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A Scalable “Junction Substrate” to Engineer Robust DNA Circuits Xianbao Sun, Bing Wei, Yijun Guo, Shiyan Xiao, Xiang Li, Dongbao Yao, Xue Yin, Shiyong Liu, and HaoJun Liang J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 12, 2018

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Journal of the American Chemical Society

A Scalable “Junction Substrate” to Engineer Robust DNA Circuits Xianbao Sun, Bing Wei, Yijun Guo, Shiyan Xiao,* Xiang Li, Dongbao Yao, Xue Yin, Shiyong Liu, 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, 230026, P. R. China

ABSTRACT: Versatile building blocks are essential for building complex and scaled-up DNA circuits. In this study, we propose a conceptually new scalable architecture called the “junction substrate” (J-substrate) that is linked by pre-purified double-stranded DNA molecules. As a proof-of-concept, this novel type of substrate has been utilized to build multi-input DNA circuits, offering several advantages over the conventional substrate (referred to as the “linear substrate”, L-substrate). First, the J-substrate does not require long DNA strands, thus avoiding significant synthetic errors and costs. Second, the traditional PAGE purification method is technically facilitated to obtain high-purity substrates, whereby the initial leakage is effectively eliminated. Third, the asymptotic leakage is eliminated by introducing the “junction”. Finally, circuits with the optimized J-substrate architecture exhibit fast kinetics. We believe that the proposed architecture constitutes a sophisticated chassis for constructing complex circuits.

which focused on improving the quality of DNA substrates, such as introducing "clamp" domains12 or mismatches26 to block active terminal sites of DNA substrates, increasing energy barriers of leakage through four-way branch migration,27 or developing advanced purification methods.19 Recently, a strategy based on chemical biology, which utilizes restriction endonucleases to nick double-stranded DNA templates derived from plasmids, has been confirmed as an effective method for preparing multi-stranded substrates of high purity.15 Herein, we propose a “junction substrate” (J-substrate) architecture that has been proven to effectively suppress leakage in multiinput DNA circuits.

INTRODUCTION In dynamic DNA nanotechnology, the toehold-mediated strand-displacement reaction has been widely used in programming and executing nanoscale predesigned digital and analog dynamic devices.1,2 It constitutes a very promising method due to its unique predictability and programmability in the manufacture of autonomous molecular devices, which currently receive broad applications, ranging from smart therapeutics to complex dynamic biological event mimicking, including assistance to self-assembly of DNA tiles,3 gold nanoparticles,4,5 and proteins,6 construction of autonomous oscillators,7,8 motors,9 and walkers,10,11 programming for computation algorithms of digital logic circuits12,13 and chemical reaction networks (CRNs),14,15 molecular diagnostics of microRNA16 and proteins,17 and prototype mimicking of the adaptive immune response.18 The main challenge in this area is the undesirable leakage reaction, which occurs simultaneously with the desired reaction during the implementation of DNA circuits. This leakage becomes more severe in a scaled-up circuit due to the complex biomolecular interactions among the increased number of participating DNA strands. This creates a substantial nonspecific background that not only limits the performance of DNA circuits,15,19-21 but also impedes the manufacture of complex circuits with multiple inputs and/or outputs. Circuit leakage can be roughly classified into two categories: initial leakage and asymptotic leakage.19,20 Initial leakage is an early burst of signal primarily due to poor purity of the DNA substrate,12,22 while asymptotic leakage represents a slow but continuously growing signal in the absence of the trigger strand, which is mainly caused by the spontaneous conformational fluctuations of the DNA substrate, including end-to-end stacking23,24 and “breathing” of DNA helices.25 Considerable efforts have been exerted to prohibit circuit leakage, most of

RESULTS AND DISCUSSION Behaviors of “linear-substrate”. Conventional DNA substrate (referred to as “linear substrate”, L-substrate) structurally contains an intact long DNA strand hybridized with multiple short protector strands, and has been widely utilized to build multi-input AND gates in complex DNA circuits.15,28 In the pioneering work of Seelig et al., multi-input AND gates have been created a decade ago using the structures different from the L-substrate.29 Later, based on the theoretical design,7,14,30 Chen et al. have adopted the L-substrate to realize the multi-input AND gates, in which they used chemical biological methods instead of the traditional PAGE method to achieve high-purity L-substrate.15 In order to illustrate the necessity of our current design, we first study the conventional method for constructing multiinput circuits by designing triple- (S ), quadruple- (S ) and quintuple-stranded (S ) substrates, which are correspondingly used to fabricate two-, three-, and four-input circuits (Figure 1). These substrates are prepared in the conventional way and purified by non-denaturing PAGE after one-pot thermal an-

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nealing, which is a routine procedure of purification12,19,31,32

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with the advantages of simplicity and availability.

