On Switchâ in the Toehold-Mediated Strand

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What Controls the “off/on switch” in the Toehold-mediated Strand Displacement Reaction on DNA Conjugated Gold Nanoparticles? Dongbao Yao, Bei Wang, Shiyan Xiao, Tingjie Song, Fujian Huang, and Haojun Liang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b01671 • Publication Date (Web): 09 Jun 2015 Downloaded from http://pubs.acs.org on June 14, 2015

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What Controls the “off/on switch” in the Toehold-mediated Strand Displacement Reaction on DNA Conjugated Gold Nanoparticles? Dongbao Yao,† Bei Wang,† Shiyan Xiao,∗,† Tingjie Song,† Fujian Huang,† and Haojun Liang∗,‡,¶ CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China., CAS Key Laboratory of Soft Matter Chemistry, iChEM, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China., and Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, Anhui, 230026, P. R. China. E-mail: [email protected]; [email protected]

Phone: +86 (0)551 63607824. Fax: +86 (0)551 63607824

∗ To

whom correspondence should be addressed Key Laboratory of Soft Matter Chemistry ‡ CAS Key Laboratory of Soft Matter Chemistry, iChEM ¶ Hefei National Laboratory for Physical Sciences at Microscale † CAS

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Abstract In DNA dynamic nanotechnology, toehold-mediated DNA strand-displacement reaction has demonstrated its capability in building complex autonomous system. In most cases, the reaction is performed in pure DNA solution that is essentially a one-phase system. In the present work, we systematically investigated the reaction in a heterogeneous media, in which the strand that implements displacing action is conjugated on gold nanoparticle. By monitoring the kinetics of spherical nucleic acid (SNA) assembly driven by toehold-mediated strand displacement reaction, we observed significant differences, abrupt jump in behavior of an “off/on” switch, in reaction rate when invading toehold was prolonged to eight bases from seven bases. These phenomena are attributed to the effect of steric hinderance arising from high density of invading strand conjugated to AuNPs. Based on these studies, an INHIBIT logic gate presenting good selectivity was developed.


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Introduction Since the pioneering works by Mirkin, 1 Alivisatos and Schultz 2 published in 1996, polyvalent nucleic acid nanoparticle conjugates, especially spherical gold nanoparticles (AuNPs) with densely functionalized and highly oriented oligonucleotides (DNA) covalently tethered to their surfaces, have been extensively utilized for manufacturing nanomaterials with well-defined structures and functions. Given the novel physical and chemical characteristics of inorganic metal NPs, such as their optical, catalytic, plasmonic, scattering, and quenching properties, and the powerful programmable capability of nucleic acids, 3 DNA-functionalized AuNPs (DNA-AuNPs) have been applied in various fields, including biodiagnostics, medicine, and biosensors in terms of colorimetricbased detection, 4 electronic-based detection, 5 scanometric-based detection, 6 Raman-based detection, 7 intracellular gene regulation, 8 and so forth. DNA-AuNPs were recently used as basic building blocks for assembling superlattices, 9–11 an undertaking that opens a door for rational designs of general periodic structures of NPs. 12 These impressive advances in nanotechnology can be attributed to a “programmed assembly” strategy 1 that uses complementary DNA strands as linkers to direct the self-assembly of DNA-AuNPs; and such a strategy provides the possibility of controlling the fabrication of DNA-AuNPs in terms of the DNA length, sequence, and structure of the DNA, and the size and composition of the NPs. 3,13 In most previous works on the the assembly of DNA-AuNPs, the “direct-linker-addition” strategy was employed, in which DNA-AuNPs and oligonucleotide linkers were simultaneously introduced to a reaction system to obtain DNA-linked NP aggregates through thermal annealing. By combining the structural aspect of DNA-AuNPs and the dynamics aspect of DNA nanotechnology, 14–23 Song et al. recently introduced the concept of DNA-fueled molecular machines 14–16 to the DNA-AuNP assembly whereby DNA-AuNP assembly into explicit geometrical structures is triggered and mediated via a series of strand displacement reactions 24,25 with the assistance of toehold domain (i.e., a short single-stranded overhang region). This new strategy provides the opportunities for programming NPs to self-assembly into lattice or engineering specific self-assembly reactions or structural changes 23 through rational design and precise control of isothermal dynamic 3 ACS Paragon Plus Environment

