Facile Strategy for Visible Disassembly of Spherical Nucleic Acids

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Biological and Medical Applications of Materials and Interfaces

A Facile Strategy for Visible Disassembly of Spherical Nucleic Acids Programmed by Catalytic DNA Circuit Bing Wei, Dongbao Yao, Bin Zheng, Xiang Zhou, Yijun Guo, Xiang Li, Chengxu Li, Shiyan Xiao, and HaoJun Liang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02107 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 14, 2019

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ACS Applied Materials & Interfaces

A Facile Strategy for Visible Disassembly of Spherical Nucleic Acids Programmed by Catalytic DNA Circuit Bing Wei,†,# Dongbao Yao,*,†,# Bin Zheng,‡ Xiang Zhou,† Yijun Guo,† Xiang Li,† Chengxu Li,† Shiyan Xiao,† and Haojun Liang*,† †Hefei

National Laboratory for Physical Sciences at the Microscale, CAS Key Laboratory of Soft Matter Chemistry, iChEM (Collaborative Innovation Center of Chemistry for Energy Materials), Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China ‡School

of Chemistry and Chemical Engineering, Hefei Normal University, Hefei, Anhui 230061, P. R. China #B.W.

and D.Y. contributed equally to this work.

*Correspondence

should be addressed to [email protected]; [email protected]

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Abstract The

programmable

toehold-mediated

DNA-strand-displacement

reaction

has

demonstrated its extraordinary capability in driving spherical nucleic acids assembly. Here, a facile strategy of integrating a DNA-strand-displacement-based DNA circuit with a universal spherical nucleic acid aggregates system was developed for visible disassembly of spherical nucleic acids. This integrated system exhibited rapid colorimetric response and good sensitivity in disassembly reaction and it was also demonstrated its capability in the application of single nucleotide polymorphism discrimination. Moreover, an OR logic gate used for multiplex detection was constructed through combining the fixed spherical nucleic acids disassembly system with two DNA circuits. This strategy will have great potential in fabrication of portable low-cost DNA diagnostic kit, and it is also a very promising method to be used in other applications, such as complex DNA networks and programmable phase transformation of nanoparticle superlattices.

Keywords: spherical nucleic acids, DNA circuit, visible disassembly, DNA-stranddisplacement, single nucleotide polymorphism discrimination, colorimetric detection

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Introduction The field of dynamic DNA nanotechnology has seen a surge of interest since the wellknown toehold-mediated strand-displacement reaction (TMSDR) was emerged in 2000.1 During the past nearly two decades, a variety of sophisticated DNA nanomachines with refined designs have been constructed using TMSDRs as building blocks, including entropy-driven catalytic reaction networks,2,3 enzyme-free DNA dynamical systems,4 nucleic acid molecular gears,5 digital computing circuits,6-9 DNA walkers that move along predesigned tracks,10-15 and DNA cascade systems for high signal amplification.16-18 In most of these systems, input DNA strands that act like catalysts could accelerate the running of the nanomachines through a series of TMSDRs in an efficient manner. In another field, spherical nucleic acid (SNA) conjugate,19 a polyvalent DNA covalently functionalized gold nanoparticle (AuNP) that composed of a gold core and highly oriented oligonucleotide shell, was first synthesized by Mirkin et al in the mid1990s.20 Possessing both distinct physical and chemical properties of the inside AuNP core and powerful programmable and algorithmic capabilities of the outside nucleic acids shell, SNA conjugate has demonstrated its superiorities in various fields by a number of attractive applications, such as programmable nanomaterials with superlattice structures,21-23 in vivo and in vitro diagnostics,24-26 intracellular microRNA imaging,27,28 colorimetric biosensors,29-31 gene regulation,32 cancer therapy,33,34 and drug delivery.35,36 In the traditional direct-linker-addition approach, the process for assembly of SNA conjugates is implemented through simply linking two types of SNA conjugates together by a complementary target strand.37 Recently, a delicate design for the programmable SNA conjugates assembly driven by TMSDR-based catalytic DNA circuit was first advanced by our group,38 which has demonstrated its extraordinary capability in the constructions of DNA logic gates,38-40 cascade self-assembly networks,41 single nucleotide polymorphism (SNP) discrimination,42 and pHresponsive colorimetric sensor.43 Although the established DNA circuit-controlled

