively. The error bars indicate the standard deviation of three parallel measurements for each concentration of target DNA.

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Acknowledgments This work was supported by Ministry of Science and Technology of the Poeple's Republic of China (2015CB931804), National Natural Science Foundation of China (NSFC) (Grant No.: 21275002 and 81273548), and Independent Research Project of State Key Laboratory of Photocatalysis on Energy and Environment (No. 2014CO1).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.05.045.

References Fig. 5. Specificity evaluation of the DNA nanomachine for the detection of mutant target DNAs against the wild-type gene. The recorded value, (Fm  F0)/(Ft  F0)  100%, is used to evaluate the optical response induced by mutant target DNAs, where Ft, Fm and F0 are the fluorescence peak at 518 nm corresponding to wild-type p53 gene, mutant target DNAs and blank, respectively. The concentration of p53 gene is 100 nM, and its fluorescence response is defined as 100%. The standard deviation was acquired from three repeated measurements.

other three imaginary mutant target DNAs with two, three or four mutation points were interrogated under identical conditions to accurately assess the target recognition specificity of DNA nanomachine. The comparative analyses are shown in Fig. 5. If the fluorescence response of the fully complementary target DNA (T) is defined as 100%, the signals corresponding to MT1, MT2, MT3, and MT4 are 78%, 70%, 22% and 9%, respectively. Namely, the mutant p53 gene with a mutation point screened often in clinical cancer specimens is successfully identified, and target DNA with more mismatched bases can be more easily detected, reflecting a desirable detection specificity for the potential application in a reliable screening of genetic diseases.

4. Conclusion In the present study, we have developed a cascade DNA nanomachine for sensitive detection of p53 gene via the dual-CNDP exponential amplification. Compared to the already known DNA nanomachines, the intelligent DNA nanomachine possesses several remarkable advantages. First, the RP, machine track, serves as recognition element, replication template and primer, leading to a detectable signal in a very straightforward fashion. During the cascade operation of DNA nanomachines, not only is the requirement of exogenous reporting probe avoided, but also the possible signal loss encountered in an indirect signal conversion is circumvented, contributing to the simplification of target detection and the improvement of analytical capability. Second, via incorporating RT-CNDP and RN-CNDP into DNA nanomachine operation, a synergistic effect responsible for exponential amplification was achieved. Third, besides the high sensitivity, a high detection specificity was obtained, and the recognition of mutation point in target DNA can be implemented. All in all, the unique operation mechanism and excellent assay capability exhibit great promise for biomolecule analysis and relevant studies, and the innovative concept of dual-CNDP exponential amplification offers valuable insight to guide the development of novel DNA nanomachines.

Bi, S., Cui, Y., Dong, Y., Zhang, N., 2014. Target-induced self-assembly of DNA nanomachine on magnetic particle for multi-amplified biosensing of nucleic acid, protein, and cancer cell. Biosens. Bioelectron. 53, 207–213. Bykov, V.J., Issaeva, N., Shilov, A., Hultcrantz, M., Pugacheva, E., Chumakov, P., Bergman, J., Wiman, K.G., Selivanova, G., 2002. Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound. Nat. Med. 8 (3), 282–288. Chen, Y., Lee, S.H., Mao, C., 2004. A DNA nanomachine based on a duplex–triplex transition. Angew. Chem. Int. Ed. 43 (40), 5335–5338. Chen, Y., Xiang, Y., Yuan, R., Chai, Y., 2015. A restriction enzyme-powered autonomous DNA walking machine: its application for a highly sensitive electrochemiluminescence assay of DNA. Nanoscale 7 (3), 981–986. Dittmer, W.U., Kempter, S., Rädler, J.O., Simmel, F.C., 2005. Using gene regulation to program DNA‐based molecular devices. Small 1 (7), 709–712. Guo, Q., Yang, X., Wang, K., Tan, W., Li, W., Tang, H., Li, H., 2009. Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction. Nucl. Acids Res. Li, H., Wu, Z.-S., Shen, Z., Shen, G., Yu, R., 2014. Architecture based on the integration of intermolecular G-quadruplex structure with sticky-end pairing and colorimetric detection of DNA hybridization. Nanoscale 6 (4), 2218–2227. Marras, S.A., Kramer, F.R., Tyagi, S., 2002. Efficiencies of fluorescence resonance energy transfer and contact‐mediated quenching in oligonucleotide probes. Nucl. Acids Res. 30 (21), e122-e122. Modi, S., Swetha, M., Goswami, D., Gupta, G.D., Mayor, S., Krishnan, Y., 2009. A DNA nanomachine that maps spatial and temporal pH changes inside living cells. Nat. Nanotechnol. 4 (5), 325–330. Nigro, J.M., Baker, S.J., Preisinger, A.C., Jessup, J.M., Hosteller, R., Cleary, K., Signer, S. H., Davidson, N., Baylin, S., Devilee, P., 1989. Mutations in the p53 gene occur in diverse human tumour types. Nature 342 (6250), 705–708. Qiu, L.-P., Wu, Z.-S., Shen, G.-L., Yu, R.-Q., 2011. Highly sensitive and selective bifunctional oligonucleotide probe for homogeneous parallel fluorescence detection of protein and nucleotide sequence. Anal. Chem. 83 (8), 3050–3057. Seeman, N.C., 2003. DNA in a material world. Nature 421 (6921), 427–431. Simmel, F.C., Dittmer, W.U., 2005. DNA nanodevices. Small 1 (3), 284–299. Song, C., Wang, Z.G., Ding, B., 2013. Smart nanomachines based on DNA self‐assembly. Small 9 (14), 2382–2392. Tyagi, S., Kramer, F.R., 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308. Wang, Z.-G., Elbaz, J., Willner, I., 2010. DNA machines: bipedal walker and stepper. Nano Lett. 11 (1), 304–309. Wen, Y., Xu, Y., Mao, X., Wei, Y., Song, H., Chen, N., Huang, Q., Fan, C., Li, D., 2012. DNAzyme-based rolling-circle amplification DNA machine for ultrasensitive analysis of microRNA in Drosophila larva. Anal. Chem. 84 (18), 7664–7669. Wu, Z.-S., Lu, H., Liu, X., Hu, R., Zhou, H., Shen, G., Yu, R.-Q., 2010. Inhibitory effect of target binding on hairpin aptamer sticky-end pairing-induced gold nanoparticle assembly for light-up colorimetric protein assay. Anal. Chem. 82 (9), 3890–3898. Xie, S.-J., Zhou, H., Liu, D., Shen, G.-L., Yu, R., Wu, Z.-S., 2013. In situ amplification signaling-based autonomous aptameric machine for the sensitive fluorescence detection ofcocaine. Biosens. Bioelectron. 44, 95–100. Xu, J., Dong, H., Shen, W., He, S., Li, H., Lu, Y., Wu, Z.-S., Jia, L., 2015. New molecular beacon for p53 gene point mutation and significant potential in serving as the polymerization primer. Biosens. Bioelectron. 66, 504–511. Zhao, C., Song, Y., Ren, J., Qu, X., 2009. A DNA nanomachine induced by singlewalled carbon nanotubes on gold surface. Biomaterials 30 (9), 1739–1745.