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Versatile and Programmable DNA Logic Gates on Universal and Label#Free Homogeneous Electrochemical Platform Lei Ge, Wenxiao Wang, Ximei Sun, Ting Hou, and Feng Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02584 • Publication Date (Web): 01 Sep 2016 Downloaded from http://pubs.acs.org on September 3, 2016

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Analytical Chemistry

Versatile and Programmable DNA Logic Gates on Universal and Label‑ ‑Free Homogeneous Electrochemical Platform

Lei Ge, Wenxiao Wang, Ximei Sun, Ting Hou, and Feng Li*

College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao, 266109, People’s Republic of China

*Corresponding author: Feng Li E-mail: [email protected] Telephone: +86-532-86080855 1

ACS Paragon Plus Environment


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ABSTRACT: Herein, a novel universal and label-free homogeneous electrochemical platform is demonstrated, on which a complete set of DNA-based two-input Boolean logic gates (OR, NAND, AND, NOR, INHIBIT, IMPLICATION, XOR, and XNOR) are constructed by simply and rationally deploying the designed polymerization/nicking

machines

without

complicated

sequence

DNA

modulation.

Single-stranded DNA is employed as the proof-of-concept target/input to initiate or prevent the DNA polymerization/nicking cyclic reactions on these DNA machines to synthesize numerous intact G-quadruplex sequences or binary G-quadruplex subunits as the output. The generated output-strands then self-assemble into G-quadruplexes that renders remarkable decrease to the diffusion current response of methylene blue and, thus, provides the amplified homogeneous electrochemical readout signal not only for the logic gate operations but also for the ultrasensitive detection of the target/input.

This

electrochemical

system

logic

represents

operation.

the

first

Importantly,

example the

of

homogeneous

proposed

homogeneous

electrochemical logic gates possess the input/output homogeneity and share a constant output threshold value. Moreover, the modular design of DNA polymerization/nicking machines enables the adaptation of these homogeneous electrochemical logic gates to various input and output sequences. The results of this study demonstrate the versatility and universality of the label-free homogeneous electrochemical platform in the design of biomolecular logic gates and provide a potential platform for the further development of large-scale DNA-based bio-computing circuits and advanced biosensors for multiple molecular targets. 2


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INTRODUCTION Chemical computation, mimicking conventional microprocessors to perform Boolean functions through the manipulation of elementary molecular Boolean logic gates, is a promising substitute for the traditional silicon-based information technologies.1-3 Recently, considerable efforts have been dedicated to construct unconventional molecular Boolean logic gates as ideal candidates for the development of more powerful and efficient chemical computations. Bio-computation,4-9 belonging to a subarea of unconventional chemical computations, employs biomolecules or biomaterials to realize higher complexity of logic gates operation while using much simpler biochemical methodologies. Moreover, bio-computation possesses the potential of integrating both the processing and sensing of biomolecular inputs to generate autonomous programmed readouts and actions due to their capability of intelligent judgment,10-12 thus, not only offering a new paradigm for future chemical computation technology, but also holding great promise for biomolecular devices and biomolecular detections. In the past decade, researchers have challenged themselves to utilize various biomolecules/biomaterials, such as proteins/enzymes,13 DNA,14 RNA,15 and even whole cells,16 to build biomolecular logic gates for computational operations with elegant design strategies. Of particular interest are biomolecular computations constructed and operated by DNAs, due to their predictable and combinatorial structures, easy chemical synthesis, reactivity toward enzymes (e.g. endonuclease and exonuclease), and sequence-controlled functional features (e.g. aptamers and DNAzymes), providing sufficient theoretical and experimental bases for 3