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SNA assembly strategy has achieved many desired functions that were inaccessible in the traditional direct-linker-addition approach, the assembly of SNAs still need to be initiated by a specially designed target strand. In addition, other DNA strands that were involved in the DNA circuit also shared a same partial domain with the DNA strands that grafted on SNA surface. These drawbacks undoubtedly limited the application universality of the synthesized SNA conjugates since the processes to synthesize new SNAs specific to different targets over and over again are time-consuming and expensive.37 To overcome these drawbacks, we have recently also proposed an updated strategy through integrating an upstream TMSDRs-based catalytic DNA circuit with a downstream self-assembly of SNAs system.44 In the updated strategy, assembly of the downstream SNA conjugates was triggered by a linker strand released from the upstream DNA circuit, and these two subsystems could be optimized and operated separately. However, the DNA complexes, which were complementary to the DNA strands on SNA surface, still need to be changed according to the changes of the target strands. In addition, it took a long time (more than 12 h) to complete the whole assembly process to form visible SNA aggregates under relative low concentrations (at the level of 10 nM) of input strands for our TMSDRs-based catalytic assembly approaches (also including the conventional DNA target programmed SNAs assembly strategy), since it has been reported that the assembly of SNA conjugates is an intrinsically slow process.45,46 According to previous reports, dissociation of SNA aggregates is a relatively rapid process compared to assembly of SNA conjugates.47-49 Thus, developing a more convenient and rapid strategy of SNAs-based catalytic DNA circuit is urgently needed to better meet the demand of SNAs-based colorimetric assays for possible usage in point-of-care (POC) testing and other applications. Herein, we demonstrate a facile strategy for visible disassembly of SNA aggregates programmed by an entropy-driven catalytic DNA circuit. In this design, the integrated system is composed of a downstream SNAs disassembly-based colorimetric signaling subsystem and an upstream free DNA circuit. The SNA aggregates in the downstream system is formed through self-assembly of only one type of SNA conjugate 4


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by adding a duplex DNA complex as linker (one end of the duplex linker can hybridize with SNA, while the other end is a self-complementary region, see below for the detailed design principle). The initiation of the visible disassembly of SNA aggregates relies on a trigger strand released from the upstream DNA circuit through successive TMSDRs. It is worth noting that the SNA aggregates in our design could be compatible with any upstream circuit without changing any DNA strand in the downstream system, which will cost less and avoid wasteful duplication of efforts for preparation of new SNA aggregates every time. This universality enables our SNA aggregates-based colorimetric signaling system to be prepared and stored in large quantities and used for specific detection of arbitrary DNA targets based on the changes of upstream circuits. Moreover, this facility and universality of our strategy will show great potential in POC testing without need for any instrumentation.

Figure 1. Schematic of the strategy for catalytic DNA circuit programmed visible disassembly of SNA aggregates.

Results and Discussion Design Principle of the Strategy for Disassembly of SNA Aggregates Programmed by Catalytic DNA Circuit. As shown in Figure 1, the integrated system in this strategy comprises two subsystems, an upstream TMSDRs-based catalytic DNA circuit and a downstream SNA aggregates-based disassembly subsystem. These two 5



subsystems can be connected together by a single DNA strand, called Trigger T, released from the upstream circuit. Inspired by the formation mechanism of facecentered cubic (fcc) SNA crystal structures of Mirkin,21 only one type of SNA conjugate and a double-stranded DNA complex (called duplex linker) are employed to form SNA aggregates in our SNAs disassembly-based subsystem (the lower panel of Figure 1). Figure S1 of the Supporting Information (SI) displays the DNA structures of these two components and the aggregation principle for the construction of SNA aggregates. First, duplex linker is formed by hybridization of a pair of single strands, called h-SNA and p-SNA, respectively (Figure S1a), and the structure of it mainly consists of four functional domains: (i) the anchor domain h that can hybridize with the DNA molecule modified on SNA surface; (ii) the toehold domain e that can be recognized by trigger T; (iii) the strand branch migration domain formed by hybridization of domain d in h-SNA and domain d* in p-SNA; (iv) the sticky end domain f that is composed of a palindromic sequence with six bases. Second, as shown in Figure S1b, SNA aggregates can be prepared through employing duplex linker as a bridge. On one hand, one end (domain h) of duplex linker can hybridize with the DNA strands on SNA surface to form a SNA-duplex linker polyvalent conjugate at first; on the other hand, the sticky end with six bases (domain f) at the other end of duplex linker can interact with each other directly to connect free SNA molecules together to form a huge aggregation network on account of cooperative interactions between these neighboring sticky ends on SNA surface.21,37 The DNA sequence length of the crosslink domain in our SNAs aggregation network is only six bases compared to previously reported SNA aggregates that formed through hybridization of relatively longer DNA strands.48,49 This innovative design should be able to greatly increase the ease and flexibility of disassembly of SNA aggregates. The upper panel of Figure 1 displays the DNA structures and reaction process of the TMSDRs-based catalytic DNA circuit which is slightly modified based on the classical DNA circuit developed by Zhang et al.2 The triple-stranded substrate S is constituted by three single strands through hybridization: linker L, protector P, and trigger T. Another long single strand called fuel F acts as fuel to accelerate TMSDRs 6


