Multiresponsive Rolling Circle Amplification for DNA Logic Gates

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Multi-Responsive Rolling Circle Amplification for DNA Logic Gates Mediated by Endonuclease Weidong Xu, Ruijie Deng, Lida Wang, and Jinghong Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac501726s • Publication Date (Web): 11 Jul 2014 Downloaded from http://pubs.acs.org on July 14, 2014

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Analytical Chemistry

Multi-Responsive Rolling Circle Amplification for DNA Logic Gates Mediated by Endonuclease

Weidong Xu, Ruijie Deng, Lida Wang, and Jinghong Li* Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China.

*to whom corresponding should be addressed. Tel and Fax: +86-10-62795290 Email: [email protected]

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ABSTRACT Rolling circle amplification (RCA), an efficient isothermal amplification method allowing the polymerase-mediated generation of long single-stranded DNA molecules made of tandem repeats, has been widely used in biomedical and nanotechnology fields due to structural and compositional versatility of its components. In this work, we confer multi-responsiveness to RCA reactions by designing dumbbell-shaped DNA templates and hairpin probes containing different endonuclease cleavage sites. Endonucleases trigger the release of RCA primers or the cleavage of DNA templates, which controls subsequent RCA reactions. A set of one-input and two-input DNA logic gates, which use endonucleases or hairpin probes as inputs, including YES, NOT, AND, OR, NOR, and INHIBIT, are constructed based on our proposed multi-responsive RCA reactions. We demonstrate flexibility and scalability of these logic gates by integrating them to fabricate more complex three-input logic circuits (AND-OR and NOR-AND circuits). Moreover, our strategy is used to construct an assay system for endonuclease activity. Our proposed method might be applicable in the multi-channel detection of endonucleases, nucleic acids, and other biomolecules.

KEY WORDS multi-responsiveness, rolling circle amplification (RCA), logic gate, endonuclease

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INTRODUCTION Rolling circle amplification (RCA) is an isothermal enzymatic process that allows the replication of a circular template primed by a single-stranded nucleic acid to generate extremely long DNA strands.1-4 Owing to structural and compositional versatility of its typical components (a DNA/RNA primer and a circular DNA template), RCA can be utilized to fabricate DNA nanostructures including nanotubes,5,6 nanoribbons,7,8 nanoflowers,9 origami,10 and DNA metamaterials,11 which hold great potentials for applications in drug delivery, biosensors, bioimaging, and electronic circuits. As a highly tunable and versatile technique, RCA has been explored to develop sensitive detection methods, in which RCA can respond to a variety of “stimuli” including nucleic acids,12-18 proteins,19-24 small molecules,25 and cells.26,27 Aided by finely designed primers and templates, RCA has been conferred environmental responsiveness. 12-23,25-29

In most previous reports, however, RCA only responded to a single “stimulus” in a

monotonous way, which limited the applications of RCA in multi-parameter analysis and bioelectronics. It remains a challenge to confer multi-responsiveness to RCA reaction. Endonucleases, which exist in most organisms, can recognize specific sites and cleavage the phosphodiester bonds within polynucleotide chains.30,31 Endonucleases play important roles in many biological processes, such as DNA replication, repair, and recombination.32-34 Due to their ability to cut DNA strands reproducibly in the same places, endonucleases have been widely used in gene cloning, genotyping, gene mapping, PCR assays, and nanostructures fabrication.35-41 Herein, we built multi-responsive RCA reactions by designing hairpin probes that “hid” primers via adjacent domain containing endonuclease cleavage site corresponding to specific

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endonucleases, and dumbbell-shaped templates where endonuclease cleavage sites were flanked by two DNA loops. Various combinations of primers and templates with different endonuclease cleavage sites enabled the multi-responsiveness of RCA reactions. Using these reactions, we constructed a set of DNA logic gates mediated by endonucleases. A Logic gate is an idealized or physical device that implements a Boolean function by performing a logical operation on one or more logical inputs and producing a single logical output.42 Nucleic acid-based logic gates have been demonstrated to be able to emulate Boolean operations and have potential uses in molecular computing machines, smart sensory devices, and drug delivery systems.43-52 Compared to previously reported fabrication of RCA-based logic gates,29 which used metal ions as inputs, our approach has better scalability on account of the variability of endonucleases. In the present work, we designed one-input and two-input logic gates based on our proposed multi-responsive RCA reactions, followed by integrating some of them to fabricate three-input logic circuits, which showed their ability to be integrated as part of a larger circuitry. Furthermore, our strategy was used to construct an assay system for endonuclease activity as a proof of application. EXPERIMENTAL SECTION Logic operations: DNA sequences are listed in Table S1 in Supporting Information. The concentrations of oligonucleotides used in logic gates/circuits are described in Supporting Information. Firstly, 1 μL of oligonucleotides was mixed with 2 μL of 10  NEBuffer (500 mM Potassium Acetate, 200 mM Tris-acetate, 100 mM Magnesium Acetate, 1 g/mL BSA, pH 7.9 at 25 C), 5 μL of dNTPs (10 mM for each of dATP, dGTP, dCTP, and dTTP), and inputs (endonucleases, 0.1 U/μL, or hairpin probe, 0.03 μM), followed by adding ddH2O to a final volume of 19 μL, to perform cleavage reaction at 37 C for 30 min. Next, 1 μL of Bst DNA

