Nonlinear Regulation of Enzyme-free DNA Circuitry with

molecular mechanism is particularly important in large-scale CRNs with complex ... opportunities for further development in molecular diagnostics,2 sm...
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Nonlinear Regulation of Enzyme-free DNA Circuitry with Ultrasensitive Switches Wei Lai, Xiewei Xiong, Fei Wang, Qian Li, Li Li, Chunhai Fan, and Hao Pei ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00208 • Publication Date (Web): 28 Aug 2019 Downloaded from pubs.acs.org on August 28, 2019

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Nonlinear Regulation of Enzyme-free DNA Circuitry with Ultrasensitive Switches Wei Lai,1 Xiewei Xiong,1 Fei Wang,2 Qian Li,2 Li Li,1 Chunhai Fan2 and Hao Pei1* 1Shanghai

Key Laboratory of Green Chemistry and Chemical Processes, School of

Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai, 200241, P. R. China 2School

of Chemistry and Chemical Engineering, and Institute of Molecular Medicine,

Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China *Correspondence:

[email protected] (H. Pei)

Abstract DNA is used to construct synthetic chemical reaction networks (CRNs), such as inorganic oscillators and gene regulatory networks. Nonlinear regulation with simpler molecular mechanism is particularly important in large-scale CRNs with complex dynamics, such as bistability, adaptation and oscillation of cellular functions. Here, we introduce a new approach based on ultrasensitive switches as modular regulatory element to nonlinearly regulate DNA-based CRNs. The nonlinear behavior of the systems can be finely tuned by programmable regulation of the linker length and the ligand binding sites, of which the Hill coefficients (nH) are in the range of 1.00~2.32. By integrating two different strand displacement reactions with low-order nonlinearities (nH=~1.44 and~1.54), we could construct a CRNs with high-order nonlinearities of that the Hill coefficient up to ~2.70. In addition, it could provide an efficient approach for designing CRNs at will with complex chemical dynamics by incorporating our design with previously developed enzyme-free DNA circuits. 1

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KEYWORDS: Nonlinear regulation, Hill coefficient, ultrasensitive switch, chemical reaction networks Living organisms exploit intricate chemical reaction networks (CRNs) to perform highly sophisticated tasks such as self-organization and navigation.1 The development of synthetic CRNs with such information-processing functions could open opportunities for further development in molecular diagnostics,2 smart therapeutics,3,4 active separation,5 and adaptive materials.6 Enzyme-free DNA reaction systems are a highly attractive substrate for molecular programming due to their predictable noncovalent interactions that are facilitated by the constrained chemical diversity and Watson-Crick hydrogen-bonding rules.7 Beyond static binding, toehold switches allow the occurring of strand displacement reactions (SDRs) through a branch migration (BM).8-10 Nucleic acid circuitry is composed of multiple SDRs that interact via massaction kinetic processes to achieve desired functions.4,11,12 The termed dynamic DNA nanotechnology has been actively exploited to develop intelligent nano-devices,13-15 image processing,16 gene regulation,17 Boolean logic circuits,18 and neuron-like computing.19 However, the scope of dynamic behaviors established by DNA-only reaction networks has not yet approached the sophistication and complexity of living systems.20 This is because DNA displacement reactions poorly approximate sharp transitions as their rate changes linearly with reagents, whereas biological circuits exhibit sharper nonlinearities.21 Recently, researchers have devoted to build nonlinear dynamical nucleic acid circuitry to systematically implement formal CRNs, which could simulate complex mathematical models and derive analog functions.22-26 For example, Sawlekar et al used three elementary abstract idealized reactions, including catalysis, annihilation, and degradation, to theoretically realize the ultrasensitive functions.27 Montefusco et al proposed a multistep signaling strategy combined with a negative feedback to simulate yeast osmoregulatory response network.28 Ultrasensitive switches are critical components in eukaryotic cellular signaling networks that produce high-order dynamical functions,29 such as oscillation, 2

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amplification, and toggling behavior.30,31 These switches provide a nonlinear input/output (I/O) control for reaction networks, which small input change gives large change in output behavior.32 In living cells, ultrasensitive switches connect upstream and downstream components into a bigger network.33,34 The nonlinearity of such cascaded networks are much larger than that of the isolated reactions. Inspired by this mechanism, we herein experimentally demonstrate a simple and modular strategy with ultrasensitive I/O control for nonlinear regulation of DNA-only CRNs, which could provide a new approach to construct artificial CRNs with complex behavior.