Figure 1. Multi-input DNA circuits based on conventional linear substrate (L-substrate). The two- (A), three- (B), and four-input (C) circuits are constructed with the L-substrates S , S and S , respectively. These substrates sequentially consume the input strands to ultimately release the signal chain (P1) that illuminates the Reporter's fluorescence through the toehold-mediated strand displacement reaction (D). The corresponding kinetics under varied concentrations (solid curves) of trigger input (I2(T), I3(T), or I4(T)) and the leakage (dashed curves) are illustrated in (E), (F), and (G). The subscript “(T)” of I2(T), I3(T), or I4(T) represents the trigger input, and the subscript “(L)” of P1(L), P2(L), P3(L), P3′(L), or P4(L) denotes the protector strands in L-substrates. The length of the toehold on L-substrates for the trigger input is 8-nt, and that for other inputs is 9-nt. [S ] = [S ] = [S ] = 1× = 15 nM, and [I1] = [I2] = [I3] = [Reporter] = 2×. The 1.0 unit fluorescence corresponds to 15 nM of triggered Reporter. Experiments were run at 25 °C in Tris-acetate-EDTA buffer containing 12.5 mM Mg2+ (1× TAE/Mg2+).

In the experiment, the S -based circuit exhibits good leakage resistance and low leakage kinetics, thereby generating an explicit positive signal in the presence of trigger input I2(T) (Figure 1E). In contrast, the S -based circuit produces high leakage (Figure 1F). In the S -based circuit, the high leakage signals nearly override the expected signals to make them almost indistinguishable (Figure 1G). These results indicate that PAGE purification is effective when used for triplestranded substrates (e.g., S ), which has been proven by previous reports.8,22,32 However, our experiments demonstrate that PAGE purification is not acceptable in the case of substrates consisting of more than three strands (e.g., S and S ). In principle, annealing multiple strands to form substrates is prone to suffer concomitant production of undesired detrimental byproducts, i.e., imperfect isoform hybridization structures (Figure S1, Supporting Information), which may result from imperfect stoichiometry due to experimental limits, such as pipetting defect, dilution inaccuracy, and concentration errors.33 These by-products, whose species increase with the number of

DNA chains in the substrate, are difficult to purify, thereby causing severe initial leakage. Obviously, our current circuits do not perform as well as those in several reports8,12,15 in kinetics and leakage profiles. This is mainly due to the unfavorable toehold setting (invading/incumbent = 8/9) for the forward step of the rate-limiting toehold-exchange reaction38 and limitations of the experimental conditions used because the process is controlled by a variety of factors, including concentrations, salts, pH, equipment and materials. Despite these limitations, we conducted experiments under the same conditions to ensure fair assessment of the improvement of the J-substrate relative to the Lsubstrate.

Design of “J-substrate” and initial leakage analysis. To circumvent this difficulty, we propose a concept of the “junction substrate” (J-substrate), a structure concatenating a set of short double-stranded motifs (Figure 2A). We designed and constructed the J-substrate by assuming that the conventional triple-stranded L-substrate was cut into two parts at the con-


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ceptual nick position. Then, two complementary singlestranded arms (i.e., , and ) were grafted at the nicked positions to create two double-stranded motifs (i.e., M1, and M2). Experimentally, each double-stranded motif was prepared separately by conventional annealing and PAGE purification. Then, the corresponding arm strands were hybridized by incubating the two purified motifs together. Considering that two short linker strands (i.e., LP1 and LP2 in Figure 2A) of the Jsubstrate replace the long DNA linker strand (i.e., L2 in Figure 2A) of the L-substrate, it should greatly reduce the synthesis cost and error because these two items rise with the increase of the DNA strand length. More importantly, due to its simple structure, the double-stranded motifs of the J-substrate are technically much easier to purify by the traditional PAGE method than the L-substrate, which is a multi-stranded complex. This improved purity benefits the inhibition of initial leakage for the J-substrate-based DNA circuit.

advantages of the J-substrate design in overcoming the asymptotic leakage, we first briefly explain the origin of such leakage in a conventional L-substrate-based two-input circuit shown in Figure 1A. There are three potential asymptotic leakage reactions due to inevitable instantaneous fraying (arising from thermal energy) of the terminal domain of strand P2 and strand P1 (Figure 2B). In the case of Type I, once strand I1 is recruited onto the frayed domain, the domain will quickly transform into a toehold, thereby causing strand I1 to displace strand P1 via toehold-mediated strand displacement. For Type II, strand I1 binds a few bases on the frayed domain, which is not sufficiently stable to serve as a toehold, but the binding portion may extend to the left and right, and eventually develop into a usable toehold for displacing strand P1. Type III has the same reaction mechanism as Type II except for displacement events from right to left. This type of leakage can be substantially reduced via adding a “clamp” at the right end,12 which will not significantly affect the kinetics of the displacing reaction.34

