Cooperative Toehold: A Mechanism To Activate DNA Strand

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Cooperative Toehold: A Novel Mechanism to Activate DNA Strand Displacement and Construct Biosensors Pan Hu, Mengmeng Li, Xijiao Wei, Bin Yang, Ye Li, Chun-Yan Li, and Jun Du Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01202 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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Analytical Chemistry

Cooperative Toehold: A Novel Mechanism to Activate DNA Strand Displacement and Construct Biosensors Pan Hu, Mengmeng Li, Xijiao Wei, Bin Yang*, Ye Li, Chun-Yan Li, Jun Du

Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan 410005, P. R. China

* To whom correspondence should be addressed. E-mail: [email protected]

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract Toehold-mediated DNA strand displacement has proven powerful in the construction of various DNA circuits, DNA machines, and biosensors. So far, many new toehold activation mechanisms have been developed to achieve programmed DNA strand displacement behaviors. However, almost all those toeholds are inflexible via either a covalently attached manner or a complementary hybridization strategy, which limit the versatility of DNA devices. To solve this problem, we developed a new toehold, named “cooperative toehold”, to activate DNA strand displacement. Based on a base stacking mechanism, the cooperative toehold is comprised of two moieties with completely independent DNA sequences between each other. The cooperative toehold enabled to continuously tune the rate of DNA strand displacement, as well as more sophisticated strand displacement reactions. The cooperative toehold has also been employed as a universal signal translator for biosensors to qualitatively determine RNA and ATP. Moreover, as a novel specific PCR monitoring system, cooperative toehold-mediated DNA strand displacement can detect the pUC18 plasmid in genomic DNA samples with an aM detection limit.


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Introduction Toehold-mediated DNA strand displacement is a process that two DNA strands hybridize with each other, and displace one prehybridized strand from the double-stranded substrate.1 The reaction route can easily be predicted, because the displacement is always initiated at a short single-stranded“toehold” domain and further carried out in a fast “base-by-base” programmable manner. This simple yet powerful principle has enabled to develop a dreamy set of DNA devices, including logic gates,2 DNA machines,3 chemical amplifiers,4,5 and high-order circuits.6,7 Moreover, biosensors based on this principle were targeted at proteins, synthetic nucleic acids,8 microRNA,9 small molecules10 and metal ions. The resulting non-enzymatic DNA devices have also been employed in various intracellular bio-assay applications, such as a nanoflare for RNA imaging, immunofluorescence labeling,11 a molecular automata for markers evaluation,12 and biomedical diagnostics in vivo.5 In the design of a DNA device based on the above-mentioned principle, a key consideration is the programmed and controllable activation of a toehold.1 This goal is usually achieved by first hiding a toehold in an inter- or intramolecular DNA duplex, which prevents the toehold from reacting to its intended target sequence. In the presence of external stimuli or toehold-exchange reactions, the toehold was then reactivated and participated in the following DNA strand displacement.1 Enriching the toolbox of DNA strand displacement with alternative strategies for toehold activation will not only help to improve the programmability of this process, but also be beneficial to developing DNA devices with higher complexity. Therefore, many researchers have made efforts to develop various new toeholds, including hidden toehold,13 metallo-toehold,14 DNA tetraplexes-based toehold,15 photocontrollable toehold,16 pH-Controlled toehold,17 remote toehold,18 associative toehold,19 allosteric toehold.20 Although powerful and robust, these strategies also have several drawbacks which limit the possible architecture of DNA circuits and the versatility of DNA devices. On the one hand, the linkage between the toehold and the branch migration domain is usually covalently “hardwired” during DNA synthesis.1318

Because of this inflexibility, it is difficult to employ an additional external control for further

regulation during the execution of DNA circuits. On the one hand, most of existing toeholds are either ACS Paragon Plus Environment