Figure 1. Design principle of nonlinear I/O controller based on cooperative allosteric strategy. Potential steps in the evolution of a nonlinear I/O controller includes: 1) an unregulated I/O controller produces constant output signaling; 2) an allosteric I/O controller with single binding site produces linear output signaling; 3) a cooperative allosteric I/O controller with two binding sites produces nonlinear output signaling.

In principle, an ultrasensitive switch with the nonlinear I/O behavior could be built using multiple cooperative allosteric transitions (Figure 1). Whereas an allosteric I/O controller with single binding site would produce linear output signaling,35 a cooperative allosteric I/O controller with multiple binding sites might generate nonlinear output signaling. The proposed mechanism in cooperative processes is that the occurring of one binding event energetically increases the favorability of the 3

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subsequent binding events, which is analogous to that used by biological cooperative signaling proteins such as hemoglobin.36

RESULTS AND DISCUSSION Construction of allosteric cooperative DNA I/O controllers with nonlinear regulation. Our approach to the rational design of DNA I/O controllers with nonlinear signal-processing behavior involves the introduction of a cooperative allosteric DNA switch as modular regulatory element for regulating the kinetics of toehold-mediated SDRs.37 We designed a series of DNA switches with multiple binding sites (cocaine aptamer, DNA-based receptor, Figure S4a) that connect toehold and BM domain with two termini. Their individual states are responsive to the ligands with nonlinear behavior due to cooperativity. In the initial state, the DNA switches maintain a disorder conformation which is unfavorable for the substrate (S) to react with input strand (I). When inputting ligands, the conformational motion of these switches occurs upon the first ligand binding event facilitating the pre-organization of the second receptor architectures. This improves the affinity of the subsequent binding events, thus resulting in a cooperative allosteric folding (Figure S1, SI).38 Molecular switches use such modular cooperative mechanism, which would change the distance between toehold and BM domain, could provide a convenient means to nonlinearly regulate the kinetics of the downstream SDRs. Here, we introduced two key parameters to describe the nonlinearities of the DNA I/O controllers, including Hill coefficient and dynamic range. Especially, Hill coefficient (nH) is used for quantifying the cooperative nonlinearities,39 and dynamic range reflects the steeper dependence on concentration (Part II of SI). As shown in Figure 2, we designed three kinds of DNA I/O controllers, whose architectures contain zero, one, and two ligand binding sites, respectively. Initially, the reaction starts with hybridization of the two toehold domains, follows by a reaction limited internal diffusion step (controlled by the molecular switches) and undergoes the branch migration process, then the output strand (O) is released (Figure S5). In the case of unregulated controller that has no ligand binding 4

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site, the SDRs produce a virtually unchanging output strands (O) and the rate constants (k) show no response to ligand concentrations (k = 2.8 × 105 M-1·s-1). In the case of allosteric controller that has one ligand binding site, the O yield and k of the SDRs show a linear response to the ligand concentration; while the cooperative controller that has two ligand binding sites generates nonlinear output signaling as evidenced by the sigmoid growth profile of the O yield or the k constants (The rate constant fitting is modelled as instantaneous, second-order process, see Part II of SI). The SDRs with three kinds of DNA controllers exhibited a nH of 0, 1.01 ± 0.02, and 1.57 ± 0.07, respectively. Together these parameters describe our cooperative DNA I/O controllers.

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Figure 2. Construction of a DNA I/O controller. (a) Schematic illustration of three kinds of DNA I/O controllers, whose architectures contain zero, one, and two ligand binding sites, respectively. (b) The yield of output (O) of SDRs using different controllers in response to ligands. (c) The rate constant (k) of SDRs using different controllers in response to ligands. The O yield and k were calculated from the fluorescence results in Figure S5. [S] is 10 nM and [I] is 50 nM in this experiment. The DNA I/O controller was labeled with a fluorophore-quencher pair (Cy3-BHQ2) for tracking the reaction process.