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in the circuit. The initiation of the upstream circuit starts by a target strand, named disassembler D, that can bind with the dangling toehold domain a at the end of L in S. After that, P can be displaced by D through TMSDR, and a triple-stranded intermediate is generated simultaneously. Then, F can interact with a new generated toehold (domain c) in intermediate and trigger a new round of TMSDR to displace the hidden T and D, and then yields a double-stranded waste W composed of L and F. The displaced D will participate in new rounds of TMSDR cycles repeatedly, thus playing a role of catalyst in the circuit. Subsequently, the other released single strand T will react with the DNA molecules on surface of SNA to disassemble the SNA aggregates. As can be seen in the lower panel of Figure 1, free single strand T can easily enter into the SNAs aggregation network and bind with the toehold (domain e) on duplex linker. Then, p-SNA, which is prehybridized with h-SNA, can be displaced by T through TMSDR and a new DNA complex will be formed on SNA surface. Thus, the crosslink point composed of selfcomplementary domain f will be dissociated to induce the disconnected SNA molecules to be redispersed in the supernatant, and finally result in complete disassembly of SNA aggregates with the increased amount of T released from the upstream catalytic circuit. Due to the distinct optical properties of SNA conjugates, the disassembly process of SNA aggregates can be easily detected by the naked eye through a visible color change of the supernatant (from colorless to red). Assessment of the Downstream SNAs Disassembly Subsystem. First, in order to obtain stable SNA aggregates to be used in the downstream subsystem, the formation condition of SNA aggregates was optimized through simply adding different amounts of duplex linker to the solution containing dispersed SNA conjugates. The experiment was performed by taking photos using a cellphone at different times to observe the SNA aggregation degree from color change of the SNA solution. From Figure S2, it can be found that the SNA aggregation rate accelerated with the increase of the molar ratio of the SNA conjugate to duplex linker (from 1:10 to 1:35) after 2 h of reaction, while the concentration of SNA was fixed at 8 nM. When the molar ratio of the SNA conjugate to duplex linker increased up to 1:20, the SNA conjugates could assemble completely 7



in 8 h (visible aggregates were formed at the bottom of the plastic tube), and the corresponding color of the SNA solution changed from red to colorless. Therefore, in order to ensure both the stability of the SNA aggregates and the disassembly reaction sensitivity, the molar ratio of SNA conjugate to duplex was fixed at 1:20 for preparation of SNA aggregates in all subsequent experiments. Additionally, the melting temperature (Tm) of the prepared SNA aggregates was determined using UV-vis spectrophotometer. As shown in Figure S3, the aggregates formed by the SNA conjugate and duplex linker in a molar ratio of 1:20 exhibited a Tm of ∼43 °C, which demonstrated that the prepared SNA aggregates is fairly stable at room temperature and could be used for the following SNA aggregates-based disassembly experiments.

Figure 2. Trigger-induced disassembly of SNA aggregates. (a) Photos of the SNA aggregates disassembly reaction taken at different times with the direct addition of varied amounts of trigger strand. [SNA] = 8 nM, [duplex linker] = 160 nM. (b) UV-vis absorption spectra of the corresponding supernatants of the samples in (a) after 4 h of reaction.

Then, the performance of the downstream SNAs disassembly subsystem was tested by the direct addition of T in varied concentrations (Figure 2). As can be seen from Figure 2a, it was clear that the disassembly rate of SNA aggregates accelerated with the increasing concentration of T, since the reaction samples with addition of higher concentrations of T showed more pronounced red color in the supernatants (Figure 2a). In addition, as shown from the corresponding UV-vis absorption spectra of the supernatants of the samples after 4 h of reaction (Figure 2b), the absorbance increased with the increase in concentration of T, and this non-catalytic direct-triggeraddition system was able to detect T at a concentration of 20 nM. However, only when

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trigger concentration was higher than 40 nM, color difference could be distinguished by the naked eye for the samples with and without addition of T after 4 h of reaction (visual limit of detection: 40 nM). Moreover, the SNA aggregates cannot be completely disassembled in 4 h when the concentration of T was lower than 320 nM (the concentration of duplex linker was 160 nM) in this direct-trigger-addition test.