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the rational design of extremely effective biomolecular logical operations.17-19 Therefore, on the basis of the aforementioned features of DNAs, DNA-based logic gates has been widely applied not only in the fields of medical diagnostics,20 sensing,21 drug delivery,22 but also in development of complex logic devices,23-27 demonstrating its wide application potential. Up to now, various signal transducers for logic outputs have been used to fabricate diverse logic gates, such as colorimetry,28,29 chemiluminescence,30,31 fluorescence,32-34 electrochemistry,35-37 electrochemiluminescence,38-40 and biofuel cell.41,42 In this respect, electrochemical strategy holds great promise as powerful signal transducer for logic outputs and exhibits several noticeable advantages in chemical computations, such as simple instrumentation, low cost, rapid response, high sensitivity, and ease of integration into miniaturized devices for scale-up and multiplexed chemical- or biochemical-electronic computations. However, all the reported electrochemical logic gates rely on the compulsory labeling of electroactive substances, enzymes, or nanomaterials onto biomolecules and/or the immobilization of them onto solid electrode surface, which play significant roles in the performances of these electrochemical logic gates but also show some intrinsic drawbacks: (i) the labeling and immobilization processes inevitably increase the cost and limit the routine use of electrochemical logic gates, especially, in multiplexed logic devices with multiple outputs, (ii) the chemical labeling/immobilization procedures may affect the activity of the biomolecules in the logic gates, and (iii) the existence of local steric hindrance, induced by the labels or electrodes, may lead to relatively low 4


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binding/reaction efficiency, limiting the chemical computing rate. Therefore, an electrochemical transducer that can directly implement chemical computations in homogeneous solution without any labeling and immobilization process would greatly facilitate the development of rapid, simple, cost-effective, and reliable chemical computations. Until now, no report has been made on the use of homogeneous electrochemical transducer to construct biomolecular logic gates for chemical computational applications. To engineer a series of fundamental logic gates based on homogeneous electrochemical transducer, a signal transfer mechanism should be established in logic gates to allow the signals to transfer from input to output. In most of the DNA-based homogeneous electrochemical strategies, the output signal is obtained from the diffusion current variation of electroactive labels.43-50 One significant approach to trigger the variation of electrochemical signal in homogeneous solutions is the intercalation of methylene blue (MB) within G-quadruplex.51,52 Upon the intercalation of MB into G-quadruplex, the formed G-quadruplex/MB complex greatly inhibited the electrochemical diffusion current of MB, which is even more effective than that caused by double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA),51 generating an unambiguous

electrochemical signal output.

In this study,

target/input-switched DNA polymerization/nicking (TISD-P/N) machineries, capable of numerous generations of G-quadruplexes, are designed as the input convertors and signal amplifiers in our homogeneous electrochemical logic gates. Upon input introduction, hybridization of the inputs to the DNA machines triggers (switch-on 5



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machines) or prevents (switch-off machines) the DNA polymerization/nicking circular reaction, which transduces the input recognition into the variation of the electrochemical diffusion current of MB. These machines provided amplified homogeneous electrochemical output not only for the ultrasensitive detection of the two inputs, but also for the development of the YES gate, NOT gate, OR gate, and NAND gate. It has been reported that G-quadruplex can be split into two fragments that show no binding ability to PPIX and hemin, but upon template-assisted formation of intact G-quadruplex from the split fragments, the binding ability is restored.53 This mechanism has attracted substantial research efforts to integrate such a binary G-quadruplex strategy into molecular logic design for the construction of various colorimetric54 and fluorescent25,27,55 logic gates/devices. By the integration of binary G-quadruplex

into

the

designed

TISD-P/N

machineries,

homogeneous

electrochemical AND gate, NOR gate, INHIBIT gate, IMPLICATION gate, XOR gate, and

XNOR

gate,

are

realized

in

this

work

through

the

rational

combination/permutation of different TISD-P/N machines, further demonstrating the versatility and universality of this TISD-P/N-based homogeneous electrochemical approach. Thus, our study contributes to establishing a complete set of two-input logic gates

for

homogeneous

information

processing

between

biological

and

electrochemical systems, and sheds light on designing and developing intelligent homogeneous electronic bio-computation and sensing applications, such as biosensing, food safety, and medical diagnosis. 6