Fine-tuning of the nonlinearities of DNA I/O controller. Next, we demonstrate that fine-tuning of the nonlinearities of DNA I/O controllers can be realized by programming the linker domain that connects binding sites (T-T mismatch, Figure S4b). 6

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In the presence of ligand (Hg2+), the DNA switch form into a hairpin structure through a form stable T-Hg2+-T base pairs. First, a series of cooperative allosteric S with different linker lengths were constructed. As shown in Figure 3a, we designed four kinds of DNA I/O controllers with linker length varying from 10 to 40 nt (SL-10, SL20, SL-30, and SL-40). Then, we studied the nonlinear I/O behavior with different linker lengths by calculating rate constant (k) from the fluorescence curves (Figure S6). It is clear from Figure 3b that SDR generates nonlinear output signaling as evidenced by the sigmoid growth profile of the rate constants. It is worth noting that the ultrasensitive response region shifts to a higher ligand concentration with increasing the linker length, and the corresponding k’s range changes from 1.13 × 105 M-1·s-1 ~ 1.19 × 106 M-1·s-1 to 8.35 × 104 M-1·s-1 ~ 1.03 × 106 M-1·s-1. This reduces the dynamic range from 49-fold to just 26-fold (Figure 3c), significantly increasing sensitivity to small changes in ligand concentration. The nonlinear I/O behavior was further characterized using cooperativity (nH). As displayed in Figure 3c, nH increases from 1.08 ± 0.02 to 1.44 ± 0.04 accompanying with dynamic range decreasing from 49 to 26 when the linker length increases from 10 to 40 nt. In addition, the number of binding site also contributes to the nonlinearities of DNA I/O controller. Here, we engineered five kinds of DNA I/O controller, in which the number of binding sites was set as 2 (SB-2), 3 (SB-3), 4 (SB-4), 5 (SB-5), and 6 (SB6), as illustrated in Figure 3d. The effect of the number of binding site on the signal’s nonlinearities of DNA controllers were studied by calculating the rate constant (Figure S7). Figure 3e plots the rate constant of SDR vs. the ligand concentration, which shows a sigmoid-growth profile with increasing the ligand concentration. Moreover, the ultrasensitive response region shifts to a higher ligand concentration due to lowered affinity for ligand when increasing the binding site from 2 to 6. Subsequently, we investigated the nonlinear I/O behavior by calculating nH and dynamic range (Figure 3f). It should be noted that nH reaches a maximum value (2.42 ± 0.07) and dynamic range reaches a minimum value (6-fold decrease from SB-2 to SB-5) in the case of SB-5; which is likely due to lengthened diffusion distance for BM process. Further 7

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increasing the binding site to 6 (SB-6) leads to a substantial decrease in nH, indicating that the switch becomes difficult to activate due to increased individual domain affinities. Taken together, by modulating the linker length and ligand binding site, we can thus finely tune the nonlinear I/O behavior in terms of both the degree of cooperativity and dynamic range.

Figure 3. Fine regulation of nonlinear I/O behavior of SDRs with DNA controllers. The DNA switch was constructed through the high specificity between the ligand (Hg2+) and T-T mismatches. (a) Fine-tuning of the nonlinear I/O behavior of SDRs by adjusting the length of linker in DNA controllers (from 10 to 40 nt, corresponding to SL-10, SL-20, SL-30, and SL-40). (b) Effect of linker length on the nonlinear behaviors of the ligand concentration-dependent kinetics. The nonlinear response region (marked by the shaded areas, 10-90% of the maximum response) shifts to high ligand concentration when increasing the linker length. (c) Effect of linker length on nH and dynamic range. The nH increases and dynamic range decreases with increasing the linker length. (d) Fine-tuning of the nonlinear I/O behavior of SDRs by adjusting the ligand binding site (from 2 to 6, corresponding to SB-2, SB-3, SB-4, SB-5, and SB-6). (e) Effect of the binding site on the nonlinear behaviors of the ligand concentration-dependent rate constant (k). The nonlinear response region (marked by the shaded areas, 10-90% of the maximum response) shifts to the high ligand 8

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concentration when increasing the binding sites. And, the nonlinear I/O behavior for 5 binding sites (SB-5) is the most significant with k ranging from 7.45 × 104 M-1·s-1 to 3.79 × 105 M-1·s-1. (f) Effect of the binding site on the nH and dynamic range. The nH increases and dynamic range decreases by increasing the binding site from 1 to 5. Note that a turning point occurs for SB-5. [S] is 10 nM and [I] is 50 nM in this experiment.