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limited to the operation with DNA double-stranded hybridization19 or based on the molecular recognition of specific targets.13,15 The resulting sequence-dependent feature limits the possible complexity of DNA devices and the versatility of DNA scaffold toward the control of DNA strand displacement. For example, allosteric toehold splits an input DNA strand into a toehold domain and a branch migration domain, allowing the flexible regulation of strand displacement.20 But the toehold domain and the branch migration domain have the same sequence of seven bases, and these two domains cannot be optionally and dependently designed. As a result, it is ideal to create new general toehold with independent sequence with the DNA branch migration domain, allowing for more flexible controlling DNA strand displacement reactions. In this study, we developed a novel type of toehold, named “cooperative toehold”, to activate DNA strand displacement. In the cooperative toehold, two DNA moieties with completely independent sequences could cooperatively activate DNA strand displacement reaction via a base stacking mechanism. The cooperative toehold was proved to not only continuously tune the rate of DNA strand displacement, but also work together with other toehold principle such as remote toehold. microRNA and ATP were successfully determined using the cooperative toehold as a universal biosensing strategy. Moreover, the cooperative toehold was demonstrated as a novel specific PCR monitoring system to ultra-sensitive detection of pUC18 plasmid. EXPERIMENTAL SECTION Materials and Instrumentation. DNA oligonucleotides were synthesized by Sangon Biotechnology Co., Ltd (Shanghai, China) and purified by high-performance liquid chromatography (HPLC). The DNA sequences were listed in Table S1 (Supporting Information). Water was doubly purified from a Ultrapure Milli-Q system. Tris/Mg2+ buffer (40 mM Tris-hydrochloric acid, 1 mM EDTA, 12.5 mM MgCl2, pH balanced to 8.0) was used as the reaction medium. Stains-all was obtained from BBI life sciences and other reagents of electrophoresis were obtained from Sangon Biotechnology. Real-time fluorescence measurements were carried out at 25 °C on a QM40 Fluorescence Spectrophotometer (PTI, USA). The fluorescence was excited at 480 nm and detected at 520 nm (optimal signal for FAM ACS Paragon Plus Environment

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fluorophore) with a slit of 2 nm. Real-time Fluorescence Measurements. Samples were formed via slow annealing: the mixture solution was heated to 95 °C for 5 min and then slowly cooled to room temperature. Real-time fluorescence measurements were conducted in a reaction volume of 100 µL. Taking the data in Figure 1 for example, 95 µL annealed mixture (50 nM F, 50 nM Q and varying concentrations of T-1) as added to a cuvette, and the real-time fluorescence intensity was monitored. Then 5 µL 100 nM R was introduced using micro-syringe, and the sample was incubated until the fluorescence signal reached an equilibrium. For other experiments, real-time fluorescence measurements were carried out in a similar way. Error bars show the standard deviation of three experiments. The relative fluorescence response was defined as the fluorescence intensity divided by the initial one. The end-point time for fluorescence measurements was 1 hour. Fluorescence Normalization. In Figure 1, fluorescence intensity was normalized so that 1 unit (n.u.) of fluorescence corresponded to 1 nM of an unquenched F (the FAM-labeled strand). This normalization is based on the different fluorescence levels of F using a positive control (P.C.) containing 50 nM F, 50 nM T-1, 50 nM R and a negative control (N.C.) containing 50 nM F, 50 nM T-1, 50 nM Q. The the effective rate constant k can be estimated by the second-order rate equation:

1 [ R ][ FQ ]0 ln = kt [ R ]0 − [ FQ ]0 [ R ]0 [ FQ ] [R]0 and [FQ]0 are initial concentrations of oligonucleotides R and FQ,

[R] and [FQ] are the

real-time concentrations of oligonucleotides R and FQ at time t.