Figure 3. The performance of the upstream catalytic DNA circuit. (a) A detailed schematic of the reaction mechanism of the upstream catalytic DNA circuit. (b) A double-stranded fluorescent reporter used for signaling. (c) Fluorescence kinetic curves of the upstream catalytic DNA circuit with varied concentrations of disassembler strand. In this experiment, [S] = 100 nM, [F] = 200 nM, and [reporter] = 300 nM.

Examination of the Upstream Catalytic DNA Circuit. A fluorescence experiment was executed to examine the performance of the upstream catalytic DNA circuit. A more detailed running mechanism of the TMSDRs-based DNA circuit is illustrated in Figure 3a. T released from the catalytic DNA circuit can initiate a onestep TMSDR with a double-stranded reporter (hybridized by the reporter-F strand and the reporter-Q strand) to release fluorescence signal to characterize the catalytic performance of the DNA circuit (Figure 3b). The corresponding real-time fluorescence kinetics experiment results displayed in Figure 3c indicated that the upstream catalytic DNA circuit exhibited excellent stability without the presence of D (the black line in Figure 3c). The fluorescence intensity had a remarkable rise with the increasing concentration of D added in the catalytic reaction, and the fluorescence signal of D as low as 0.1 nM could be detected in 1 h in this TMSDRs-based system. In consequence,

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the upstream catalytic DNA circuit was proved to be stable and efficient enough to release T to be used to disassemble the SNA aggregates in the downstream subsystem.

Figure 4. Optimization of the catalytic DNA circuit integrated with SNA aggregates disassembly. (a) Image results for addition of various concentrations of substrate and fixed concentration of fuel of 7 μM in the integrated system after 4 h of reaction. The upper photo was for the stability test of the system in the absence of disassembler; the bottom photo showed the disassembly degrees of the SNA aggregates with addition of 2 nM of disassembler. (b) The corresponding UV-vis spectra of (a) were measured to confirm the substrate dependent disassembly efficiency after 4 h of reaction. (c) Image results of the integrated system for addition of different concentrations of fuel and fixed concentration of substrate of 1.5 μM in the reaction after 4 h of reaction. The upper photo was for the stability test of the system without adding disassembler; the bottom photo indicated the disassembly degrees of the system with the presence of 2 nM of disassembler. (d) The corresponding UV-vis spectra of (c) were measured to confirm the fuel dependent SNA disassembly results after 4 h of reaction. [SNA] = 8 nM, [duplex linker] = 160 nM.

Optimization of the Integrated System. After examining the two subsystems separately, the upstream catalytic DNA circuit was integrated with the downstream SNA aggregates disassembly subsystem for further optimization of the catalytic disassembly reaction conditions. We first investigated the influence of the concentration of S on the SNA disassembly reaction (Figure 4a,b). The release amount of T should be dependent on the amount of S since T is initially hidden within S. In this experiment, the concentration of S was varied from 1.0 μM to 2.5 μM, the 10


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concentrations of F and D were fixed at 7 μM and 2 nM, respectively. We noticed that the disassembly rate increased with the increase in the concentration of S from the UVvis spectra of the supernatants of the reaction samples after 4 h of reaction (Figure 4b). However, the control samples without the presence of D presented an obvious growth in the absorbance value at 520 nm when the concentration of S was increased from 1.5 μM to 1.8 μM (Figure 4b), and the bottom color image shown in Figure 4a also showed an obvious color change from colorless to light red when the concentration of S was increased from 1.5 μM to 1.8 μM. Hence, the concentration of S should be lower than 1.8 μM in the SNA disassembly experiment, otherwise the leakage induced by high concentration of S will conceal the effective signal of reaction. Second, the concentration of F on the effect of the SNA aggregates disassembly was also examined using a similar method (Figure 4c,d). Similarly, we found that the disassembly reaction rate increased when the concentration of F increased from 5.0 μM to 9.0 μM, however, a visible signal leak was detected in the integrated system when the concentration of F increased up to 8.0 μM. Therefore, on basis of considering the higher concentrations of S and F induced both the system instability and the faster SNA disassembly reaction rate, the concentrations of S and F were finally determined as 1.5 μM and 7.5 μM respectively.

Figure 5. Investigation of the sensitivity of the catalytic DNA circuit programmed visible disassembly of SNA aggregates with varied concentrations of disassembler under the optimal reaction condition. (a) Color images of the integrated system taken after different time intervals. (b) The corresponding UV-vis absorption spectra after 4 h of reaction. In this experiment, [S] = 1.5 μM, [F] = 7.5 μM, [SNA] = 8 nM, and [duplex linker] = 160 nM.