Reversibly Dynamic regulation of the nonlinearities of DNA I/O controller. Our designed DNA I/O controller can realize reversible switch by introducing a specific acceptor to compete binding the ligand with DNA modular regulatory element. As schematically illustrated in Figure 4a and 4b, the key step is to design the core component of the nonlinear I/O controller-cooperative allosteric center. Here, we employed L-cysteine (Cys) as a specific acceptor, which can preferably binds ligand (Hg2+). The addition of ligand triggers the conformational motion of S, resulting in a cooperative allosteric folding from a disorder conformation (S) into a hairpin conformation (S-Dual ligand). Since rates of the reaction between Cys and ligands are fast, this process can be reversed by adding Cys, which releases ligand through cysteine complexation (dotted box in Figure 4b) and unfolds S-Dual ligand back to disorder conformation (S). This reversible ligand loading/release process was monitored by time-course reaction rate measurement with alternating addition of Cys and Hg2+. Figure 4c plots the reaction rate derived from the fluorescence tracks shown in Figure S8. Initially, the reaction pool contains S and I, leading to a modest reaction rate. When we added the ligand at 5 min, the reaction rate increased instantly to 0.20 nM∙s-1 and then decayed over time. On the other hand, the reaction rate is kept at a low value after adding Cys at 12 min. In the presence of ligand, the cooperative S folds into the active conformation (S-Dual ligand) by binding to ligands, which is unfavorable for the downstream SDR and results in a fast reaction rate. In the presence of Cys, the ligand is extracted from the modular regulatory element to bind with the acceptor, resulting in a slow reaction rate. Interestingly, temporal cycling of the nonlinear networks can be achieved by repeating the above operation multiple times. Note that the reaction rate gradually decreases with increasing number of cycles. This is likely due to some 9

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irreversible ligand binding in the DNA I/O controller, which was similarly observed in complexation between pyrene-thymine and mercury ions.40 Control experiment have also been conducted by adding buffer without Cys.41

Figure 4. Dynamic reversible regulation of nonlinear I/O behavior of SDR with DNA controller. (a) The schematic illustration of reversible regulation of nonlinear I/O controller. The key step is to design the core component of the nonlinear I/O controller-cooperative allosteric center (black box). (b) Reversible regulation of nonlinear I/O DNA controller. We introduced a specific acceptor (L-cysteine, Cys) to extract Hg2+ ion from T-Hg2+-T of S through a Ligand-Cys process (dotted box). (c) Dynamic reversible regulation of nonlinear I/O reaction rate with DNA controller. In the presence of ligand, the cooperative S folds into the active conformation (S-Dual ligand) by binding to ligands, leading to a fast reaction rate. In the presence of Cys, the ligand is extracted from the modular regulatory element to bind with Cys, resulting in a slow reaction rate. Here, the ligand 10

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loading/release to/from the hairpin structure is monitored by time-course reaction rate measurement obtained in a solution of 100 nM S and 20 nM I with the addition of ligand (12.5 nM) and Cys (20 nM). Notably, Cys were not added in the control experiment.

Figure 5. Nonlinear regulation of DNA-CRN. (a) Simplified schematic diagram of a nonlinear DNA-CRN. The DNA-CRN consists of two inputs (I1 and I2) and a complex substrate (CS); the conformation of CS is regulated by binding ligands. (b) Illustration of DNA-CRN. Node 1 and 2 represent the cooperative binding of ligand 1 (L-1) and ligand 2 (L-2), respectively. Node 3 to 6 are different nonlinear CRNs by controlling combinations of ligands. (c) Effect of the combination of the ligand on the nonlinear I/O behavior of CRN. L-1, L-2, and Dual are represented in the presence of only L-1 or L-2, both L-1 and L-2, respectively. The rate constant (k) was derived from the fluorescence response shown in Figure S9. Here, the concentration of both [I1] and [I2] was 10 nM, [CS] was 50 nM, the amount of ligands was set to be equal when adding different ligands. The CS was labeled with a fluorophore-quencher pair (Cy3-BHQ2) for tracking the reaction process. 11