Native Gel Electrophoresis. The 12% polyacrylamide gels were prepared using TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA). A volume of 10 µL of reaction mixture with loading buffer (1×) was carefully added to each lane and the resulting native polyacrylamide gels were run at 110 V for 100 min. Then the gels were stained with Stains-all solution and finally pictures were obtain after the purple colour in the gels faded. ACS Paragon Plus Environment

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Symmetric PCR on pUC18 plasmid. Symmetric PCR on the pUC18 plasmid was carried out with a forward primer (M13M3) and a reverse primer (M13RV). The reaction was performed with a final concentration of 1 µL of each primer, 20 µL Tag PCR Master (2×), and varying concentration of pUC18 plasmid in a final volume of 40 µL. The reaction protocol was 95℃ for 1 min, and 35 cycles of 95℃ for 10 s, 55℃ for 30 s, and 72℃ for 30 s on a TC1000-G PCR Thermal Cycler (SCILOGEX).

RESULTS AND DISCUSSION Principle of cooperative toehold. As shown in scheme 1A, cooperative toehold contains two oligonucleotides of independent sequence which can cooperatively regulate DNA strand displacement reaction via a base stacking mechanism. Due to the short (e.g., 4 nucleotide) length of toehold motif (domain 2*), invading strand R can't efficiently displace strand Q away (Scheme 1A). By employing a prehybridization step with oligonucleotide T-1 complementary to the single-stranded motif of F (domain 3) and adjacent to the toehold motif (domain 2), coaxial adjacent stacking at the nick between T-1 and R brings additional stability of the toehold (Scheme 1A, red brackets). Base stacking is the dipole-dipole interaction between the planar aromatic bases in two adjacent nucleotides, and it is among the two essential and important parameters which contribute to the major forces for DNA duplex stability (Scheme 1B).21,22 This additional stability can activate the toehold which further regulate DNA strand displacement reaction to displace strand Q away (Scheme 1A). In DNA sequences design, every individual strand was completely unstructured, and different domains also possess minimal crosstalk. The resulting cooperative toehold mechanism can be expressed as the following reactions: Reaction 1: FQ + T-1 → (T-1)FQ Reaction 2: (T-1)FQ + R →(T-1)FR + Q The net reaction of the cooperative toehold system is thus T-1 + FQ + R → (T-1)FR + Q

(Scheme 1) ACS Paragon Plus Environment

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Scheme 1. (A) Principle of cooperative toehold-mediated DNA strand displacement. (B) Schematic illustration of coaxial base stacking at the nick in the red brackets of (A). Firstly, the real-time fluorescence response in varying concentrations of T-1 from 0 nM to 200 nM was monitored. In Figure 1A, very slow DNA strand displacements are observed without T-1 (black curve), indicating that the short (e.g., 4 nt) length of toehold motif (domain 2*) in R is not strong enough to efficiently initiate the strand displacements with FQ. The initial rates was determined to be 1.2×10-12 M/s (Figure 1B), and the effective rate constant k was 4.0×102 M 1 s 1 by fitting the kinetic data into the −

−

second-order rate equation (Figure S1). When the concentrations of T-1 were increased from 0 to 200 nM, the strand displacement initial rates gradually increased to 1.78×10-10 M/s (Figure 1A and 1B). This system was also served as a biosensor for DNA detection, and the detection limit was estimated to be 0.5 nM (signal-to-noise ratio >3), with a linear range of 1−10 nM, R2 =0.997 (Figure 1B). The effective rate constant k of cooperative toehold-mediated strand displacements was 5.8×104 M 1 s 1 with a two orders of −

−

magnitude change (Figure 1C). Furthermore, we employed native gel electrophoresis to demonstrated the response process: the higher concentrations of T-1 resulted in an decrease of the FQ and R-d band, while more reaction product appeared (Figure 1D).

(Figure 1)

Figure 1. (A) Real-time fluorescence monitoring in solutions containing 50 nM FQ, 100 nM R, and varying concentrations of T-1 from 0 nM to 200 nM. R was added at 3 min to initiate the DNA strand displacement. (B) Quantitative relationship between initial rates and the concentrations of T-1. (C) Determination of the effective rate constant k by fitting the data using least-squares linear regression (200 nM T-1). (D) Native polyacrylamide gel electrophoresis (PAGE). R was extended with an additional 20-nt poly-dT domain for the gel separation and visualization (named as R-d). Lanes a, b and c were the corresponding

oligonucleotides standard. 1 µM FQ, 2 µM R-d, and varying concentrations of T-1: 0 µM (Lane 1), 0.01 µM (Lane 2), 0.1 µM (Lane 3), 0.2 µM (Lane 4), 1 µM (Lane 5), 2 µM (Lane 6).