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EXPERIMENTAL SECTION Chemicals and Materials. All oligonucleotides were obtained from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). All the synthetic oligonucleotides were HPLC purified and freeze-dried by the supplier. Their sequences were listed in Supporting Information. All oligonucleotides were received as powders and centrifuged so that they would reside at the bottom of the containers. The powder was then dissolved with 10 mM phosphate-buffered saline (PBS, pH 7.4) to give stock solutions of 100 µM. Klenow fragment polymerase (KF polymerase), deoxynucleotide solution mixture (dNTPs), and Nt.AlwI nicking endonuclease were purchased from New England BioLabs, Inc. SYBR Green I was obtained from Xiamen Bio-Vision Biotechnology Co. Ltd. (Xiamen, China). All reagents are analytical grade and solutions were prepared using ultrapure water (specific resistance of 18 MΩcm, Milli-Q Gradient System, Millipore, Bedford, MA). Apparatus. All homogeneous electrochemical experiments were carried out on an Autolab electrochemical workstation (Metrohm, Netherland) at room temperature using a conventional three-electrode system comprising a bare indium tin oxide (ITO) working electrode (Φ = 5 mm), an Ag/AgCl reference electrode, and a platinum wire counter electrode. Before each electrochemical measurement, the ITO electrodes used was sequentially sonicated in an Alconox solution (8 g Alconox in 1 L water) for 15 min, propan-2-ol for 15 min, and twice in water for 15 min. After that, the ITO electrode was immersed in 1 mM NaOH solution for 5 h at room temperature and then sonicated in ultrapure water for 15 min to obtain a negatively charged electrode 7



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surface. The cleaned ITO electrodes were stored in ultrapure water until use. The images of nondenaturating polyacrylamide gel electrophoresis (PAGE) were scanned by the Gel Doc XR+ Imaging System (BIO-RAD, America). Homogeneous Electrochemical Logic Gate Operations. The operation procedures for these homogeneous electrochemical logic gates could be briefly described as follows: Prior to logic operation, all the switch-off machines are refolded into hairpin structure through heating to 95 °C for 3 min and then allowed to cool to room temperature to form the desirable stem-loop DNA structure. For the operation of homogeneous electrochemical logic gate, corresponding TISD-P/N machine configurations were first subjected to the oligonucleotide inputs, DNA1 and/or DNA2 with different combinations. Then, this obtained logic solution was added into the reaction mixture (pH 7.9), consisting of 10 U KF polymerase, 10 U Nt.AlwI nicking endonuclease, 500 µM dNTPs, and 1× KF polymerase buffer (10 mM Tris−HCl, 50 mM NaCl, 20 mM KCl, 10 mM MgCl2, and 1 mM dithiothreitol), for the time interval of 60 min at 37 °C in a constant temperature incubator. For homogeneous electrochemical readout, MB solution diluted with the same buffer was added into the above mixture to a final concentration of 5.0 µM, followed by incubation at room temperature for another 40 min. Finally, the resulting mixture are pipetted onto the ITO working electrode for differential pulse voltammetry (DPV) scanning with the potential window from −0.4 V to −0.2 V (vs Ag/AgCl). The parameters applied for DPV scanning were 500 ms interval time, 50 ms modulation time, 25 mV modulation amplitude, 5 mV step potential, and 10 mV·s-1 scan rate. All 8


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DPV curves are baseline-corrected using the Nova 1.1 software embedded in the Autolab electrochemical workstation.

RESULTS AND DISCUSSION Figure 1A elucidated the design of the proposed homogeneous electrochemical switch-on machine, and the mechanism of which is confirmed by nondenaturating PAGE in Supporting Information. As shown in Figure 1A, the switch-on machine is a single-stranded DNA, which consists of three regions: domain-a, domain-b, and domain-c. Domain-a acts as a recognition probe for specific input recognition. Domain-b is a specific sequence (3′-CCTAGNNNN-5′, denoted in orange in Figure 1A) for the recognition site of Nt.AlwI (Nt) nicking endonuclease. Domain-c represents the complementary sequence for T30695 G-quadruplex. In the absence of input (DNA1 or DNA2, proof-of-concept inputs in this work), no DNA polymerization/nicking reaction occurs on this switch-on machine. Upon input introduction, DNA input/target could hybridize to the domain-a of switch-on machine, and then acts as a primer to induce DNA polymerization in the presence of KF polymerase and dNTPs according to the sequence of domain-b and domain-c. Upon polymerization, domain-b yielded a double-stranded nicking site for the Nt.AlwI nicking endonuclease, and domain-c generated the T30695 sequence (domain-c*). The nicking of the formed dsDNA produces a 3′-OH end that allows the next polymerization on the switch-on machine from this nicking site. As the next polymerization processes, the previously generated domain-c*, the T30695 sequence, is displaced, resulting in an autonomous polymerization/nicking circular reaction that 9