Asymptotic leakage analysis. Next, we analyze the asymptotic leakage in the J-substrate-based system that occurs in the absence of trigger input I2(T). In order to understand the

Figure 2. Design strategy and theoretical analysis of the DNA “junction substrate” (J-substrate). (A) Schematic of a J-substrate (SJ) created from a conventional L-substrate (SL). (B) Three types of the asymptotic leakage (Type I, Type II, and Type III) in the S -based doubleinput circuit shown in Figure 1A. (C) Corresponding asymptotic leakage reactions in the S -based two-input circuit. (D) Intuitive energy landscape (IEL) of the Type I asymptotic leakage in both circuits, derived from the model proposed by Srinivas et al.35 The S -based Type I asymptotic leakage reaction depicted in (B) starts from fraying of a base pair at the 3′ end of P2 (state A), allowing the binding of input I1 onto strand L2 (state B). After nucleation (state C), the red toehold domain is formed between I1 and L2 (state D) and then proceedingly represented by the “sawtooth amplitude” in the free energy profile. Afterwards, I1 disrupts the first base pair of the 5′ end of hybridized P1 and initiates the subsequent strand displacement. If there is a three-way junction (called “junction”) as shown in (C), the input I1 requires an energy penalty of ∆G ‡ = 4.26 kcal/mol to cross the “junction”. Adapted from the calculations of Srinivas et al.,35 ∆G = -1.7 kcal/mol, ∆G = 11.9 kcal/mol, ∆G = 1.2 kcal/mol, ∆G = 2.6 kcal/mol, ∆G ∆G ∆G .

From the perspective of structure, the J-substrate has an extra three-way junction (called “junction”) compared to the Lsubstrate (Figure 2A). The “junction” acts as an obstacle that induces two effects: (i) preventing strand I1 from crossing the “junction” (Figure 2C, left panel); and (ii) keeping the binding portion from extending toward the left to develop into a longer

toehold (Figure 2C, middle and right panel). Both effects are conducive to resisting asymptotic leakage. When a strand displacement reaction occurs, the “junction” may be approximately (not exactly) considered as a mismatch on the Lsubstrate, causing additional energy barriers. Jiang et al.26 have recently developed a method for suppressing the asymptotic



leakage of catalytic hairpin assembly (CHA) circuits by introducing mismatched base pairs to block active sites adjacent to the ends of hairpin duplexes, resulting in an enhanced signalto-background ratio up to 100-fold.

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action on P1 before it dissociates from the bound toehold of length & (see text S4 of Supporting Information for details). Compared to the L-substrate, the “junction” causes an excess barrier of ∆G ‡ in the J-substrate, which corresponds to a decrease of exp %+∆, ‡ /RT' in the overall reaction rate during displacing strand P1 by I1. According to the estimations of Chen36 and Genot,37 it may exhibit more than three orders of magnitude at the energy barrier of ∆, ‡ = 4.26 kcal/mol. Thus, the asymptotic leakage should be effectively suppressed by the introduction of this “junction”. For Type II and Type III leakage, there is no discrepancy between L-substrates and the corresponding J-substrates. More specifically, these two leakage are zero-toehold-mediated strand displacement reactions, which have been experimentally measured to be extremely low.38 During “J-substrate” optimization, one base is exposed (t3 domain in Figure 3B) to serve as a sticky point to facilitate the desired reactions. Although the introduction of t3 would also contribute to the Type II leakage for the J-substrate, this effect is negligible. Our simulations show that complex Jsubstrate-based circuits containing more “junctions” are able to result in greater degrees of leakage suppression than the corresponding L-substrate-based circuits (see text S4 of Supporting Information for details), indicating that our strategy offers considerable advantages in manufacturing multi-input circuits that are prevalent in large-scale complex networks. In brief, the J-substrate-based approach not only facilitates the manufacturing process and inhibits initial leakage, but also effectively suppresses asymptotic leakage, revealing the merit of this design.