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Sensitivity Investigation of the Integrated System. In the field of nucleic acidbased diagnostics, numerous techniques have been employed for the detection of those known disease-related oligonucleotide sequences. Owing to the distinct optical property produced by the gold core, our SNAs disassembly-based colorimetric method should be one of the competitive approaches for nucleic acid detection. Of course, possessing low limit of detection is extremely important for our strategy, especially for its potential application in POC assays. Therefore, the detection sensitivity of the visible disassembly of SNA aggregates programmed by catalytic DNA circuit was investigated under different concentration conditions of D based on the above optimizations. As shown in Figure 5a, a distinguishable color change produced by the disassembled SNA aggregates could be detected by the naked eye after only 20 min of reaction with the presence of 2 nM of D. Besides, the disassembly degree of the downstream SNA aggregates was seen a clear growth when the concentration of D was higher than 2 nM under the catalysis of the upstream TMSDRs-based catalytic DNA circuit, which demonstrated the extraordinary capability of our DNA circuit programmed SNA aggregates disassembly system. As time progresses, visible color changes in the supernatants of the samples with the presence of lower concentrations of D started to appear. Finally, the concentration of D as low as 0.2 nM could be detected by the naked eye after 4 h of reaction (visual limit of detection: 0.2 nM). Furthermore, the corresponding UV-vis absorption spectra after 4 h of reaction also proved the detection sensitivity of the integrated system (Figure 5b), and the reaction signal with addition of as low as 0.15 nM of D could be distinguished from the control signal (limit of detection: 0.15 nM). Compared with the detection sensitivity of the trigger-induced disassembly of SNA aggregates shown in Figure 2 that was used to imitate the traditional directlinker-addition approach, the visual limit of detection of the TMSDRs-based catalytic integrated system was significantly increased by 200 folds. Moreover, the disassembly reaction progress of this facile strategy is much faster and more efficient than our previous catalytic DNA circuit programmed SNA assembly systems, which could only detect the signal produced by ~10 nM of the target DNA in 4 h.38,44

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Besides, our SNAs disassembly-based colorimetric method showed a similar detection sensitivity and a comparable reaction rate with the fluorescence reporterbased catalytic DNA circuit that employ free DNA strands in solution (red line of 0.1 nM D in Figure 3c). To compare our SNAs disassembly-based DNA circuit and the fluorescence reporter-based catalytic DNA circuit more intuitively, a control experiment was performed to investigate the performance of the upstream catalytic circuit coupled with the fluorescence reporter at the same conditions as the overall SNAs disassembly system. As can be seen in Figure S4a, the catalytic circuit was leaky with the absence of D due to the leak of T under high concentrations of S (1.5 μM) and F (7.5 μM). However, our SNAs-disassembly based DNA circuit system could remain stable in 4 h with very low signal leakage in the presence of 1.5 μM of S and 7.5 μM of F since our SNA reporting strategy acts as a threshold against this noise on some level as we have demonstrated in our previous DNA circuit driven SNA assembly system.41 Additionally, our SNAs disassembly-based colorimetric assay was also carried out in the reaction buffer containing 15% serum. As can be seen from the color images in Figure S5, visible color changes could be detected after 4 h of reaction in the supernatants of the samples containing 15% serum with the addition of different amounts of D (0.5 nM, 2 nM, 5 nM, and 10 nM), and the color of the supernatants gradually deepened with the increase of the concentration of D. This phenomenon was consistent with the sensitivity investigation of the integrated system reaction in clean buffer in Figure 5, which proved that our assay was satisfactory in target DNA detection in clinically relevant media and it is of potential in POC testing.

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Figure 6. Specificity investigation of the integrated system in the application of SNP discrimination. Sequence of the correct target and different positions (labeled in red frames, respectively) with a single-base changes for mismatch (a), insertion (b), and deletion (c) that lead to different types of the spurious targets. (d) Color images of the integrated system after 4 h of reaction with the presence of the spurious targets which have a single base changes of mismatch (d), insertion (e), and deletion (f), respectively, and the corresponding UV-vis absorbance values at 520 nm of the supernatants of all samples. [D] = 2 nM, [S] = 1.5 μM, [F] = 7.5 μM, [SNA] = 8 nM, and [duplex linker] = 160 nM.