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leads to the generation of numerous T30695 sequences, which can then form the G-quadruplexes with the aid of K+-ions. As a result, MB molecules are firmly “locked” into the formed G-quadruplexes51,52 and thus show remarkably decreased diffusivity toward the ITO electrode surface. Moreover, the electrostatic repulsion between the negatively charged phosphate backbone of G-quadruplexes and the negatively charged ITO electrode surface further prevented the “locked” MB molecules from reaching the electrode surface. Thus, the diffusion current of MB (IMB) would be inhibited significantly, which is proportional to the concentration of input/target (Figure 1B). As expected, the calibration curve in the inset of Figure 1B, made by plotting ∆IMB (the IMB difference between the absence and presence of DNA1) versus the DNA1 concentration (as an example), shows good correlations over a wide dynamic range of 0.1 pM to 6.5 nM. The resulting linear regression equation was obtained as ∆IMB(nA) = 167.51lg[cDNA1(pM)]+196.84 (r = 0.9972) with a directly measured detection limit of 0.1 pM, which clearly demonstrated that the proposed switch-on machine could be applied in sensitive detection of oligonucleotides.

10


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Figure 1. (A) Schematic illustration of the switch-on machine for homogeneous electrochemical transducer. The sequence of DNA molecules were described in terms of encoded domains, each of which represents a short fragment of DNA sequence. Complementarity between encoded domains is denoted by an asterisk. (B) DPV responses of the proposed switch-on machine to different concentrations of DNA1 (from a to m: 0, 0.1, 0.2, 0.5, 1.0, 3.0, 10, 30, 100, 300, 1000, 3000, and 6500 pM. Inset: the linear relationship of ∆IMB versus the logarithm of DNA1 concentration. The final concentration of switch-on machine was 0.5µM.

Based on the mechanism of TISD-P/N machineries, ten elementary logic gates, are realized on this homogeneous electrochemical platform. The logic input for each homogeneous electrochemical logic gate in this work are one or two oligonucleotides, DNA1 and/or DNA2, of which concentrations of 0.5 µM are defined as logic input value of 1, respectively. The absence of the respective input is defined as logic input value of 0 for these logic gates. Furthermore, for each homogeneous electrochemical logic gate, the variation of IMB induced by the logic gate operations (denoted as ∆IGate 11



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= I0 – IGate, I0 corresponds to the IMB of the MB solution without any TISD-P/N machine, KF polymerase, dNTP, nicking endonuclease, and input; IGate is the resulting IMB of the reaction mixture after the respective logic gate operation) greater than 800 nA is assigned a true operating output of 1, while the ∆IGate less than 800 nA is designated as a false output of 0. YES gate and NOT gate Obviously, the aforementioned DNA1 switch-on machine (DNA1-on) can be directly operated as single-input YES gate. As shown in Figure 2A, no T30695 G-quadruplex sequence is generated in the absence of input (input = 0). In this case, the observed slight IMB decrement (Figure 2B, IGate(0), ∆IGate < 800 nA) is mainly attributed to the interaction of MB with the added DNA1-on, KF polymerase, dNTP, and nicking endonuclease. The presence of input DNA1 (input = 1) leads to the polymerization/nicking circular reaction and, thus, the significant inhibition of IMB (∆IGate > 800 nA). From Figure 2A, one could find that, in this single-input logic gate, input 1 produces output 1; input 0 produces output 0. Thus, based on the relationship between input and output in the truth tables, homogeneous electrochemical YES gate is realized.