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DNA-fueled molecular machines, and could potentially be applied in the biomedical technology. 26 Besides, toehold-mediated strand displacement reaction has been utilized to improve the selectivity and sensitivity of DNA sensing, such as the graphene oxide (GO)-based FRET assays 27,28 or quartz crystal microbalance (QCM)-based biosensor. 29 The ability for DNA strands from solution anchored to AuNPs to hybridize to their complementary oligonucleotides in a robust and efficient manner is crucial to AuNPs assembly, 30 and DNA hybridization occurring at interfacial environments differs greatly from the solution-phase hybridization. 31–33 Proceeding of this process needs the DNA target from solution to first penetrate into the dense layer of oligonucleotide strand, implying the crucial role of properties of oligonucleotide layer in the DNA hybridization on AuNP surfaces. Thermodynamic studies performed by Lytton-Jean and Mirkin demonstrate that nanoparticle probes with spherical nucleic acid (SNA) have a higher sensitivity of assays compared with their molecular counterparts. 34 Further experimental and theoretical analyses 30 reveal that SNA binding and hybridization are regulated by electrostatic effects (enthalpic penalty) and collective conformational changes (entropic penalty) of the dense monolayer, and each binding event destabilizes subsequent binding events. The efficiency of duplex formation drops from 100% to 10% as the probe density on the planar gold surface increased from ∼ 3 × 1012 to ∼ 5 × 1012 molecules/cm2 . 35 Takeda et al. have shown that the hybridization rate of bulky probes having the complementary sequence at the center of the target ssDNA was found to be smaller than that of bulky probes having the complementary sequence at the terminus. 36 The steric effect on the hybridization was also confirmed by Liu et al., whose experiments demonstrate that introducing overhangs to the linker DNA in the assembly of DNA-AuNP results in a significant drop in the melting temperature. 13 The DNA hybridization at the interface also depends on the charge density 37,38 and curvature of the solid surface. 39 In contrast to the extensive studies on the DNA hybridization at the interfacial environments, only a few studies tried to understand the toehold-mediated DNA strand displacement reaction in the solid phase. For instance, the experiments of Song et al. 24–26 indicate that the toeholdmediated strand displacement reaction at the interfacial environments differs greatly from that


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in solution phase. More specifically, a toehold of longer than seven bases is needed for the fuel oligomer on the second AuNPs to initiate the strand displacement reaction and replace the protector strand of the linker-protector complex on the first AuNPs, whereas a toehold with five bases is sufficient to provide a high rate of toehold-mediated strand displacement reaction in solution phase. Xu, Li and their coworkers 40,41 have shown that toehold exchange reaction on the chip surface is enhanced compared with their molecular counterparts, with the efficiency value increased from 24.9% in solution to 86.1% on the chip surface. However, the kinetics of toehold-mediated strand displacement reaction at the solid phase is still not well characterized, and the impact of SNA properties on this reaction needs to be systematically investigated. In the present work, we characterized the kinetics of DNA toehold exchange at the DNA-AuNP surface (Fig. 1), and carefully studied the factors that affect the efficiency of toehold exchange reaction. After that, we built an INHIBIT logic gate for silver ion (Ag+ ) detection. Our study contributes to establishing a complete understanding of toehold exchange reaction at surface, and shades light on designing and developing nanostructures or systems based on the assembly of AuNP driven by dynamic DNA-fueled molecular machines.

Experimental section Preparation and functionalization of AuNPs. The DNA oligonucleotides used in this study were synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China). AuNPs with a diameter of 13 nm were prepared according to previous literature. 4 The measured surface plasmon resonance maximum of AuNPs is λmax = 520 nm. The molar concentration of AuNPs was measured using a Cary 300 UV-Vis spectrophotometer. AuNPs were functionalized with thiol-modified DNA as previously described. 42 3.85 µ M of thiol-modified DNA (treated with TCEP beforehand) was added in 11 nM AuNPs solution. Then, the solution was stored for 16 hours. After that, 10 mM PB buffer was added for 12 hours. Then 0.1 M NaCl, 0.2 M NaCl and 0.3 M NaCl was added in the solution step by step for every 8 hours. All these procedures above were performed at 4◦ C. Lastly,