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Secondly, we fixed the concentration of T-1 to be 100 nM and investigated the effect of R concentration on the cooperative toehold-mediated strand displacements. The initial rates V increased

from 0 to 4.75×10-10 M/s in a linear quantitative relationship with the concentrations of R varying from 0 to 250 nM (Figure S2). In terms of its logic function, the cooperative toehold can be viewed as an AND gate with T-1 and R as inputs, which have completely irrelevant DNA sequences and function with a novel

base stacking mechanism. This new toehold was also employed to analyze several different single nucleotide mismatched DNA at the terminus, including three nucleotide substitutions (T-C, T-A, T-T), one nucleotide insertion (T-iG) and one nucleotide deletion (T-dG). In the results of both real-time fluorescence (Figure S3) and gel electrophoresis (Figure S4), all these mismatched DNA induced nearly

no response compared to the background (no target) signal. Because single nucleotide mismatches at the terminus usually have little effect on the thermodynamics of DNA double strands, it is difficult to discriminate by previous probes such as molecular beacon.23 In this way, our cooperative toeholdmediated system served as a new strategy to solve this problem. These results also demonstrated that the principle of cooperative toehold mainly relied on the coaxial adjacent stacking at the nick between T-1 and R. Thirdly, the effect of the toehold length of R (domain 2*, Scheme 1A) on the cooperative toeholdmediated strand displacements was characterized. When the toehold motif (domain 2*, Scheme 1A) was 3 nt, the strand displacement initial rates changed from 8.17×10-14 M/s to 4.82×10-11 M/s (Figure S5A). Another 5 nt toehold motif (domain 2*, Scheme 1A) resulted in an rates increase from 1.97×10-11 M/s to 1.16×10-8 M/s (Figure S5B). The above result was comparable with the previous toehold,24 so the

combination of cooperative toehold with conventional toehold allowed another additional fine-tuning of DNA strand displacement kinetics. Moreover, we further studied the adaptability of the cooperative toehold with toehold exchange systems, as toehold exchange is among the most powerful mechanisms to design complex DNA devices and circuits.24 In Figure S6, it was found that the cooperative toehold was successfully used to trigger toehold exchange reactions when the reverse toehold length of R ranged


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from 0 to 10 nt. A gradual shift of reaction equilibrium toward the reactants was occurred as the reverse toehold length increasing, and this reversible feature was similar to the conventional toehold-mediated toehold exchange.24 In a word, we have tuned the rate of the cooperative toehold-mediated strand displacements via four independent parameters: concentrations of T-1 and R, toehold and reverse toehold lengths of R. In the cooperative toehold, T-1 and R are not either linked by covalent bond or functioned by a doublestranded hybridization manner, but are completely sequence independent. In comparison, the previous associative toehold was developed by connecting the toehold and branch migration domains through a complementary hybridization process.19 The resulting sequence dependence between the two domains limited the design versatility of DNA devices. The cooperative toehold allows flexible controlling DNA strand displacement reactions and powerful regulation of DNA devices.

Dynamic regulation of an advanced DNA device and activation of a remote toehold. The cooperative toehold could be quenched by a conventional toehold-mediated strand displacement for dynamic regulation of an advanced DNA device.20 When T-2, with a additional 6 nt toehold (domain 4*), is part of the cooperative toehold, an inhibitor T-2C can decrease the effective concentration of T-2 via a toehold-mediated strand displacement process (Figure 2A). When T-2C was added in excess, the cooperative toehold-mediated strand displacement between R and FQ was largely turned off (Figure 2B). The process was also restored when T-2 was re-added in excess, and this real-time dynamic regulation was reversible for several times upon the alternate addition of T-2 or T-2C. As cascade reaction of DNA strand displacement are the basis of complex DNA devices and circuits, our dynamic regulation method is useful for the design of advanced molecular devices and circuits. (Figure 2)