Figure 3. Optimization and behavior of the J-substrate S in the two-input circuit. (A) Schematic of the operation of the S -based two-input circuit: the trigger input I2(T) initiates the reaction and releases the signal strand P1(J) with the attendance of input I1 to light the fluorescence of the Reporter displayed in Figure 1D. Herein, the subscript “(J)” of P1(J) or P2(J) denotes the protector strands in J-substrates. (B) Illustration of parameters (t1, t2, t3, t4, t5, Tn, and l) of S for optimization. (C-F) Circuit kinetics versus different variables, including “junction” length l, bulged thymidine number Tn, incubation time, and ratio of motifs ([M2]/[M1]). (G) List of the optimized toehold lengths (Figure S4, Supporting Information). (H) Kinetics of the optimized S -based circuit. (I) Background-to-signal (B/S) ratios of the S - and S -based twoinput circuits, calculated by dividing the output of the leakage reaction (S only, or S + I1) by the signal output of the desired reaction (S + I1 + 15 nM I2(T)) at 1 h. For simplicity, S and S are referred to as S in the reaction expressions. (J) Signal levels (yields of P1) at 1 h for different amounts of input I2(T). The dotted line indicates correct reaction stoichiometry. In these experiments, [S ] = [S ] = 1× = 15 nM, [I1] = [Reporter] = 2×, and 1.0 unit corresponds to 15 nM of triggered Reporter. Experiments were run at 25 °C in Tris-acetate-EDTA buffer containing 12.5 mM Mg2+ (1× TAE/Mg2+).

“J-substrate” optimization. In the experiment, we constructed a two-input circuit based on J-substrate (S ) through concatenating two double-stranded motifs M1 (P1(J)/LP1) and M2 (P2(J)/LP2) shown in Figure 3A (the sequences of strands are listed in Table S1 of Supporting Information). To ensure its stability and reactivity, the S -based circuit has been optimized according to the following parameters, as indicated in Figure 3B: “junction” length l; bulged thymidine number Tn; incubation time; motif ratio ([M2]/[M1]); and toehold lengths (uncovered toeholds t1, t2, and t3, and covered ones t4 and t5). First, when the “junction” length l is longer than 15 bp, we observed an undifferentiated kinetics change (Figure 3C), indicating that 15 bp is a good choice to balance stability, reactivity, synthesis correctness, and economy. In the case of 0-bp “junction” length, M1 and M2 cannot form an integrated Jsubstrate, and the recorded fluorescence signal represents the leakage between I1 and M1. Considering that several bulged bases at the terminal of the “junction” (Tn in Figure 3B) facilitate the stabilization of the three-way junction structure39,40 and the acceleration of the entire strand-displacement reaction,36 two bulged thymidines were finally chosen after several trials, and good kinetics and yield were obtained (Figure 3D). A similar kinetic profile of incubation for 10 and 40 min denotes a rapid hybridization reaction between M1 and M2 motifs (Figure 3E). Similar kinetic profiles for different concentration ratios of M2 to M1 of 1 : 1, 1.1 : 1, and 1.2 : 1 suggest an ingredient of an equal amount of M1 and M2 motifs (Figure 3F). Finally, to ensure circuit reactivity, the toehold lengths (t1, t2, t3, t4, and t5 shown in Figure 3B) were optimized (Figure S4, Supporting Information), and the corresponding optimal values are listed in the table of Figure 3G.

On the basis of the model proposed by Srinivas et al.,35 we theoretically analyze the asymptotic leakage reactions by deriving the initiative energy landscape (IEL) (Figure 2D and Figure S2 of Supporting Information). The leakage reaction comprises two steps: (i) a bimolecular reaction in which input I1 binds to the frayed bases of strand L2; and (ii) a unimolecular displacement of strand P1 through I1. The rate constant of Type I leakage in the L-substrate (Figure 2B) is estimated using !"#| %&' , where is the rate constant of the collision between strands I1 and L2 during terminal fraying of the hybridization of P2 and L2, and !"#| %&' represents the probability that strand I1 exerts a displacement

Two-input circuit. With the optimized J-substrate, a twoinput circuit (S -based) was rebuilt to characterize its perfor-


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mance. The corresponding L-substrate-based two-input circuit (S -based) presented in Figure 1A was compared in parallel. The leakage reactions in the absence of trigger input I2(T) in both S -based and S -based circuits show very slow kinetics, as depicted by black dashed curves (S only) and wine dashed curves (S + I1) in Figure 3H and 1E, respectively. The background-to-signal (B/S) ratios of the leakages (S only, and S + I1) to the desired signal (S + I1 + 15 nM I2(T)) in the S -based circuit at 1 h are estimated to be 0.07 and 0.12, respectively, which are only half of those in the S -based circuit (0.13 and 0.26, respectively) (Figure 3I), indicating an improved leakage resistance of the J-substrate-based circuit.