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the solution was centrifuged three times at 15000 rpm for 30 min and the oily precipitate was redispersed in 0.1 M PBS (10 mM PB, pH = 7.4, with 0.1 M NaCl) and stored at 4◦ C for further use. Note that, the (no-modifided) oligonucleotides and DNA-AuNPs used in the steric effect experiments were resuspended and stored in 0.1 M PBS buffer (10 mM PB, pH = 7.4, with 0.1 M NaCl). The cysteine, metal-ion solutions, (non-modified) oligonucleotides, and DNA-AuNPs used in experiments of INHIBIT logic gate were resuspended and stored in another type of PBS buffer with a concentration of 0.1 M (10 mM PB, pH = 7.4, with 0.1 M NaNO3 ). Preparation of double-stranded DNA complex. The linker and protector oligomers were mixed at a 1:1 molar ratio, and the fluorophore and quencher oligomers were mixed at a 1:1.2 molar ratio. All oligomers were kept at 90◦ C for 15 min and then slowly cooled down to room temperature for over 2 h. Preparation of linker-protector complex functionalized AuNPs. The linker-protector complex was mixed with thiol-modified AuNPs at a 27:1 molar ratio and kept at 25◦ C for at least 6 h. The solution was centrifuged twice at 15,000 rpm for 30 min to remove excess DNA complexes and single strand DNAs. The DNA-AuNP-2 obtained was resuspended in 0.1 M PBS buffer. Preparation of AuNPs with decreased density of functional oligomers. To synthesize DNA functionalized AuNPs with approximately halved probe density on its surface, 1.93 µ M of thiolmodified DNA was added in solution with 1.93 µ M of thiol-modified poly-T10 to 11 nM AuNPs, and the functionalization process was carried out as described above. For DNA functionalized AuNPs with approximately one third of surface probe density, it is prepared by adding 1.28 of µ M thiol-modified DNA and 2.56 µ M of thiol-modified poly-T10 together to 11 nM AuNPs solution. Based on previous studies of Storhoff et al. 43 that almost the same stability and surface coverage of DNA-Modified AuNPs can be achieved using spacer oligomers (poly-Tx ) ranges from 10 bases to 20 bases, it is suspected that oligomers with the same spacer but different probe oligonucleotides will present similar loading ability of oligonucleotides onto AuNP surface. Thus, it is rational to prepare AuNPs with decreased density of functional oligomers by controlling the relative concentrations of DNA and diluent in solution, and the probe density on particle surface can be estimated


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using the relative concentration of DNA and diluent in solution. For DNA-AuNPs, a spacer region of approximately 10 nucleotides (poly-T10 , about 3 nm) is used to extend the active recognition portion of the oligonucleotide sequence away from the positively charged gold surface in the fabrication of DNA-AuNPs. 3 Although the loading of oligonucleotide may differ slightly for probe oligonucleotide and diluent, the effect of this slight difference in local probe density contributes little on the DNA hybridization on the surface due to the long spacer oligomer.

Results and discussion Kinetics of DNA toehold exchange reaction on AuNPs. The mechanism of toehold exchange reaction on AuNPs for rate measurements adopted herein is similar to that of Song et al., 24 with no catalyst strand involved in the assembly of DNA-AuNPs. As shown in Fig. 1, a multi-stranded DNA molecule, which is the linker-protector complex, is modified on one of two types of AuNPs (AuNP-2), and a single strand serving as the invading strand is modified on the surface of another type of AuNPs (AuNP-1). The toehold exchange reaction is initiated by binding the invading strand onto the active toehold region (hereinafter referred to as the invading toehold in this study; heavy green domain, αm ) of the linker strand on AuNP-2, and then undergoes a branch migration process to reveal the occluded toehold (hereinafter referred to as the incumbent toehold in this study; light blue domain, βn ). Consequently, two events may be noted, namely, cross-linking aggregation of DNA-AuNPs and protector-strand release into the solution. The released protector-strand subsequently binds to the five-base toehold domain of the fluorophore strand to undergo another cycle of toehold exchange with the quencher/fluorophore complex, releasing the fluorophore to enable spectroscopic monitoring of the reaction. These cyclic reactions enable DNA-AuNPs to crosslink into larger clusters with visible deposits in solution (inset in Fig. 2). During the experiments, βn was set to five bases, and αm was varied from five to ten bases to investigate the effects of invading toehold on the assembly of AuNPs. Considering that the steric hinderance of SNA on the curved surface of AuNP correlates with the distance between the site and the particle center, we


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also studied the effects of βn domain on the AuNP assembly. Fig. 2 shows variations in fluorescence intensities as a function of reaction time. At a constant

βn length of five bases, the toehold exchange reaction proceeds at a negligible rate when αm is assigned a short length of ≤7 bases (Fig. 2a). However, as αm is lengthened from seven bases to eight bases, that is, only a single base longer, a remarkable enhancement of the reaction rate has been observed. This “off/on switch” behavior in reaction rate observed herein may be attributed to two reasons. First, the length difference between αm and βn . In the toehold exchange reaction, the elongation of the active invading toehold domain αm and the reduction of the incumbent toehold domain βn continuously speed up the kinetics of the forward reaction. Thus, an optimal length difference between αm and βn , such as that between systems with seven and eight bases, may induce a sharp increase in reaction rate. To verify this assumption, the reaction kinetics for different toehold length differences must be investigated by varying βn . Second, the intrinsic properties of DNAAuNPs, such as the probe density on the AuNPs, and the relative distance between the two types of nanoparticles, may affect reaction rate significantly. We then focused on the incumbent toehold βn to determine how its length affects the assembly of DNA-AuNPs. The kinetics of toehold exchange reaction is considered in the system containing different lengths of βn but with a fixed length of αm . As shown in Fig. 2b, for a constant αm length of ten bases, the reaction rates continuously increase when βn is shortened from eight bases to four bases. These results are consistent with the behaviors of toehold exchange reaction in solution. 44 Evidently, the characteristic sharp increase or “off/on switch” behavior observed in the reaction above (Fig. 2a) cannot be obtained by simply regulating the length of βn . According to the experimental results and our analyses, the “off/on switch” behavior of the reaction rate is most possibly attributed to the intrinsic properties of the DNA-AuNPs. The interaction strength of αm with its complement determines the hybridization rate. 44 In our study, to perform hybridization reactions at the solid interface, these two types of AuNPs should first move themselves close to each other, and then the invading strand grafted onto AuNP-1 must penetrate