Figure 2. Dynamic regulation of strand displacement using the cooperative toehold. (A) Schematic illustration of the inhibition of the cooperative toehold-mediated DNA strand displacement using an inhibitor strand T-2C. (B) Real-time fluorescence monitoring with the alternate addition of 200 nM T-2 or T-2C. (50 nM FQ, 100 nM R) ACS Paragon Plus Environment

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The cooperative toehold was also employed to activate a remote toehold-mediated strand displacement system,18 which has allowed increased control of DNA strand displacement kinetics. In Figure 3A, the length of remote toehold (domain 3b* of R-3, 7 nt ) is too short to trigger the displacement reaction by R-3 alone, when the toehold (domain 3b* of R-3) and displacement domains (domain 1* of R-3) are separated by a spacer with 10-nt poly-dT (domain 5* of R-3). The cooperative toehold generated in the presence of T-3 resulted in the continuous fine-tuning of reaction rates, which increased from 8.2×10-12 to 7.74×10-11 M/s (Figure 3B). It was another example of the excellent adaptability of the cooperative toehold with other DNA strand displacement system. (Figure 3)

Figure 3.

(A) Schematic illustration of activation of a remote toehold using the cooperative toehold.

(B) Real-time fluorescence monitoring with varying concentrations of T-3.

(50 nM FQ, 100 nM R, from

down to up: 0, 2, 5, 10, 20, 50, 100, 200 nM T-3)

Detection of microRNA and ATP.

The cooperative toehold was also employed as a universal

signal translator to construct biosensors. MicroRNAs have important roles in many fundamental cellular processes and are important biomarkers of serious human diseases such as cancers.25 Figure 4A showed the biosensor to detection of microRNA let-7a: QF-2 was served as a universal signal translator and had independent DNA sequences with let-7a. The microRNA let-7a hybridized with P-RNA, and then initiated a cooperative toehold-mediated strand displacement, leading to the increase of fluorescence signal (Figure 4A). In Figure 4B, a gradual increase in fluorescence signal was observed when the concentration of microRNA let-7a was increased from 0 to 500 nM. According to a signal-to-noise ratio >3, the limit of detection was calculated to be 2 nM, with a linear range of 0−20 nM, R2 =0.990 (Figure 4B). In Figure S7, native gel electrophoresis results were in good agreement with the fluorescence data, which further confirmed the let-7a concentration-dependent mechanism. Moreover, we found that this biosensor could distinguish between let-7a and the other two RNA sequences, which are only one nucleotide removed from let-7a at the terminus (Figure S8). The discrimination ability ACS Paragon Plus Environment

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would be further improved by optimizing the sequence of probe P-RNA. These results in turn demonstrated that the coaxial adjacent stacking at the nick between DNA (F-2) and RNA (let-7a) triggered the cooperative toehold-mediated strand displacements. The proposed biosensor was able to detect let-7a in complex samples containing 100-fold genomic DNA (from salmon sperm), demonstrating its good anti-interference ability (Figure S9). Separating recognition domain from signal domain is beneficial to achieve the parallel activation of a cooperative toehold-mediated strand displacement using different targets. To further demonstrate the practicability of this design, we built a logic gate with OR function using RNA (let-7a) and DNA (TDNA) strands as inputs. In Figure S10, P-RNA and P-DNA was designed to contain the specific domain (domain 3*) for target recognition, but they shared the same strand displacement domain (domain 1*2*) relative to QF-2. As indicated by the time-dependent fluorescence changes (Figure S11), either input let7a or T-DNA could activate the fluorescence signal via a parallel cooperative toehold-mediated strand displacements. In this way, we have successfully developed a logic gate with OR function using QF-2 as a universal signal translator. (Figure 4)