circuit (Figure 3H) were significantly faster than those in the S -based circuit (Figure 1E). This result indicates the unique merit of this architecture. It is well known that suppressing leakage in DNA-based circuits should facilitate the measurement of the desired signal. In practice, however, it is difficult, if not impossible, to prevent leakage reactions. Accordingly, researchers often use some compromise strategies to control leakages at the expense of reaction speed,5,26,27 achieving a trade-off between leakage and kinetics. In other words, it is highly desirable to eliminate leakage while maintaining or promoting fast kinetics. Kotani and Hughes have designed the “multi-arm junction” catalytic circuits in which the energy barrier of the leakage pathway was elevated through a fourway branch migration, while the energy barriers of catalytic pathway remained at the level of conventional three-way branch migration.27 Compared with the corresponding linear substrates, they realized a high ratio of the catalytic to the leakage rate constants (kcat/kleak). However, for multi-input AND gate, such a method does not yet exist. Now, this problem is solved by this J-substrate design.

We next examined the performance of the S -based circuit in the presence of the trigger input I2(T). By measuring the output at 1 h, the signal amount generated from the S -based circuit is close to the amount of input strand I2(T) (Figure 3J), exhibiting the correct stoichiometry characteristics. The S based circuit, however, does not produce good stoichiometric behavior. In terms of reaction kinetics, we simply expected that this J-substrate would not exert a severe adverse effect on the desired reactions so that the kinetics might not be too slow. Surprisingly, we observed that the kinetics in the S -based

Figure 4. Behaviors of three- (A) and four-input (B) circuits fabricated with J-substrates S and S , respectively. For each circuit, panel (I) shows the schematic. The inputs bind to the substrates sequentially, and finally release the signal strand P1(J) through toehold-mediated strand displacement to light the fluorescence of the Reporter (Figure 1D). Corresponding L-substrate-based circuits are shown in Figure 1. Panel (II) presents the circuit kinetics profiles: solid and dashed curves correspond to the reactions in the presence and absence of the trigger input (I3(T) or I4(T)), respectively. Panel (III) shows background-to-signal (B/S) ratios, calculated in a manner that is consistent with that in Figure 3. For simplicity, S and S are referred to as S. In these experiments, [S ] = [S ] = 1× = 15 nM, [I1] = [I2] = [I3] = [Reporter] = 2×, and 1.0 unit corresponds to 15 nM of triggered Reporter. Experiments were run at 25 °C in Tris-acetate-EDTA buffer containing 12.5 mM Mg2+ (1× TAE/Mg2+).

In fact, the key to the design is the introduction of the “junction” into the J-substrate. It plays an important role in suppressing both initial and asymptotic leakage as mentioned above. Conceivably, it could also be a stumbling block to the expected toehold-mediated strand-displacement reaction. This is true for the pure “junction” case, which leads to a very slow kinetics as shown in black curve (0T) in Figure 3D. However, when adding two bulged T bases at the terminal of “junction”, a significant improvement in the reaction, or fast kinetics is obtained (red curve (2T) in Figure 3D). This fast kinetics was

also observed in Chen's earlier report of the “associative toehold” strategy, where the introduction of 2-T bases bulge is thought to enhance three-way junction through the coaxial stacking of two of the three duplexes as indicated by Figure S6a in reference,36 thereby accelerating the toehold-mediated strand displacement reaction. Now it is very clear and interesting: as shown in Figure 2C, before complete binding of the red-domain of strand input (I1) with its corresponding complementary red-domain in J-substrate, the three-way junction has not yet formed and at this stage the “junction” simply play



a role of a “mismatch” to suppress asymptotic leakage. However, upon totally exposing the red-domain in J-substrate after displacing protector P2(J) by input (I2(T)) as shown in Figure 3A, red-domain in input (I1) will quickly hybridize onto the Jsubstrate to form a three-way junction structure, thus accelerating the reaction by the two-T bases bulge structure. On the other hand, the particular architecture of the J-substrate allows several exposed bases (t2 in Figure 3B) in the region near the “junction” (occurring in the L-substrate will cause severe leakage), which in turn result in a shortened incumbenttoehold that exerts a relatively weak resistance against the forward displacement reaction, and ultimately gain an accelerated reaction. For example, the protector strand P2(J) in S (Figure 3A) allows three-base shorter than its counterpart in S (P2(L) in Figure 1A) without yielding severe leakage. Thus, we reasonably conceived that the two-T base bulge and this particular shortened incumbent-toehold are a critical factor responsible for the fast reaction.38