12


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Figure 2. Schematic presentation, DPV responses, bar presentation of output signals, and truth table of homogeneous electrochemical YES gate (A), NOT gate (B), OR gate (C), and NAND gate (D). 5.0 nM DNA1-on and 5.0 nM DNA1-off is employed for YES gate and NOT gate, respectively. Concentrations for each TISD-P/N machine in OR gate and NAND gate are all 2.5 nM.

If a Boolean output value should be represented contrary to the input, a NOT gate is necessary. To realize this function, we designed a DNA1 switch-off TISD-P/N machine (DNA1-off), which consists of a stem-loop structure at its 3’-end and an overhang ssDNA segment at its 5’-end. As shown in Figure 2B, domain-a* and domain-b* in the stem-loop structure of DNA1-off corresponds to the recognition probe for input DNA1. Domain-e at the 5’-end of the overhang ssDNA segment is complementary to the T30695 G-quadruplex sequence. Domain-d of the overhang ssDNA segment includes a sequence-specific domain that, upon formation of a duplex 13



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structure, yields the nicking domain to be cleaved by Nt.AlwI nicking endonuclease. Moreover, an 18-carbon spacer (domain-c) is inserted between domain-a and domain-b* to prevent the DNA polymerization from the 3’-end of input. In the absence of input DNA1, domain-a* in the stem of DNA1-off acts as a primer to initiate autonomous self-polymerization that synthesizes a large amount of G-quadruplexes through the polymerization/nicking circular reaction. In contrast, the input DNA1 opens the stem-loop structure of DNA1-off via toehold-mediated strand displacement and, thus, prevents the DNA1-off from autonomous self-polymerization. When the input is 1, high IMB intensity is observed (Figure 2B), giving an output of 0 (∆IGate < 800 nA). The absence of input, which represents an input of 0, leads to low IMB intensity, so that the logic output is 1 (∆IGate > 800 nA). This behavior is consistent to the NOT gate. OR gate and NAND gate Starting from the single-input homogeneous electrochemical YES gate and NOT gate, two-input homogeneous electrochemical OR gate and NAND gate are constructed based on the combinatorial different switch-on/off machines. The inputs of OR gate are two oligonucleotides, DNA1 and DNA2. It can be seen from Figure 2C that the homogeneous electrochemical OR gate included two switch-on TISD-P/N machines, DNA1-on and DNA2-on. Although the two machines included similar domains that yielded the nicking site and the T30695 G-quadruplex sequence, they differ in the recognition sequences that are complementary to the two inputs, DNA1 and DNA2, respectively. 14


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As can be seen from the experimental results in Figure 2C, in the presence of either DNA1 input (1,0) or DNA2 input (0,1), or both DNA1 input and DNA2 input (1,1), one or both of the two switch-on machines is/are activated, leading to the autonomous synthesis of numerous T30695 G-quadruplex sequences and, thus, to the remarkable decline of IMB. While in the absence of both the inputs (0,0), no T30695 G-quadruplex sequence is produced to bind MB molecules, and thus, the IMB intensity remained high. Therefore, the output will be 1 (∆IGate > 800 nA) when either DNA1 input or DNA2 input is 1. In the opposite case, when neither DNA1 input nor DNA2 input is 1, the output will be 0 (∆IGate < 800 nA). This corresponds to the OR gate. Using a similar approach, as shown in Figure 2D, the homogeneous electrochemical NAND gate is designed by integrating two switch-off TISD-P/N machines, DNA1-off and DNA2-off. The homogeneous electrochemical NAND gate is designed in such a way that prior to its recognition of the DNA1 and DNA2, mute state (0,0), both the DNA1-off machine and DNA2-off machine undergo the autonomous polymerization/nicking circular reaction, yielding a large amount of T30695 G-quadruplexes and thus obvious decrease of the IMB in homogeneous solution is observed. Addition of either DNA1 input (1,0) or DNA2 input (0,1) individually cannot prevent the synthesis of T30695 G-quadruplex sequence and the output ∆IGate of the gate still remains high. Conversely, only when both DNA1 input and DNA2 input are recognized (1,1), no T30695 G-quadruplex sequence is generated to inhibit the output IMB. As shown in Figure 2D, this logic gate offers a true 1 (∆IGate > 800 nA) output in the absence of either input (0,1; 1,0) or in the absence of both input (0,0) 15



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except for the (1,1) input state, in which case the IMB intensity is high (∆IGate < 800 nA), yielding a false 0 output. The resulting truth table reveals the characteristics of a NAND gate.