Figure 4. (A) Schematic illustration of microRNA detection. (B): (a) Real-time fluorescence monitoring with varying concentrations of microRNA let-7a. (b) Quantitative relationship between fluorescence

intensity and the concentrations of let-7a. (50 nM QF-2, 100 nM P-RNA) Aptamers are DNA or RNA oligonucleotides with unique affinity to various targets, and serve as ideal recognition elements for sensing systems because of their advantages over antibodies.26 Here, we also employed ATP and its DNA aptamer to demonstrate the feasibility and generality of the abovementioned universal signal translator. The probe P-ATP in Figure 5A was designed to be partially complementary with ATP aptamer. They can cooperatively activate the fluorescence signal of QF-2, via a cooperative toehold-mediated strand displacement. ATP induces dehybridization of the hybrid between the ATP aptamer and probe P-ATP, which inhibits the DNA strand displacement process (Figure 5A). In the presence of 0-500 µM ATP, the strand displacement rate was gradually reduced in a 11 ACS Paragon Plus Environment


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concentration-dependent way (Figure 5B). The fluorescence signal change was linear from 0 to 50 µM ATP and the detection limit was estimated to be 5 µM. This results were similar with the sensing performance of structure-switching signaling aptamer,27 but our biosensor design was more universal for different target by separating the sensing and signal domains. Moreover, the biosensor showed a high specificity for ATP, because both ATP analogues, CTP and GTP, induced no change in the fluorescence signal (Figure S12). In Figure S13, native gel electrophoresis has been employed to characterize the response process, and the result was also in good agreement with the fluorescence data.

(Figure 5)

Figure 5. (A) Schematic illustration of ATP detection. (B): (a) Real-time fluorescence monitoring with varying concentrations of ATP. (b) Quantitative relationship between fluorescence intensity and the concentrations of ATP. (50 nM Q, 50 nM F-2, 100 nM P-ATP, 100 nM ATP aptamer)

As reported, the free-energy of the coaxial stacking interaction depends on the type of nearest neighbor bases X and Y at the nick.28 It is useful to characterize the effect of base type at the nick on the cooperative toehold-mediated strand displacement. We have employed intra-molecular hairpin probes with unique stem-loop structure to simplify the probe design (Figure S14). As indicated in the real-time fluorescence measurement (Figure S15), the ability of different bases to activate the cooperative toehold-

mediated strand displacements decreased in the following order (neighbor bases at the nick: X/Y): A/A > C/A > C/G >A/G > C/T > C/C >A/T. This information is beneficial for further application of the cooperative toehold in advanced DNA devices. Finally, we compared the different influence of hairpin DNA (R-C/C, with a cooperative toehold) with linear DNA (R, without a cooperative toehold) on DNA strand displacement (Figure S16). Only the hairpin DNA with a cooperative toehold obviously increased strand displacement rate (Figure S17). As the hairpin DNA R-C/C has no other possible interaction with QF-2, it is clear that the cooperative toehold relies on the coaxial adjacent stacking at the nick.

PCR monitoring system. Polymerase chain reaction (PCR) is a popular technique for bio-analysis and has various applications in genotyping, virus detection, sequencing and so on.29 Here we focused on an alternative new way to monitor PCR amplification using the above-mentioned universal signal translator. ACS Paragon Plus Environment

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As shown in Figure 6, probe P-PCR was designed to detect M13M3 via a similar mechanism for microRNA detection (Figure 4A), while M13M3 is one of the PCR primers for pUC18 plasmid.30 After PCR process, the amount of M13M3 will decrease with increasing concentrations of pUC18 plasmid, resulting in the inhibition of the cooperative toehold-mediated strand displacement with QF-2.