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through the dense layer of linker-protector oligonucleotide strands on the AuNP-2 surface. Therefore, toehold exchange reaction could be regulated by the oligonucleotide density on the surface of AuNPs or the relative position of AuNP-1 and AuNP-2. In order to addreess why the reaction rate undergoes the “off/on switch” increase as the elongation of αm from seven to eight bases, we designed the following systems. System design. To study the effects of properties of SNA on the reaction rate of toehold exchange on the surface of AuNPs, six methodologies were designed as illustrated in Fig. S2: 1. Scheme A (SA), the linker-protector complex is left free in solution rather than tethered to the AuNP-2 surface. 2. Scheme B (SB), the invading strand is left free in solution instead of attached to AuNP-1. 3. Scheme C (SC), similar to the mechanism displayed in Fig. 1 but the density of invading strands on AuNP-1 is halved. 4. Scheme D (SD), the density of invading strands on AuNP-1 is one third of that in the mechanism of Fig. 1. 5. Scheme E (SE), the density of linker-protector complex on AuNP-2 is halved. 6. Scheme F (SF), the spacer domain (γ and δ ) is elongated by 28 bases in total (poly-T28 ). It should be noted that the amounts of invading strand or linker-protector complex are the same in all investigated systems above. Kinetics of DNA toehold exchange reactions in the designed systems. Fig. 3 and Fig. S3 present fluorescence intensities as a function of time in systems designed (SA ∼ SF) with different set up of αm and βn . In scheme A (Fig. S2), the rate of toehold exchange reaction continuously increased with either elongation of αm (Fig. 3a) or truncation of βn (Fig. S3a). By contrast, the real-time fluorescence readouts in SB are highly close to each other in all systems with different

αm and βn lengths (Fig. 3b and S3b). As well, the “off/on switch” behavior was not observed yet.


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In SA, the experimental results show that either longer αm or shorter βn can accelerate the reaction. These results can be understood according to the theory proposed by Zhang and Winfree 44 for toehold exchange reaction, saying that stronger binding strength of αm to its complementary domain can enhance the ability to initiate the strand displacement reaction for the invading strand, and shorter βn means weaker binding strength thus stronger ability for the protector to dissociate from the linker strand then benefit the overall reaction. Meanwhile Zhang and Winfree predicted that in free DNA systems in solution, a six-base αm domain could result in maximum rates of toehold-mediated strand displacement reactions. However, our experimental results show that the net reaction rate in SA continuously increases for αm ranges from 5 to 10 bases (Fig. 3a). That is, these results are still consistent with Zhang and Winfree’s rules even though αm in SA is larger than six bases. We attribute these phenomena to the increased enthalpic impediment arises from the higher electrostatic surface charge density and steric hinderance of the dense layer of invading oligonucleotides on the AuNP-1 surface, in which the linker strand must penetrate into the dense oligonucleotide layer to achieve toehold binding and the following strand displacement. The increased difficulty for strand displacement makes the rules of Zhang and Winfree 44 function well for the systems in SA with αm domain larger than six bases. In SB, the rate of toehold exchange reaction changed in a relatively small scape as the lengths of both αm and βn varied from short to long. These results are in accordance with those observed in the experiments of free DNA toehold exchange reaction in solution, in which toehold exchange reactions exhibit almost the same rate constant for the systems with a invading toehold longer than six bases, implying that the linker-protector complexes tethered on the AuNP-2 surface behave similar to the free DNA complexes in solution. Considering that the linker-protector complex in duplex formation is mixed with thiol-modified AuNPs at a 27:1 molar ratio in the preparation of DNA-AuNP-2, the probe density on AuNP-2 is much smaller than that on AuNP-1. Additionally, we believe that the longer spacer between the linker-protector complex and AuNP-2 particle may also help to reduce the effects of steric hindrance of the AuNP-2 oligonucleotide layer on the efficiency of αm binding to its complementary domain; thus, the reaction rates are nearly in-