Figure 3. (A) Schematic illustration of the binary G-quadruplex system; (B) Homogeneous electrochemical responses of (a) MB alone, (b) MB with G4+auDNA, (c) MB with G8+auDNA, (d) MB with auDNA, (e) MB with G8+G4, (f) MB with G8+G4+auDNA in 1× KF polymerase buffer. The concentration of MB is 5 µM. Concentrations for each DNA strand are 2.0 µM.

AND gate and NOR gate The aforementioned homogeneous electrochemical OR gate and NAND gate were designed based on the synthesis of intact G-quadruplex sequence, while the design of AND gate, NOR gate, INHIBIT gate, IMPLICATION gate, XOR gate and XNOR gate in this work took advantage of the concept of binary G-quadruplex. The mechanism of the binary G-quadruplex-based homogeneous electrochemical strategy is schematically depicted in Figure 3A. In this case, the T30695 G-quadruplex sequence is split into two segments, 5′-GGGTGGGTGG-3′ (G8, domain-p) and 5′-GTGGGT-3′ (G4, domain-q), each of which is linked with an anchoring arm, named as domain-m and domain-n respectively. An auxiliary single stranded DNA

16


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(auDNA) is employed to guide the formation of binary G-quadruplex. The auDNA included two functional domains, the domain-n* and domain-m*, which are complementary to the anchoring arm of G4 and G8, respectively. As shown in Figure 3B, the absence of any component of the binary G-quadruplex (curve-b to curve-e) results low IMB decrease. When G8, G4, and auDNA are all presence, the binary G-quadruplex is formed since G8 and G4 get close to each other through their hybridizations with auDNA.53 Thus, the diffusion current of MB is dramatically decreased (curve-f) after its binding to the binary G-quadruplex, indicating that the affinity between MB and the G-quadruplex is largely dependent on the integrity of G-quadruplex structure. With this mechanism in hand, as illustrated in Figure 4A, the homogeneous electrochemical AND gate is achieved through the combination of two interdependent switch-on machines, DNA1 switch-on machine for the synthesis of G8 (DNA1-on@G8) and DNA2 switch-on machine for the synthesis of G4 (DNA2-on@G4), both of which also comprise nicking domain (marked in orange) and the recognition domains (marked in mazarine and wathet for the respective DNA1 input and DNA2 input). Nevertheless, the domains for DNA polymerization are encoded to complementary to the two G-quadruplex subunits, G8 and G4 respectively. Thus, through modulating the generation of two G-quadruplex subunits by the two inputs, DNA1 input and DNA2 input respectively, the homogeneous electrochemical AND gate is constructed. As shown in Figure 4A, when only one of the inputs is 1, DNA1 input or DNA2 input, it activates its corresponding switch-on machine in the 17



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logic gate, leading to the synthesis of only one type G-quadruplex subunit, either G8 or G4, respectively. However, if the other input is absent (1,0 or 0,1), there is no generation of another G-quadruplex subunit, and hence, no binary G-quadruplex is formed, exhibiting low IMB decrease. When treating the system with both inputs (1,1), both the DNA1-on@G8 and DNA2-on@G4 are activated, leading to the autonomous synthesis of G8 and G4 simultaneously. The two G-quadruplex subunits are synergistically stabilized with the aid of auDNA to form the binary G-quadruplex through the formation of the duplexes between the G-quadruplex subunits and the auDNA, and thus the IMB response is greatly inhibited. Obviously, no G-quadruplex subunits are generated in the absence of both input (0,0). Thus, significant decrease of IMB (∆IGate > 800 nA) occurs only when both inputs are recognized, resulting in an AND gate. Likewise, two interdependent switch-off machines, DNA1-off@G8 and DNA2-off@G4, constitute the homogeneous electrochemical NOR gate. Figure 4B depicts the logic NOR gate operation with four combinations of inputs: 0,0; 0,1; 1,0; and 1,1. In the absence of any input (0,0), each machine synthesizes only one of the two G-quadruplex subunits, G8 or G4, respectively. The synthesized two halves of the G-quadruplex then join together and form the binary G-quadruplex upon the auDNA binding. However, any input (DNA1 input or/and DNA2 input) could induce the inactivation of either or both of the two switch-off machines, which prevents the generation of corresponding G-quadruplex subunit and, thus, the formation of binary G-quadruplex. Therefore, it produces an output of 1 (∆IGate > 800 nA) only if both 18