(Figure 6)

Figure 6. Illustration of PCR monitoring system using the universal signal translator. Firstly, we performed native polyacrylamide gel electrophoresis to confirm the PCR process, and more band corresponding to PCR product was appeared with high concentrations of pUC18 plasmid (Figure S18). Then the change in the real-time fluorescence after PCR was measured with increasing concentrations of pUC18 plasmid: the strand displacement rate was decreased in a concentration-

dependent manner (Figure 7A(a)). The plot of the ogarithmic of pUC18 concentrations and fluorescence intensity showed a linear relation in the presence of 400 aM-400 pM pUC18 (Figure 7A(b)). Combined with PCR amplification, the universal biosensor has a potential for a quantitative and real-time analysis of DNA with an aM detection limit. The anti-interference ability was evaluated by perform the PCR process

in the presence of a large excess of genomic DNA (from salmon sperm) and then real-time fluorescence was monitored in a similar manner. In Figure S19, 10000-fold genomic DNA had only negligible effect on the fluorescence response, indicating the possibility of bio-analysis in complex biological sample. The

universal biosensor for another target T-DNA was also monitored in the solution of the above PCR product to characterize the specificity. T-DNA is not the primer for the above PCR of pUC18 plasmid and didn’t consumed during the PCR process. The cooperative toehold-mediated strand displacement

triggered by T-DNA was largely unaffected by the PCR product (Figure S20). These results clearly indicated that the PCR monitoring system was specific based on the selective recognition of a primer for desired PCR system.

(Figure 7)

Figure 7. (A): (a) Real-time fluorescence monitoring with increasing concentrations of pUC18 plasmid: 0, 400 aM, 4 fM, 40 fM, 400 fM, 4 pM, 40 pM, 400 pM. (b) The standard plot showing a linear correlation ACS Paragon Plus Environment

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between the ogarithmic of copy number and fluorescence intensity. (B): (a) Real-time fluorescence monitoring with increasing PCR cycles (400 pM pUC18 plasmid). (b) The standard plot showing a linear correlation between the PCR cycles and fluorescence intensity. In addition, PCR samples after designating PCR cycles were also analysed by real-time fluorescence measurements. Before PCR amplification, the fluorescence intensity was quickly activated by M13M3 via the cooperative toehold-mediated strand displacement (Figure 7B). After 10-20 PCR cycles, the fluorescence response was largely inhibited due to the exhaustion of primer M13M3 in the PCR procedure.The fluorescence intensity was only 30% of the initial intensity after 40 cycles, and so this biosensor could be used to monitor the PCR process of one plasmid sample. This PCR monitoring system has at least two obvious advantages. Firstly, the widely-used dye for PCR, SYBR® Green, is convenient via use of natural unmodified DNAs, but it is unspecific and difficult to discriminate the nonspecific PCR products from the desired one.31 By contrast, our PCR monitoring system is better choice as it is based on the selective recognition of a specific primer for a DNA target. Secondly, our PCR monitoring system is also more economic and convenient in terms of use of natural non-chemically modified DNAs when it is compared with other specific PCR probes, such as TaqMan® probes, molecular beacons,23 and the secondary-structure-inducible ligand probe.30

CONCLUSIONS In summary, a novel cooperative toehold was developed based on a base stacking mechanism, and it was comprised of two moieties with completely independent DNA sequences. As a new addition to the current toolbox of DNA strand displacement methods, it was adaptable to other toehold activation mechanisms, such as dynamic regulation strategy and a remote toehold, to build more advanced DNA devices. Based on the cooperative toehold, a universal biosensor was construct to selectively and sensitively detect microRNA and ATP. In addition, the universal biosensor could served as a PCR monitoring system to detect pUC18 plasmid with an aM detection limit. The cooperative toehold will enrich the toolbox with alternative approaches to improve the development of DNA nanotechnology and DNA biosensors. ACS Paragon Plus Environment

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Real-time fluorescence data; native polyacrylamide gel electrophoresis results; one table for oligonucleotides used; several schematics for probes design.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (B.Y.)

ORCID Bin Yang: 0000-0003-0425-2718 Chun-Yan Li: 0000-0002-8382-5264

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT This work was supported by National Natural Science Foundation of China (Grants 21505113), the Research Foundation of Education Bureau of Hunan Province (Grant 16C1533), The Open Project of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (Grants 2014014).

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