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distinguishable from each other if αm is longer than six bases, just as the free DNA system. As we insert a longer spacer between the linker-protector complex and AuNP-2, a larger reaction rate was observed for each βn system (SF, Fig. S3f) compared with that of its counterpart with shorter spacer (Fig. 2b). Since a longer spacer between the linker-protector complex would decrease the enthalpic impediment but increase the entropic penalty for toehold exchange, it is believed that an optimum distance between the particle and linker-protector complex play an important role in achieving maximum reaction rate for toehold exchange on the surface of AuNPs in our designed system. 34 From the results of scheme B and F described above, we suspect that the oligonucleotide layer on AuNP-2 affects little on the rate of toehold exchange reaction occurring on its surface. To test this, a type of DNA-AuNP-2 was prepared using half-decreased density of linker-protector complex (scheme E). We found that the systems with a ten-base αm but varied βn (Fig. S3e) exhibited nearly the same real-time fluorescence readout curves to that in Fig. 2b. Moreover, the “off/on switch” in the reaction rate of toehold exchange was also observed. These behaviours demonstrate that the sparsely distributed linker-protector complex on AuNP-2 contributes little to the significant difference in the reaction rate observed between systems with seven- and eight-base

αm , as described in the first section (Fig. 2a). We then studied the effects of oligonucleotide layer of DNA-AuNP-1 on the strand displacement of protector on DNA-AuNP-2 by the invading strand on AuNP-1. Two types of DNA-AuNP1 were prepared with the probe density decreased by 1/2 (scheme C) and 2/3 (scheme D), respectively, compared to the state that AuNP-1 is fully conjugated by invading oligonucleotides. Interestingly, although a significant acceleration for the reaction of system with seven-base αm in scheme C was observed, a large gap between the reaction efficiencies is still recognized in Fig. 3c. However, there is no obvious “off/on switch” behavior for the rate of toehold exchange reaction between the systems with different αm length in scheme D (Fig. 3d), that is, toehold exchange reaction is accelerated continually as the elongation of toehold domain αm , just as that of free DNA in solution. These results demonstrate that the density of oligonucleotide conjugated onto AuNP-1


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can efficiently regulate the toehold exchange reaction between DNA-AuNP-1 and DNA-AuNP-2, and conjugates with a high density on AuNP-1 can strongly suppress the toehold exchange reaction for systems with αm shorter than seven-bases thus the efficiencies of these systems are extremely low (Fig. 2a). We suspect that the attenuation of the DNA hybridization for the toehold domain is related to the large electrostatic repulsion between the negatively charge probes and the highly stretched brush-like conformation for the tethered oligonucleotide strands at high surface probe density on AuNP-1. 30,31,45 These properties induce a high enthalpic impediment for the linkerprotector complex to move close to and insert into the oliogonucleotide layer of DNA-AuNP-1. The process of toehold hybridization and the following strand displacement between the invading strand and linker-protector complex in this situation is strongly suppressed, thus a longer toehold domain, i.e., a stronger toehold binding strength, is needed to initiate this process compared with the free DNA toehold exchange reaction in solution, in which a five-base toehold is able to trigger the toehold-mediated strand displacement reaction. In summary, the “off/on switch” behavior in the rate of toehold exchange reaction on the AuNPs is attributed to the high surface oligonucleotide density of DNA-AuNP-1. INHIBIT logic gate. Based on the discussions above, we observed that the DNA-AuNP system indeed exhibits a behavior very distinct from that in a pure DNA system, i.e., the “off/on switch” behavior in the systems with a single base difference from seven- to eight-base for the invading toehold domain, during toehold exchange reaction. We thus expect that this property may be applied to construct DNA-based circuits. In an initial test, we built an INHIBIT logic gate using cytosine-Ag+ -cytosine (C-Ag+ -C) base pairs and cysteine (Cys). Metal-mediated base pairs have attracted considerable attention because of their potential application in sensing and DNA computation. 46,47 Ag+ can insert into natural C-C mismatches in DNA duplexes and form stable metal-base bonds. 48 Cys, which contains a thiol group, can also bind with Ag+ ions to form stable Ag+ -Cys complexes. The interaction between Cys and Ag+ is so strong that Ag+ ions may be dislodged from C-Ag+ -C base pairs. The basic design of our INHIBIT logic gate is presented in Fig. 4a. A linker with an eight-


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base toehold was tethered to AuNP-2 to enable good system dispersion. The αm on AuNP-1 was complementary to the linker toehold domain on AuNP-2, except for the first site highlighted in purple. In the absence of Ag+ and Cys (0, 0) or in the presence of Cys alone (0, 1), the system remained well dispersed and no aggregate could be detected (the “OFF” state). In the presence of Ag+ alone (1, 0), Ag+ can selectively interact with mismatched C bases, promote DNA displacement, and trigger aggregation of DNA-AuNP, which results in the “ON” state. The presence of Ag+ and Cys together (1, 1) induces formation of a complex of Ag+ and Cys, rather than the C-Ag+ -C base pairs, which results in the “OFF” signal. The UV-Vis spectral responses, symbols, and truth table of this logic gate are presented in Figs. 4b, 4c, and 4d, respectively. Finally, the selectivity of the logic gate was investigated using different metal ions, as shown in Fig. S4. Metal ions, such as Mg2+ , Ca2+ , Cu2+ , Pb2+ , and K+ , were used to replace Ag+ . The presence of these metal ions did not initiate DNA-AuNP aggregation. These results confirm that the C-C mismatched base pair can only capture Ag+ . As such, the logic gate presents good selectivity.