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inputs are 0. This is in accordance with a NOR logic gate behavior, which is expressed more clearly by the derived truth table.

Figure 4. Schematic presentation, DPV responses, bar presentation of output signals, and truth table of HEC AND gate (A), NOR gate (B), INHIBIT gate (C), IMPLICATION gate (D), XOR gate (E), and XNOR gate (F). Concentrations for each TISD-P/N machine in AND gate, NOR gate, and INHIBIT gate are all 2.5 nM. Concentrations for each switch-on TISD-P/N machine and switch-off TISD-P/N machine in IMPLICATION gate are 2.5 nM and 5.0 nM, respectively. Concentrations for each TISD-P/N machine in XOR gate and XNOR gate are all 1.25 nM. auDNA keeps at the concentration of 2.0 µM in all binary G-quadruplex-based logic gates. 19



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INHIBIT gate and IMPLICATION gate Figure 4C depicts the organization of the INHIBIT gate. The construction of the INHIBIT logic gate is achieved by means of assembling, as an example, DNA1-on@G8 and DNA2-off@G4. It is found that only introduction of DNA1 input (1,0) leads to a great decrease in IMB intensity (∆IGate > 800 nA) owing to the synchronous generation of both G-quadruplex subunits, G8 and G4, whereas the other three situations (0,0; 0,1; and 1,1), that lead to the absence of either G8 subunit (0,0) or G4 subunit (1,1), or even both G8 subunit and G4 subunit (0,1), only induce slight IMB decrement (∆IGate < 800 nA), which is consistent with a characteristic two-input INHIBIT gate behavior. As a result of the negation of INHIBIT gate operation, IMPLICATION gate is also an important two-input Boolean logic operation, which reads one input implies the other and is equivalent to the IF-THEN operation and the NOT operation.56 As shown in Figure 4D, the deployment of DNA1-off, DNA1-on@G8, and DNA2-on@G4 enabled the design of IMPLICATION gate. It is notable that, in the presence of both switch-on machine and switch-off machine, the 3’-end of switch-on machine should be phosphorylated (Table S-11) to avoid its elongation on the opened switch-off machine, eliminating the undesired output signal. As shown in Figure 4D, with no input (0,0) or with DNA2 input alone (0,1), a large amount of intact T30695 G-quadruplexes (and G4 subunits) could be generated and thus the system displayed low IMB intensity with an output of 1 (∆IGate > 800 nA). With DNA1 input alone (1,0), the stem-loop structure of the DNA1-off is disrupted, and thus the synthesis of intact 20


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T30695 G-quadruplexes is prevented. Although the DNA1-on@G8 is activated at the same time, the synthesis of G8 subunits cannot lead to the significant IMB decrement (∆IGate < 800 nA), giving an output of 0. When the system is subjected to the two inputs together (1,1), both the DNA1-on@G8 and DNA2-on@G4 are activated, promoting the formation of numerous binary G-quadruplexes, and the output signal is 1. From Figure 4D, it can be seen that the Boolean function of the system provides the output of 1 in all circumstances (0,0; 0,1; 1,1), except the case only DNA1 input is 1 (1,0). This input/output behavior yielded in a two-input IMPLICATION logic gate. XOR gate and XNOR gate To further demonstrate the versatility of homogeneous electrochemical TISD-P/N machines in the construction of biomolecular logic gates, we devote our ongoing effort on the assembly of challenging XOR gate, which have been previously proven to be quite difficult to achieve at the molecular level.57 The homogeneous electrochemical XOR gate is constructed by a more complicated design (Figure 4E), in which DNA1-on@G8, DNA2-on@G8, DNA1-off@G4, and DNA2-off@G4 are rationally deployed together. As shown in Figure 4E, in the presence of either DNA1 input (1,0) or DNA2 input (0,1), the two G-quadruplex subunits, G8 and G4, can be generated simultaneously, leading to the formation of binary G-quadruplex. However, the introduction of neither DNA1 input nor DNA2 input (0,0) only leads to the generation of G4 subunits. Similarly, the introduction of both DNA1 input and DNA2 input (1,1) only activates the generation of G8 subunits. Thus, the formation of binary G-quadruplex is impossible in the absence or presence of both DNA1 input and DNA2 21