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Higher-orders of nonlinearities of DNA-CRN. Multiple individual different SDRs with our strategy could be integrated into CRNs with chemical connection that exhibiting high-order nonlinearly dynamic behavior. As a proof of concept, a DNACRN was constructed by cascading two different SDRs with cooperative allosteric module (Figure 5a). In the presence of dual ligands (L-1 and L-2), the DNA-CRN can cooperatively release the output product (O1 and O2) and result in an increase of fluorescence; in the presence of single ligand (L-1 or L-2), the DNA-CRN was weakly cooperative as it was regulated by the ligand in a single reaction step; whereas in the absence of any ligand, DNA-CRN gives linear signaling with a constant reaction rate. With this design, we can choose the desired combination of ligands to achieve higherorder nonlinear behaviors of DNA-CRNs. Figure 5b gives the potential combination of ligands used to construct different nonlinear CRNs. In the absence of L-1 and L-2, DNA-CRN does not response to ligands with a rate constant of 18700 M-1∙s-1. Therefore, single and dual ligand were chosen to study the higher-order nonlinear behaviors of DNA-CRN, as plotted in Figure 5c. In the presence of L-1 or L-2, DNA-CRN’s rate constant shows a nonlinear I/O behavior with increasing the ligand concentration, of which the Hill coefficient (nH) is 1.44 ± 0.05 and 1.54 ± 0.04, respectively. It should be noted that the nonlinear I/O behavior is the most significant for the dual ligand (Dual in Figure 5c) with a more positive cooperativity (nH = 2.70 ± 0.12). The ligand concentration-dependent rate constant displays a typical sigmoid-growth profile. These results demonstrate the feasibility of our strategy in building complex DNA-CRNs with higher-order nonlinear behaviors.

CONCLUSION In summary, we have developed a nonlinear DNA I/O controller, which provides several insights to use DNA as possible molecules for synthetic CRNs with complex dynamical behavior. Notably, our proposed allosteric cooperativity strategy shows a richer and finely means of regulation by programming the DNA sequences, especially 12

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combining with the downstream could produce substantial changes to the ultrasensitive features of a sigmodal module and capable of building a highly ultrasensitive device, from an original asymmetrical and modestly ultrasensitive module (Part IV, Table S2).42 First, the nonlinear I/O behavior of cooperative allosteric DNA controller can be finely tuned by programmable regulation of the linker length and the ligand binding sites. Second, the dynamic reversible regulation of DNA controller was achieved by introducing a competitor to extract the ligand from the DNA-ligand complexes. Third, CRNs with high-order nonlinearities could be constructed by integrating multiple individual different SDRs with chemical connection. Fourth, by incorporating our design with existing design of DNA circuits, it could provide an efficient approach for designing CRNs with more complex chemical dynamics, such as DNA-based controller or mathematical operators especially logarithm.41,43,44 Lastly, the programmed CRNs with our method could exhibit dynamics at will that responses to ligands at different levels of complexity, which might open opportunities for smart molecular diagnostics and therapeutics.

MATERIALS AND METHODS DNA oligonucleotides. DNA oligonucleotides sequences were designed using NUPACK online. Temperature and salt concentrations were chosen to represent reaction conditions. DNA strands were designed to minimize secondary structures. All DNA sequences were then analyzed using NUPACK to ensure minimal crosstalk between unrelated domains. Sequences for all experiments are listed in Supplementary Table 1. DNA oligonucleotides were acquired from Sangon Biotechnology Co., Ltd. (Shanghai, China) and purified by high-performance liquid chromatography (HPLC). Samples were dispersed in 1× TE buffer to a concentration of 100 μΜ, using the manufacturer’s information on the amount of oligo per tube. For the needs of experiments, samples were diluted to 5 μΜ using 1× TE-Mg buffer. Samples were stored at 4 °C. We chose Cy3 as fluorescent labels for our experiments, with BHQ2 serving as quenchers. All reagent-grade chemicals, including Tris HCl and MgCl2 (all 13