Conclusion In this study, we studied the kinetics of DNA toehold exchange at the AuNP surface (Fig. 1), and carefully studied the factors that affect the efficiency of toehold exchange reaction. We found that the length of invading toehold domain αm acts an important function in initiating AuNP assembly. We also noted significant differences in reaction rates between systems with toehold lengths larger than seven bases and those with toehold lengths equal to or less than seven bases. This “off/on switch” behavior in reaction rate of toehold exchange on solid interface differs from that observed in free DNA systems, in which the toehold exchange reaction rate continuously increases as αm is increased. 44 To study the effects of properties of SNA on the reaction rate of toehold exchange on the surface of AuNPs, six methodologies were designed and carefully studied by monitoring the fluorescence intensities as a function of time since the start of reactions. We found that “off/on


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switch” in the reaction rate observed between systems with seven- and eight-base αm is attributed to the high surface oligonucleotide density of DNA-AuNP-1. Based on these understanding, an electronic logic gate (INHIBIT gate) with good selectivity was developed.

Acknowledgement We would like to thank the National Natural Science Foundation of China (Nos. 91127046, 21434007, 21404097, and 21404098), the National Basic Research Program of China (No. 2012CB821500), the Fundamental Research Funds for the Central Universities (No. WK2060200013), and the China Postdoctoral Science Foundation (No. 2014M551808) for their financial support.

Supporting Information Available Schematic illustration of the six designed methodologies; real-time fluorescence readouts for the toehold exchange reaction on the surface of AuNPs with varied βn for scheme A∼F, and αm is set to 10 bases; the UV-vis absorbance spectroscopy of the selectivity of the INHIBIT logic gate to different metal ions; and the ssDNA sequences used in the experiments. This material is available free of charge via the Internet at http://pubs.acs.org/.

References 1. Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 1996, 382, 607–609. 2. Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P.; Schultz, P. G. Organization of ‘nanocrystal molecules’ using DNA. Nature 1996, 382, 609– 611. 3. Cutler, J. I.; Auyeung, E.; Mirkin, C. A. Spherical Nucleic Acids. J. Am. Chem. Soc. 2012, 134, 1376–1391.


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4. Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. One-Pot Colorimetric Differentiation of Polynucleotides with Single Base Imperfections Using Gold Nanoparticle Probes. J. Am. Chem. Soc. 1998, 120, 1959–1964. 5. Park, S.-J.; Taton, T. A.; Mirkin, C. A. Array-Based Electrical Detection of DNA with Nanoparticle Probes. Science 2002, 295, 1503–1506. 6. Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Scanometric DNA Array Detection with Nanoparticle Probes. Science 2000, 289, 1757–1760. 7. Cao, Y. C.; Jin, R.; Mirkin, C. A. Nanoparticles with Raman Spectroscopic Fingerprints for DNA and RNA Detection. Science 2002, 297, 1536–1540. 8. Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K. R.; Han, M. S.; Mirkin, C. A. Oligonucleotide-Modified Gold Nanoparticles for Intracellular Gene Regulation. Science 2006, 312, 1027–1030. 9. Park, S. Y.; Lytton-Jean, A. K. R.; Lee, B.; Weigand, S.; Schatz, G. C.; Mirkin, C. A. DNAprogrammable nanoparticle crystallization. Nature 2008, 451, 553–556. 10. Nykypanchuk, D.; Maye, M. M.; van der Lelie, D.; Gang, O. DNA-guided crystallization of colloidal nanoparticles. Nature 2008, 451, 549–552. 11. Macfarlane, R. J.; Lee, B.; Jones, M. R.; Harris, N.; Schatz, G. C.; Mirkin, C. A. Nanoparticle Superlattice Engineering with DNA. Science 2011, 334, 204–208. 12. Travesset, A. Self-Assembly Enters the Design Era. Science 2011, 334, 183–184. 13. Smith, B. D.; Dave, N.; Huang, P.-J. J.; Liu, J. Assembly of DNA-Functionalized Gold Nanoparticles with Gaps and Overhangs in Linker DNA. J. Phys. Chem. C 2011, 115, 7851– 7857. 14. Yurke, B.; Turberfield, A. J.; Mills, A. P.; Simmel, F. C.; Neumann, J. L. A DNA-fuelled molecular machine made of DNA. Nature 2000, 406, 605–608. 15 ACS Paragon Plus Environment