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input. The XOR gate exhibit high IMB intensity (∆IGate < 800 nA) only if both of the two input values are true (1,1) or false (0,0). However, the other input combinations (1,0 and 0,1) sharply weakened the IMB signal (∆IGate > 800 nA). These results demonstrate that the system indeed performs the XOR gate operation with a characteristic truth table. Finally, a homogeneous electrochemical XNOR gate operation is also successfully realized using the assembled TISD-P/N machines activated by the two inputs, DNA1 input and DNA2 input, as shown in Figure 4F. The homogeneous electrochemical XNOR gate is designed starting with the integration of DNA1-on@G8, DNA2-off@G8, DNA1-off@G4, and DNA2-on@G4. The IMB intensity of the XNOR gate will drop down to a low level (∆IGate > 800 nA) only in the presence of neither DNA1 input nor DNA2 input (0,0) or both DNA1 input and DNA2 input (1,1). These logic truth outputs result from the simultaneous generation of G8 and G4. In contrast, no binary G-quadruplex is formed in the presence of either DNA1 input (1,0) or DNA2 input (0,1), leading to low IMB decrement (∆IGate < 800 nA). From the truth table, we can see that an output of 0 is obtained if and only if one of the inputs to the gate is 1; if both inputs are 0 or both are 1, an output of 1 is obtained. Evidently, this system operates as an XNOR logic gate.

CONCLUSIONS In summary, we have developed a novel label-free homogeneous electrochemical platform based on TISD-P/N-based DNA machineries. The activation or inhibition of the DNA polymerization/nicking cyclic reactions on these switch-on or switch-off 22


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machines by target/input controls the synthesis of numerous output sequences, intact G-quadruplex sequences or binary G-quadruplex subunits in this work, which provide the amplified homogeneous electrochemical readout signal. The main motivations of this study are focused on establishing a ultrasensitive and label-free electrochemical bio-sensing strategy without any immobilization/modification on electrode surface, but also on realizing a complete set of two-input DNA-based Boolean logic gates, including OR, NAND, AND, NOR, INHIBIT, IMPLICATION, XOR, and XNOR. As we know, this is the first report on the development of a logic gate system based on homogeneous electrochemical platform, which is expected to provide researchers with a simple and modular design basis for constructing complex logic gate operations at the molecular level just through rationally deploying the designed basic work units without any complicated modulation or modification. Additionally, the input and output of these logic gates is all single-stranded DNA except the last homogeneous electrochemical readout signal, which may enable them to find applications in further development of cascaded multi-level logic circuits that encompass more-complex functions. Moreover, homogeneous electrochemical transducers provide a platform that can be easily interfaced with electronics for scale-up, miniaturized, and multiplexed bio-sensing and bio-computation. Thus, this contribution will open new opportunities for the development and design of intelligent molecular computation. ASSOCIATED CONTENT Supporting Information Sequences of oligonucleotides used in this work (Table S-1 to S-13), nondenaturating 23



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PAGE analysis of TISD-P/N-based machineries (Figure S-1 to S-4). This material is available free of charge via the Internet at http://pubs.acs.org. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was financially supported by National Natural Science Foundation of China (21545005, 31501570, 21375072), Natural Science Foundation of Shandong Province, China (ZR2014BQ011), Research Foundation for Distinguished Scholars of Qingdao Agricultural University (663-1115003, 663-1113311), and the Special Foundation for Taishan Scholar of Shandong Province (ts201511052).

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