Application of the Integrated System in Discrimination of SNP. Another essential issue for DNA diagnostics is SNP discrimination, since SNPs (including single-base mismatch, insertion, and deletion) in nucleic acid sequences of human are crucial clinical biomarkers and related with various genetic diseases.50,51 Although many SNA-based colorimetric methods for discrimination of SNP have been reported, those strategies suffer from many drawbacks, such as the requirement of instrument for heating,45 long analysis time,

42,44

and non-universal design induced high cost,42,49

which may limit their applications in POC assays. Here, the facility and universality of our strategy shown in this paper should overcome these shortcomings. The examination of the integrated system was performed to demonstrate its capability in SNP discrimination (Figure 6). As can be seen in Figure 6a,b,c, the correct target D can bind with the forward invading toehold (yellow domain that was composed of six bases) in L to initiate a branch migration progress, and the TMSDR was completed via the spontaneous dissociation of the brown reverse toehold domain (composed of four bases) of L to release P, i.e., the toehold exchange strategy used for the SNP discrimination experiment was “6/4”. In this experiment, the concentration of the correct target D and all spurious targets were fixed at 2 nM, and the UV-vis absorption peak values of the supernatants of all samples were measured after 4 h of reaction by UV-vis spectrophotometer. Undoubtedly, the correct target D exhibited the strongest reaction activity in the SNA aggregates disassembly. Consistent with previous report,52 the spurious target has a mismatch on the 21th base near the 3’ end of the invading toehold exhibited a relatively small effect on the reaction rate compared to the correct target; while the mismatch points that were closer to the invading toehold domain (m17T) and posited at the middle position of the invading toehold (m19A) 14


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induced greater impediment to the strand-displacement rate than the mismatch on the 21th base (Figure 6d). Since the toehold exchange strategy used here was “6/4”, which was similar to our previous SNAs assembly-based SNP discrimination strategies42,44 and the nucleic acid hybridization probe model for SNP discrimination constructed by Zhang et al,53 our SNA aggregates disassembly-based system also exhibited strong suppression to the reaction rate for the mismatch points closer to the reverse toehold domain on L (m3T and m7C) and at the middle of the spurious target (m12G). In addition, the spurious targets possessing a single-base changes of insertion (Figure 6e) and deletion (Figure 6f) at the same positions with the mismatch points (the 12th base and 19th base) showed similar levels of weakening in disassembly reaction abilities. In order to further quantify the ability of the strategy in discriminating the correct target and those spurious targets, the term “discrimination factor (DF)” was defined as 𝐷𝐹 = 1−

𝐴𝑠𝑝𝑢𝑟𝑖𝑜𝑢𝑠−𝐴𝑏𝑙𝑎𝑛𝑘 𝐴𝑐𝑜𝑟𝑟𝑒𝑐𝑡 −𝐴𝑏𝑙𝑎𝑛𝑘

to be used here, where 𝐴𝑠𝑝𝑢𝑟𝑖𝑜𝑢𝑠 , 𝐴𝑐𝑜𝑟𝑟𝑒𝑐𝑡 , and 𝐴𝑏𝑙𝑎𝑛𝑘 denote

the absorbance values of the supernatant at 520 nm for spurious target, correct target, and control sample, respectively. Note that “DF = 0” means no discrimination is found between the correct and the spurious targets in this strategy; and “DF = 1” means the strongest discrimination degree between them. As shown in Figure S6, all chosen spurious targets could be clearly distinguished from the correct target using the SNAbased catalytic disassembly system since the values of DF varied from 0.55 to 1 for all kinds of spurious targets except for the spurious target with a mismatch at the position of the 21th base (m21G). In consequence, it was demonstrated that our TMSDRs-based catalytic DNA circuit programmed SNA aggregates disassembly system exhibited good sequence selectivity for SNP discrimination. Moreover, we also tested the target DNA detection performance of our SNAs disassembly-based colorimetric strategy in the interference of spurious target. In this experiment, 2 nM of the correct target D mixed with different concentrations of the spurious target m3T were added in the integrated system. As shown in Figure S7, the sample with the addition of only 2 nM of the correct target D exhibited a similar reaction ability over time compared to the samples with the presence of 2 nM of the correct

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target and 2 nM or 4 nM or 10 nM of the spurious target. It can be found that the reaction rate of the system exhibited a noticeable increase only when the concentration of the spurious target up to 20 nM; meanwhile the sample with addition of only 2 nM of the correct target also could produce visible signal at the same time, which illustrated that low abundance target DNA (2 nM) could still be detected in the presence of large excess of SNP target (10 times of the correct target) in our strategy. These results proved that our TMSDRs-based catalytic DNA circuit programmed SNA aggregates disassembly system could detect low amount of target DNA under the interference of high concentration of spurious target.

Figure 7. The OR logic gate. (a) Symbol for the OR logic gate. (b) Image of the visible OR gate system taken after 4 h with addition of different inputs (no target, D1, D2, D1 + D2, respectively). (c) The UV-vis absorbance of the corresponding supernatant of the OR logic gate system after 4 h of reaction. (d) The truth table of the OR gate. [D1] = [D2] = 10 nM, [S1] = [S2] = 0.75 μM, [F1] = [F2] = 3.75 μM, [SNA] = 8 nM, and [duplex linker] = 160 nM.