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15. Zhang, D. Y.; Turberfield, A. J.; Yurke, B.; Winfree, E. Engineering Entropy-Driven Reactions and Networks Catalyzed by DNA. Science 2007, 318, 1121–1125. 16. Seelig, G.; Soloveichik, D.; Zhang, D. Y.; Winfree, E. Enzyme-Free Nucleic Acid Logic Circuits. Science 2006, 314, 1585–1588. 17. Yan, H.; Zhang, X.; Shen, Z.; Seeman, N. C. A robust DNA mechanical device controlled by hybridization topology. Nature 2002, 415, 62–65. 18. Cerda, E.; Mahadevan, L. Geometry and Physics of Wrinkling. Phys. Rev. Lett. 2003, 90, 074302. 19. Soloveichik, D.; Seelig, G.; Winfree, E. DNA as a universal substrate for chemical kinetics. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 5393–5398. 20. Chen, Y.-J.; Dalchau, N.; Srinivas, N.; Phillips, A.; Cardelli, L.; Soloveichik, D.; Seelig, G. Programmable chemical controllers made from DNA. Nat. Nanotechnol. 2013, 8, 755–762. 21. Qian, L.; Winfree, E.; Bruck, J. Neural network computation with DNA strand displacement cascades. Nature 2011, 475, 368–372. 22. Qian, L.; Winfree, E. Scaling up digital circuit computation with DNA strand displacement cascades. Science 2011, 332, 1196–1201. 23. Zhang, D. Y.; Hariadi, R. F.; Choi, H. M. T.; Winfree, E. Integrating DNA strand-displacement circuitry with DNA tile self-assembly. Nat. Commun. 2013, 4, 1965. 24. Song, T.; Liang, H. Synchronized Assembly of Gold Nanoparticles Driven by a Dynamic DNA-Fueled Molecular Machine. J. Am. Chem. Soc. 2012, 134, 10803–10806. 25. Song, T.; Liang, H. Capability of DNA-fueled molecular machine in tuning association rate of DNA-functionalized gold nanoparticles. Chin. J. Polym. Sci. 2013, 31, 1183–1189.


Page 16 of 24

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26. Song, T.; Xiao, S.; Yao, D.; Huang, F.; Hu, M.; Liang, H. An Efficient DNA-Fueled Molecular Machine for the Discrimination of Single-Base Changes. Adv. Mater. 2014, 26, 6181–6185. 27. Lu, C.-H.; Li, J.; Lin, M.-H.; Wang, Y.-W.; Yang, H.-H.; Chen, X.; Chen, G.-N. Amplified Aptamer-Based Assay through Catalytic Recycling of the Analyte. Angew. Chem. Int. Ed. 2010, 49, 8454–8457. 28. Miyahata, T.; Kitamura, Y.; Futamura, A.; Matsuura, H.; Hatakeyama, K.; Koinuma, M.; Matsumoto, Y.; Ihara, T. DNA analysis based on toehold-mediated strand displacement on graphene oxide. Chem. Commun. 2013, 49, 10139–10141. 29. Wang, D.; Tang, W.; Wu, X.; Wang, X.; Chen, G.; Chen, Q.; Li, N.; Liu, F. Highly Selective Detection of Single-Nucleotide Polymorphisms Using a Quartz Crystal Microbalance Biosensor Based on the Toehold-Mediated Strand Displacement Reaction. Anal. Chem. 2012, 84, 7008–7014. 30. Randeria, P. S.; Jones, M. R.; Kohlstedt, K. L.; Banga, R. J.; Olvera de la Cruz, M.; Schatz, G. C.; Mirkin, C. A. What Controls the Hybridization Thermodynamics of Spherical Nucleic Acids? J. Am. Chem. Soc. 2015, 137, 3486–3489. 31. Ravan, H.; Kashanian, S.; Sanadgol, N.; Badoei-Dalfard, A.; Karami, Z. Strategies for optimizing DNA hybridization on surfaces. Anal. Biochem. 2014, 444, 41–46. 32. Gao, Y.; Wolf, L. K.; Georgiadis, R. M. Secondary structure effects on DNA hybridization kinetics: a solution versus surface comparison. Nucleic Acids Res. 2006, 34, 3370–3377. 33. Chen, C.; Wang, W.; Ge, J.; Zhao, X. S. Kinetics and thermodynamics of DNA hybridization on gold nanoparticles. Nucleic Acids Res. 2009, 37, 3756–3765. 34. Lytton-Jean, A. K. R.; Mirkin, C. A. A Thermodynamic Investigation into the Binding Properties of DNA Functionalized Gold Nanoparticle Probes and Molecular Fluorophore Probes. J. Am. Chem. Soc. 2005, 127, 12754–12755. 17 ACS Paragon Plus Environment