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from Sinopharm Chemical Reagent Co., Ltd.), were used as received. L-cysteine was purchased from Sinopharm Chemical Reagent Co., Ltd. Cocaine was achieved from the National Institutes for Food and Drug Control. Nanopure H2O (18.2 MΩ∙cm), purified using an Ultrapure Milli-Q water system, was used for all experiments. Buffer conditions. The buffer for all experiments was TE (10 mM Tris·HCl, pH 8.0, 1 mM EDTA), with 12.5 mM MgCl2 added. Because EDTA chelates magnesium ions, the effective concentration of Mg2+ is 11.5 mM. All experiments and purifications were performed at 25 °C. Annealing. All annealing processes were performed using a LongGene A300 Fast Thermal Cycler. The samples (typically at a final duplex concentration of 5 μΜ) were heated to 95 °C for 5 min and then gradually cooled to room temperature at a constant rate over a period of 2 h. Design rationale for the reporter system. Use of a reporter complex to measure strand displacement greatly increases the range of systems that can be experimentally tested without the need to prepare any further labeled oligonucleotides. This reporter (substrate S) consists of two strands initially bound to each other, one labeled with a fluorophore, the other with a quencher. Due to the spatial proximity of fluorophore and quencher, the fluorescence is suppressed. The free incumbent can displace the fluorescently labeled strand. Once incumbent strand has displaced the fluorescently labeled strand, the fluorophore is no longer quenched: the change in bulk fluorescence can be used to track the progress of the overall reaction. Characterization of cooperative allosteric chemical reaction networks using spectrofluorimetry. All spectrofluorometric measurements were performed at 25 °C with a Hitachi F-7000 fluorometer. For a typical allosteric chemical reaction, the reaction mixture contained 10 nM S, varying input concentrations and ligand in TE-Mg buffer. In addition, the ratio between [S] and [I] should be fixed for adapting other reaction systems. All reaction mixtures were prepared at a final volume of 1 mL in a 14

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quartz cuvette. Fluorescence was monitored in real-time at a frequency of 1 data per 5 second with excitation/emission wavelength at 550 nm/562 nm (Cy3). Fluorescence traces in all figures were normalized so that 1 normalized unit (a.u.) of fluorescence corresponded to fluorescence signal generated by 1 nM output (O). Dynamic reversible regulation of nonlinear behavior by introducing a Cys. The reaction solution contained 10 nM S in TE-Mg2+ buffer was transferred into a quartz cuvette. The fluorescence was then recorded every 5 s for 100 s. Input strand (I) and ligand (Hg2+) were then quickly added into the reaction mixture at a final concentration of 5 nM. The fluorescence was then measured every 5 s for another 500 s. After 500 s, L-cysteine (Cys) was then added at a final concentration of 10 nM and the fluorescence was recorded for another 500 s. The cycles for adding ligand and Cys were repeated for several times. Construction of nonlinear I/O chemical reaction network. The reaction solution, consisted of 50 nM CS and various combinations of ligands (none, L-1, L-2, and dual ligand), were transferred into a quartz cuvette. Input strands (I1 and I2) were then quickly added into the reaction mixture at a final concentration of 10 nM. In addition, CS labeled with a fluorophore-quencher pair (CY3-BHQ2) was used to track the reaction process. The fluorescence was then recorded every 5 s for 3600 s.

SUPPORTING INFORMATION DNA sequences, calculation of the Hill coefficient, rate constant, and fluorescence spectra of different systems.

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

AUTHOR CONTRIBUTIONS 15

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H. Pei conceived and supervised the research. W Lai designed the experiments. W. Lai, X. Xiong performed most of the experiments and data analysis. W. Lai, X. Xiong, and F. Wang participated in various aspects of the experiments and discussions. W. Lai, H. Pei, L. Li, Q. Li, and C. Fan co-wrote the paper. All authors discussed the results and commented on the manuscript.

ACKNOWLEDGEMENTS Funding Sources This work was supported by the National Science Foundation of China (Grant No. 21722502), Shanghai Rising-Star Program (19QA1403000), and Shanghai Science and Technology Committee (STCSM) (Grant No. 18490740500).

DECLARATION OF INTERESTS The authors declare no competing financial interests.

REFERENCES (1) Van Roekel, H. W.; Rosier, B. J.; Meijer, L. H.; Hilbers, P. A.; Markvoort, A. J.; Huck, W. T.; de Greef, T. F. (2015) Programmable chemical reaction networks: Emulating regulatory functions in living cells using a bottom-up approach, Chem. Soc. Rev. 44, 7465-7483. (2) Jung, C.; Ellington, A. D. (2014) Diagnostic applications of nucleic acid circuits, Acc. Chem. Res. 47, 1825-1835. (3) Benenson, Y.; Gil, B.; Ben-Dor, U.; Adar, R.; Shapiro, E. (2004) An autonomous molecular computer for logical control of gene expression, Nature 429, 423-429. (4) Zhang, D. Y.; Seelig, G. (2011) Dynamic DNA nanotechnology using strand-displacement reactions, Nat. Chem. 3, 103-113. (5) Picuri, J. M.; Frezza, B. M.; Ghadiri, M. R. (2009) Universal translators for nucleic acid diagnosis, 16

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