Three- and four-input circuits. In the following, we examined the behavior of the scaled-up circuits, such as the threeand four-input circuits (S - and S -based circuits) illustrated in Figure 4A and 4B, respectively. For the S -based circuit containing three double-stranded motifs (Figure 4A), the reaction begins once the trigger input I3(T) is added, first peeling off the protector strand P3(J) to expose the incumbent toehold, allowing two other input strands (I2 and I1) to sequentially displace strand P2(J) and signal strand P1(J). Notably, the S -based circuit achieves faster kinetics than the corresponding L-substrate-based three-input circuit (S based circuit). With 15 nM input I3(T) added, it generates an approximately 15 nM output signal in the S -based circuit (Figure 4A, II) at 1 h, but only 11.3 nM (75% of 15 nM) in the S -based circuit (Figure 1F). Analogous to the S -based twoinput circuit, this S -based three-input circuit exhibits a good stoichiometry by yielding approximately the same quantity of output signal as input I3(T) at 1h (Figure S5, Supporting Information), while the corresponding S -based circuit yields poor stoichiometry. We notice the leakages in excess of 20% of the total signal in the corresponding L-substrate-based three-input circuit after the 1 h reaction (Figure 1F). For this S -based circuit, the kinetics profiles between the desired reaction of 0.45 nM (0.03×) input I3(T) and the leakage (S + I1 + I2) are nearly the same due to the severe leakage, i.e., the output signal is completely overwhelmed by the leakage. By contrast, desired and leakage signals remain distinguishable from each other in the S -based circuit (Figure 4A, II), revealing the efficacy of the S -based circuit in suppressing the leakage. In the absence of input I3(T), the leakage outputs in the S -based circuit, which correspond to the cases of S only, S + I1, and S + I1 + I2, are 4, 11 and 13% of 15 nM input I3(T), respectively. These values are all lower than those (~30%) in the S -based circuit (Figure 4A, III). Thus, the S -based circuit composed of the three double-stranded complex can still retain good stoichiometry, fast kinetics, and low leakage. The J-substrate-based four-input circuit (S -based), driven by four inputs I4(T), I3, I2, and I1 (Figure 4B, I, sequences are shown in Table S2 of Supporting Information), exhibits slow kinetics (Figure 4B, II), which reflects the inherent characteristics of multi-cascaded toehold-mediated strand displacement reactions. This retardation feature has been observed in the S -

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based circuit system, in which the desired reactions exhibit slow kinetics similar to the leakage reactions (Figure 1G), indicating that not only the leakage reactions are suppressed, but the desired reactions are retarded as well. Despite the retardation, the S -based system still yields sufficiently high outputs after adding trigger input strand I4(T), achieving a substantial deviation between desired and leakage signals (Figure 4B, II). This deviation is much higher than that in the S -based circuit (Figure 1G). Strikingly, the S -based circuit behaves much better against leakage by suppressing all of the B/S ratios to less than 20%, while the leakage (S + I1 + I2 + I3) in the S -based circuit is 81% (Figure 4B, III).

CONCLUSIONS In the present work, we have developed a conceptually new structure called the “junction substrate” (J-substrate), which is created by hybridizing complementary dangling strands on pre-purified double-stranded DNA molecules. Considering that the scalable building module (i.e., double-stranded motif) contains only short DNA strands, it can bypass the difficulties of synthesizing long DNA strands that are prone to severe error and cost. PAGE purification, a routine skill in conventional chemistry laboratories, is technically acceptable to remove non-hybridized single strands in the annealed product, thereby helping to eliminate initial leakage. Furthermore, the introduction of the “junction” in the substrate allows versatile design of the DNA circuits. First, the “junction” can effectively impede the ambient strands (such as input I1 and I2 in Figure 4A) to bind into the frayed region near the “junction”, thus substantially suppressing asymptotic leakage. Second, the “junction” enables an introduction of two-T bases bulge at the terminal of “junction” (Tn in Figure 3B) , and a short, exposed domain near the “junction” (t2 in Figure 3B), which results in fast kinetics of the overall reaction without causing severe leakage. As a proof-of-concept, the proposed J-substrate has been utilized to build scaled-up circuits with three and four inputs. Compared with the conventional L-substrate-based counterparts, the J-substrate-based circuits achieve significant improvements with lower leakage and faster kinetics. On the other hand, as noted that after peeling off the first strand (P2(J)), the further toehold-mediated strand-displacement reaction is consistent with these of “associative toehold” strategy reported by Chen36 and the “combinatorial toehold” reported by Genot et al.41 Considering the sophisticated applications of these structures, we expect that our current strategy should be applicable for fabricating complex and scaled-up circuits to achieve autonomous dynamic behaviors. Recently, Wang et al.42 have developed a multi-stranded modular hybridization probe (M-Probe), which was also constructed with multiple functional double-stranded segments, enabling sequence-selectively binding of long, complex and repetitive nucleic acids. This result implies the feasibility of our strategy for potential applications in diagnosis.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge on the ACS Publications website. Detailed experimental methods and theoretical analyses of the initiative energy landscape (IEL) model of the involved DNA circuits, additional experimental and theoretical results, and DNA sequences (PDF)