OR Logic Gate. Multiplex detection of nucleic acids is of great significance since its convenience in simultaneously identifying multiple DNA sequences information in one assay.54 Finally, an OR logic gate was constructed by integrating two parallel upstream catalytic DNA circuits with the fixed downstream SNAs disassembly system for the purpose of multiplexed target detection to further demonstrate the facility and universality of our SNA aggregates-based disassembly strategy. The detailed system design mechanism of the OR gate is illustrated in Figure S8. The two parallel upstream DNA circuits could release the same output (trigger T) after multiple rounds of TMSDRs with the presence of two different specific input targets (disassembler 1 D1 and disassembler 2 D2). Then, as described above, the constantly accumulating T 16


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released from the upstream two circuits will cause a visible disassembly reaction of the downstream SNA aggregates. As shown in Figure 7b, the system remained steady and the supernatant of the reaction kept colorless in the absence of input targets (0, 0); while visible color change could be detected with the presence of D1 (1, 0), or D2 (0, 1), or both D1 and D2 (1, 1). In addition, the corresponding UV-vis spectra were also in accordance with the image results (Figure 7c). Therefore, our strategy showed huge advantages not only for its rapid and sensitive visual detection ability but also for its facile and universal design that is applicable to multiple DNA targets detection simultaneously in one integrated system without repreparation of new SNA aggregates.

Conclusions In summary, a facile strategy for visible disassembly of SNAs was established through integrating an upstream TMSDRs-based catalytic DNA circuit and a downstream SNA aggregates-based disassembly system. The trigger strand released from the upstream can be used to disassemble the SNA aggregates through TMSDR and also serves as a bridge to link these two subsystems together. For the construction of the downstream system, only one type of SNA molecule and a duplex linker (one end can hybridize with the DNA strand on SNA surface, and the other end is a self-complementary region composed of a palindromic sequence with six bases) are employed to form the stable SNA aggregates. This delicate design endows the downstream SNA aggregates-based system with the distinctive capability in combining with arbitrary upstream circuits without any need to change the DNA structure of the downstream SNA system, and hence it shows great potential in fabrication of facile and portable DNA diagnostic kit in a large scale. The disassembly process of the SNA aggregates in our system can be easily monitored by the naked eye through observing the color change or by UV-vis spectroscopy. After a series of optimization, this integrated catalytic system could detect a rapid color change with the presence of target of 2 nM by the naked eye in 20 min, and finally a visible signal produced by the disassembly reaction with addition of

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0.2 nM target could be detected after 4 h of reaction (visual limit of detection: 0.2 nM). In addition, our integrated system exhibited good sequence selectivity in discriminating DNA targets with single-mismatched bases. Moreover, a two-input DNA OR logic gate used for multiplex detection was constructed through integrating two different DNA circuits with a SNA aggregates disassembly system that has remained unchanged in DNA structure to prove the universality of our strategy. Compared to the traditional direct-linker-addition approach37 and our previous DNA machine-driven SNA assembly strategies,38,42,44 this catalytic SNAs disassembly strategy possesses several advantages, such as better sensitivity, shorter detection time, and cheaper and more universal design. Owing to these excellent properties, our catalytic SNAs disassembly strategy should be a very promising strategy to be applied in POC testing and other sophisticated applications of the field of DNA nanotechnology, such as complex DNA networks, and programmable phase transformation of SNA superlattices.

Experimental Section Materials. All DNA oligonucleotides (Tables S1 and S2) were ordered from Sangon Biotechnology Co., Ltd. (Shanghai, China) after careful design and analysis by NUPACK software55 to avoid any unwanted secondary structure. All unmodified oligonucleotides were firstly dissolved in ultrapure water (Millipore Co., USA) with the concentration above 100 μM and stored at -20 ℃, and then they were transferred to 0.1 M PBS buffer (10 mM PB, pH = 7.4, with 0.1 M NaCl) when using for follow-up experiments. 4S GelRed was also purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). Fetal bovine serum was ordered from Hyclone. Other chemicals were purchased from Sinopham Chemical Reagent Co., Ltd. (China) unless otherwise stated. Preparation and Purification of DNA Substrate. The triple-stranded DNA substrate was prepared by mixing linker, protector, and trigger in a molar ratio of 1:1.2:1.2, then the mixture was kept at 95 ℃ for 5 min and slowly cooled down to 25 ℃ 18