Langmuir

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35. Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. The effect of surface probe density on DNA hybridization. Nucleic Acids Res. 2001, 29, 5163–5168. 36. Takeda, Y.; Kondow, T.; Mafuné, F. Hybridization of ssDNA with a Complementary DNA Probe Tethered to a Gold Nanoparticle Effect of Steric Hindrance Caused by Conformation. J. Phys. Chem. C 2008, 112, 89–94. 37. Wong, I. Y.; Melosh, N. A. An Electrostatic Model for DNA Surface Hybridization. Biophys. J. 2010, 98, 2954–2963. 38. Ge, C.; Liao, J.; Yu, W.; Gu, N. Electric potential control of DNA immobilization on gold electrode. Biosens. Bioelectron. 2003, 18, 53–58. 39. Cederquist, K. B.; Keating, C. D. Curvature Effects in DNA: Au Nanoparticle Conjugates. ACS Nano 2009, 3, 256–260. 40. Xu, H.; Deng, W.; Huang, F.; Xiao, S.; Liu, G.; Liang, H. Enhanced DNA toehold exchange reaction on a chip surface to discriminate single-base changes. Chem. Commun. 2014, 50, 14171–14174. 41. Li, H.; Xiao, S.; Yao, D.; Lam, M. H.-W.; Liang, H. A smart DNA-gold nanoparticle probe for detecting single-base changes on the platform of a quartz crystal microbalance. Chem. Commun. 2015, 51, 4670–4673. 42. Jin, R.; Wu, G.; Li, Z.; Mirkin, C. A.; Schatz, G. C. What Controls the Melting Properties of DNA-Linked Gold Nanoparticle Assemblies? J. Am. Chem. Soc. 2003, 125, 1643–1654. 43. Storhoff, J. J.; Elghanian, R.; Mirkin, C. A.; Letsinger, R. L. Sequence-Dependent Stability of DNA-Modified Gold Nanoparticles. Langmuir 2002, 18, 6666–6670. 44. Zhang, D. Y.; Winfree, E. Control of DNA Strand Displacement Kinetics Using Toehold Exchange. J. Am. Chem. Soc. 2009, 131, 17303–17314.


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45. Irving, D.; Gong, P.; Levicky, R. DNA Surface Hybridization: Comparison of Theory and Experiment. J. Phys. Chem. B 2010, 114, 7631–7640. 46. Clever, G. H.; Kaul, C.; Carell, T. DNA-Metal Base Pairs. Angew. Chem. Int. Ed. 2007, 46, 1521–3773. 47. Liu, Q.; Wang, L.; Frutos, A. G.; Condon, A. E.; Corn, R. M.; Smith, L. M. DNA computing on surfaces. Nature 2000, 403, 175–179. 48. Ono, A.; Cao, S.; Togashi, H.; Tashiro, M.; Fujimoto, T.; Machinami, T.; Oda, S.; Miyake, Y.; Okamoto, I.; Tanaka, Y. Specific interactions between silver(I) ions and cytosine-cytosine pairs in DNA duplexes. Chem. Commun. 2008, 4825–4827.


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Figure 1: Schematic illustration of the dynamic DNA-fueled molecular machine strategy and the mechanism of DNA-AuNP assembly used for rate measurements. Reporter complex (quencher/fluorophore complex) reacts stoichiometrically with product (Protector strand) to yield increased fluorescence.


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Figure 2: Real-time fluorescence readouts for the toehold-mediated strand displacement reaction on the surface of AuNPs. (a) Effects of varying the invading toehold length (αm ) of AuNP-1 from five to ten, with incumbent toehold (βn ) of protector oligomer setting to five. The inset panel show the color change after six hours for the studied systems. The label “αm /βn ” means: αm = αm = ten bases, βn = five bases. (b) Effect of varying βn from four to eight bases, with αm setting to ten bases unchanged. [DNA-AuNP-1] = [DNA-AuNP-2] = 4 nM, [quencher/fluorophore complex] = 122 nM.


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Figure 3: Real-time fluorescence readouts for the toehold-mediated DNA strand displacement reaction on the surface of AuNPs with varied invading toehold domain (αm ). “SA-10-5” represents “scheme-A (SA)” with αm = αm = ten bases, βn = five bases. The explanation of SA (SB, SC, SD, SE, SF) can be found in the main text, the concentrations of each elements used in the experiments can be found in the Supporting Information.


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Figure 4: (a) Schematic illustration of the INHIBIT logic gate. (b) The UV-vis absorbance of the INHIBIT logic gate in the presence of different inputs (blank, Ag+ , Cys, Ag+ + Cys); the inset panels represent the corresponding color change of the INHIBIT logic gate with inputs after 6 hours;.[DNA-AuNP-1] = [DNA-AuNP-2] = 4 nM, [Ag+ ]=[Cys]=80 µ M. (c) The symbol for the INHIBIT logic gate. (d) The truth table of the INHIBIT logic gate.


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On Switchâ in the Toehold-Mediated Strand

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