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AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT We would like to thank the National Natural Science Foundation of China (Grant Nos. 21434007, 91427304, 21574122, and 51573175), the Fundamental Research Funds for the Central Universities (Grant Nos. WK3450000002 and WK2060200017), and the Open Research Fund of State Key Laboratory of Polymer Physics and Chemistry (Changchun Institute of Applied Chemistry, Chinese Academy of Sciences).

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(23) Nakata, M.; Zanchetta, G.; Chapman, B. D.; Jones, C. D.; Cross, J. O.; Pindak, R.; Bellini, T.; Clark, N. A. Science 2007, 318, 1276. (24) Maffeo, C.; Luan, B. Q.; Aksimentiev, A. Nucleic Acids Res. 2012, 40, 3812. (25) Frank-Kamenetskii, M. D. Nature 1987, 328, 17. (26) Jiang, Y. S.; Bhadra, S.; Li, B. L.; Ellington, A. D. Angew. Chem., Int. Ed. 2014, 53, 1845. (27) Kotani, S.; Hughes, W. L. J. Am. Chem. Soc. 2017, 139, 6363. (28) Zhang, D. Y.; Seelig, G. Nat. Chem. 2011, 3, 103. (29) Seelig, G.; Soloveichik, D.; Zhang, D. Y.; Winfree, E. Science 2006, 314, 1585. (30) Cardelli, L. Math. Struct. Comp. Science 2013, 23, 247. (31) Zhang, D. Y.; Winfree, E. J. Am. Chem. Soc. 2008, 130, 13921. (32) Zhang, D. Y.; Turberfield, A. J.; Yurke, B.; Winfree, E. Science 2007, 318, 1121. (33) Thubagere, A. J.; Thachuk, C.; Berleant, J.; Johnson, R. F.; Ardelean, D. A.; Cherry, K. M.; Qian, L. L. Nat. Commun. 2017, 8, 14373. (34) Machinek, R. R. F.; Ouldridge, T. E.; Haley, N. E. C.; Bath, J.; Turberfield, A. J. Nat. Commun. 2014, 5, 5324. (35) Srinivas, N.; Ouldridge, T. E.; Sulc, P.; Schaeffer, J. M.; Yurke, B.; Louis, A. A.; Doye, J. P. K.; Winfree, E. Nucleic Acids Res. 2013, 41, 10641. (36) Chen, X. J. Am. Chem. Soc. 2012, 134, 263. (37) Genot, A. J.; Zhang, D. Y.; Bath, J.; Turberfield, A. J. J. Am. Chem. Soc. 2011, 133, 2177. (38) Zhang, D. Y.; Winfree, E. J. Am. Chem. Soc. 2009, 131, 17303. (39) Leontis, N. B.; Kwok, W.; Newman, J. S. Nucleic Acids Res. 1991, 19, 759. (40) Overmars, F. J. J.; Pikkemaat, J. A.; van den Elst, H.; van Boom, J. H.; Altona, C. J. Mol. Biol. 1996, 255, 702. (41) Genot, A. J.; Bath, J.; Turberfield, A. J. Angew. Chem., Int. Ed. 2013, 52, 1189. (42) Wang, J. X. S.; Yan, Y. H.; Zhang, D. Y. Nat. Chem. 2017, 9, 1222.



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Figure 1. Multi-input DNA circuits based on conventional linear substrate (L-substrate). The two- (A), three(B), and four-input (C) circuits are constructed with the L-substrates S_L^2, S_L^3 and S_L^4, respectively. These substrates sequentially consume the input strands to ultimately release the signal chain (P1) that illuminates the Reporter's fluorescence through the toehold-mediated strand displacement reaction (D). The corresponding kinetics under varied concentrations (solid curves) of trigger input (I2(T), I3(T), or I4(T)) and the leakage (dashed curves) are illustrated in (E), (F), and (G). The subscript “(T)” of I2(T), I3(T), or I4(T) represents the trigger input, and the subscript “(L)” of P1(L), P2(L), P3(L), P3′(L), or P4(L) denotes the protector strands in L-substrates. The length of the toehold on L-substrates for the trigger input is 8-nt, and that for other inputs is 9-nt. [S_L^2] = [S_L^3] = [S_L^4] = 1× = 15 nM, and [I1] = [I2] = [I3] = [Reporter] = 2×. The 1.0 unit fluorescence corresponds to 15 nM of triggered Reporter. Experiments were run at 25 °C in Tris-acetate-EDTA buffer containing 12.5 mM Mg2+ (1× TAE/Mg2+). 165x137mm (300 x 300 DPI)