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at a rate of 0.1 ℃/s. After that, the annealed substrate was incubated with fuel in a molar ratio of 1: 0.4 at room temperature for 1 h, which was for removing of the doublestranded byproducts or misfolded triple-stranded substrate. Subsequently, the triplestranded substrate was purified by a 12% non-denaturing polyacrylamide gel electrophoresis (PAGE) as previously described.7 Finally, the purified substrate was stored at -20 ℃ for further use. Preparation of SNA. The commonly used 13 nm AuNPs were synthesized according to the previously reported method.56 The concentration of the obtained AuNPs was measured by an Agilent Cary 300 UV-vis spectrophotometer. (Santa Clara, CA, USA), and the surface plasmon resonance maximum (λmax) of AuNPs was 520 nm. Then, SNAs were prepared through functionalizing AuNPs surface by thiol-modified DNA according to previous literature.37 The prepared SNAs were finally redispersed in 0.1 M PBS buffer (10 mM PB, pH = 7.4, with 0.1 M NaCl) and stored at 4 ℃ for further use. Preparation of SNA Aggregates. At first, duplex linker was formed through hybridization of h-SNA and p-SNA in a molar ratio of 1:1.2 using the same annealing process as mentioned above. Subsequently, SNA aggregates were prepared by mixing SNA (8 nM) with duplex linker (160 nM) in a molar ratio of 1:20, and the mixture was kept at 25 ℃ for at least 8 h to obtain complete SNA aggregates (Figure S2). Melting Temperature Investigation. The melting temperature of the prepared SNA aggregates was determined by an Agilent Cary 300 UV-vis spectrophotometer. The SNA aggregates were redispersed in 0.3 M PBS buffer (10 mM PB, pH = 7.4, with 0.3 M NaCl) used for disassembly reaction at first, and then the SNA aggregates were transferred to a cuvette with addition of a magneton. Subsequently, the cuvette was placed into the UV-vis spectrophotometer equipped with a thermal control module, which could monitor the sample absorbance at 520 nm from 25 ℃ to 65 ℃ at a heating rate of 0.25 ℃/min. Typical Operating Condition for SNA Disassembly Experiment. First, the SNA aggregates were centrifuged at 6000 rpm for 30 s and the supernatants were discarded to remove excess DNA complexes and single strand DNAs. Second, the SNA 19



aggregates were redissolved in 0.3 M PBS buffer (10 mM PB, pH = 7.4, with 0.3 M NaCl and 0.01 % SDS), and followed by substrate and fuel were added. Finally, various concentrations of disassembler were added into the mixture. The disassembly process of SNA aggregates was monitored by the naked eye to observe the color change of the supernatants and color photos were taken by a cellphone at different time intervals. All samples were centrifuged at 6000 rpm for 10 s every time before taking photos. In addition, the supernatants of all samples were measured by an Agilent Cary 300 UVvis spectrophotometer after 4 times dilution to obtain the absorbance curves from 800 nm to 400 nm after 4h of reaction. The total reaction volume for each reaction sample was 30 μL, and all disassembly experiments were operated at 25 ℃. Fluorescence Experiment. To confirm the catalytic efficiency of the upstream DNA circuit, a fluorescence kinetics experiment was performed using Fluorolog-3-21 spectrofluorometer (Horiba Jobin Yvon, USA) at 25 ℃ in 0.3 M PBS buffer (10 mM PB, pH = 7.4, with 0.3 M NaCl) with addition of 100 nM substrate, 200 nM fuel, 300 nM fluorescent reporter, and different concentrations of disassembler. With the use of Cy5 fluorophore in this experiment, excitation / emission was set at 645 nm / 665 nm for kinetics characterization. Another similar fluorescence kinetics experiment was further carried out using a multi-mode microplate reader (BioTek Synergy H1, Winooski, VT, USA) with addition of 1.5 μM of S, 7.5 μM of F, and 2.0 μM of fluorescent reporter.

Supporting Information Additional experimental results and DNA sequences used in this work. This material is available free of charge via the Internet at http://pubs.acs.org/.

Acknowledgements We would like to thank the National Natural Science Foundation of China (Grant Nos. 91427304, 21434007, 21574122, and 51573175), the Fundamental Research Funds for the Central Universities (Grant Nos. WK3450000002 and WK2060200026), the 20


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Financial Grant from the China Postdoctoral Science Foundation (2018M630708 and BX20180285), and the Open Research Fund of State Key Laboratory of Polymer Physics and Chemistry (Changchun Institute of Applied Chemistry, Chinese Academy of Sciences) for their financial support.

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Graphical TOC Entry

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Facile Strategy for Visible Disassembly of Spherical Nucleic Acids

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