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Figure 2. Design strategy and theoretical analysis of the DNA “junction substrate” (J-substrate). (A) Schematic of a J-substrate (SJ) created from a conventional L-substrate (SL). (B) Three types of the asymptotic leakage (Type I, Type II, and Type III) in the S_L^2-based double-input circuit shown in Figure 1A. (C) Corresponding asymptotic leakage reactions in the S_J^2-based two-input circuit. (D) Intuitive energy landscape (IEL) of the Type I asymptotic leakage in both circuits, derived from the model proposed by Srinivas et al.35 The S_L^2-based Type I asymptotic leakage reaction depicted in (B) starts from fraying of a base pair at the 3′ end of P2 (state A), allowing the binding of input I1 onto strand L2 (state B). After nucleation (state C), the red toehold domain is formed between I1 and L2 (state D) and then proceedingly represented by the “sawtooth amplitude” in the free energy profile. Afterwards, I1 disrupts the first base pair of the 5′ end of hybridized P1 and initiates the subsequent strand displacement. If there is a three-way junction (called “junction”) as shown in (C), the input I1 requires an energy penalty of ∆G^‡ = 4.26 kcal/mol to cross the “junction”. Adapted from the calculations of Srinivas et al.,35 〖∆G〗_bp = -1.7 kcal/mol, ∆G_init = 11.9 kcal/mol, ∆G_p = 1.2 kcal/mol, ∆G_s = 2.6 kcal/mol, 〖∆G〗_(s+p)=∆G_s+∆G_p. 165x100mm (300 x 300 DPI)


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Figure 3. Optimization and behavior of the J-substrate S_J^2 in the two-input circuit. (A) Schematic of the operation of the S_J^2-based two-input circuit: the trigger input I2(T) initiates the reaction and releases the signal strand P1(J) with the attendance of input I1 to light the fluorescence of the Reporter displayed in Figure 1D. Herein, the subscript “(J)” of P1(J) or P2(J) denotes the protector strands in J-substrates. (B) Illustration of parameters (t1, t2, t3, t4, t5, Tn, and l) of S_J^2 for optimization. (C-F) Circuit kinetics versus different variables, including “junction” length l, bulged thymidine number Tn, incubation time, and ratio of motifs ([M2]/[M1]). (G) List of the optimized toehold lengths (Figure S4, Supporting Information). (H) Kinetics of the optimized S_J^2-based circuit. (I) Background-to-signal (B/S) ratios of the S_J^2- and S_L^2-based two-input circuits, calculated by dividing the output of the leakage reaction (S only, or S + I1) by the signal output of the desired reaction (S + I1 + 15 nM I2(T)) at 1 h. For simplicity, S_J^2 and S_L^2 are referred to as S in the reaction expressions. (J) Signal levels (yields of P1) at 1 h for different amounts of input I2(T). The dotted line indicates correct reaction stoichiometry. In these experiments, [S_J^2] = [S_L^2] = 1× = 15 nM, [I1] = [Reporter] = 2×, and 1.0 unit corresponds to 15 nM of triggered Reporter.



Experiments were run at 25 °C in Tris-acetate-EDTA buffer containing 12.5 mM Mg2+ (1× TAE/Mg2+). 76x91mm (300 x 300 DPI)


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Figure 4. Behaviors of three- (A) and four-input (B) circuits fabricated with J-substrates S_J^3 and S_J^4, respectively. For each circuit, panel (I) shows the schematic. The inputs bind to the substrates sequentially, and finally release the signal strand P1(J) through toehold-mediated strand displacement to light the fluorescence of the Reporter (Figure 1D). Corresponding L-substrate-based circuits are shown in Figure 1. Panel (II) presents the circuit kinetics profiles: solid and dashed curves correspond to the reactions in the presence and absence of the trigger input (I3(T) or I4(T)), respectively. Panel (III) shows background-tosignal (B/S) ratios, calculated in a manner that is consistent with that in Figure 3. For simplicity, S_J^3 and S_J^4 are referred to as S. In these experiments, [S_J^3] = [S_J^4] = 1× = 15 nM, [I1] = [I2] = [I3] = [Reporter] = 2×, and 1.0 unit corresponds to 15 nM of triggered Reporter. Experiments were run at 25 °C in Tris-acetate-EDTA buffer containing 12.5 mM Mg2+ (1× TAE/Mg2+). 165x83mm (300 x 300 DPI)


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