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polymerase (final concentration: 0.2 U/μL) was then added to perform RCA reaction at 65 C for 1 h, followed by heating at 80 C for 20 min to terminate the reaction. The fluorescent analysis was performed in PBS buffer with 1  Sybr Green I (SGI). More experimental details can be found in the Supporting Information. RESULTS AND DISCUSSION Firstly, we constructed the basic one-input YES and NOT logic gates (Figure 1). The YES gate consisted of a hairpin probe with endonuclease cleavage site in its double-stranded domain and a dumbbell-shaped DNA template without endonuclease cleavage site. EcoRI, a kind of endonuclease, was used as input of the gate. As shown in Figure 1A, the primer of RCA reaction was “hidden” from a string of double-stranded domain adjacent to its 3’ end, which prevented RCA reaction in the absence of EcoRI, in the hairpin probe. When EcoRI was present, the primer was released after the cutting of hairpin probe at the cleavage site in the doublestranded domain of the probe. Subsequently, an RCA reaction was performed in the presence of DNA polymerase/dNTPs. As a dumbbell-shaped DNA was used as template of RCA reaction, large amounts of ssDNA and dsDNA could be formed in the RCA product, which enabled the detection via the intercalating dye Sybr Green I (SGI). The fluorescent signal was used as the output of the gate. We defined the fluorescent intensity at 524 nm above the threshold of 5,000 as “1” or “true” in the logic gate. Similarly, a NOT logic gate was constructed by using an RCA primer and a dumbbell-shaped template containing an endonuclease cleavage site (Figure 1B). When EcoRI, the input of the gate, was absent, RCA reaction could be performed. In the presence of EcoRI, the dumbbell-shaped template would be cut, thus inhibiting subsequent RCA reaction. The fluorescent results of YES and NOT gates are shown in Figure 1.

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Accordingly, a set of two-input logic gates were constructed using similar strategies. Based on the principle of YES gate, AND and OR gates were designed. As shown in Figure 2A, the AND gate was composed of a dumbbell-shape template same as used in the YES gate: the hairpin probe used in the YES gate and EcoRI were used as two inputs of the gate. When either of the two inputs was present alone, RCA reaction couldn’t be performed. Only in the presence of the two inputs, could primer be generated to facilitate the following RCA reaction that resulted in a fluorescent signal. Different from the AND gate, the OR gate consisted of a dumbbell-shaped DNA template and two hairpin probes containing the same “hidden” primer but two different endonuclease cleavage sites respectively, i.e., a hairpin probe with EcoRI cleavage site and a hairpin probe with MseI cleavage site. Here, EcoRI and MseI, two kinds of endonucleases, were used as two inputs in the gate (Figure 2B). The presence of either EcoRI or MseI enabled the release of primer which could promote RCA reaction and generate a “true” output, then the OR gate was constructed. The fluorescent results and corresponding truth tables are also shown in Figure 2. For the above AND and OR gates, all the outputs of the ground state (0, 0) of the gates are “false”. In Boolean logic gates, however, a “true” output in the ground state is needed to meet the requirements of other logic gates, e.g., NOR gate. Based on the principle of the NOT gate, we designed a NOR gate. The NOR gate was also composed of a RCA primer and a dumbbellshaped template containing endonuclease cleavage sites. Unlike the NOT gate, however, the template used in the NOR gate included two different endonuclease cleavage sites (EcoRI and MseI cleavage sites) in its double-stranded domain (Figure 3A). EcoRI and MseI were used as two inputs in the gate. When either EcoRI or MseI was present, the dumbbell-shaped template would be cut at the corresponding cleavage site, which would inhibit subsequent RCA reaction,

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resulting in a “false” output. Only in the absence of two inputs, could RCA reaction be performed, which resulted in a “true” output. The symbolization, truth table, and fluorescent results of NOR gate are shown in Figure 3A. Another notable type of logic gate is INHIBIT gate, which is essentially a two-input AND gate with one input carrying a NOT gate. As shown in Figure 3B, the INHIBIT gate consisted of a hairpin probe including a “hidden” primer adjacent to an MseI cleavage site and a dumbbell-shaped DNA template containing an EcoRI cleavage site. Correspondingly, two inputs of the gate were EcoRI and MseI. In the INHIBIT gate, one input, EcoRI, served as a veto which had the power to disable the whole system due to its ability to cut the dumbbell-shaped template at the cleavage site, which would prevent the following RCA reaction, resulting in a “false” output, no matter whether primer, which would be released in the presence of MseI, was present or not. Only when MseI, which could cut the hairpin probe and induce the release of primer, was present alone, could RCA reaction proceed, which resulted in a “true” output; otherwise the output was “false”. Apart from fabrication of logic gates, achieving scalability of the gates is another challenge in DNA computing. The aforementioned logic gates were easily cascaded due to the consistency of materials used in the gates and the variability of endonucleases, which were used as inputs in the gates. To demonstrate the flexibility and scalability of the proposed logic gates, we combined these gates into three-input logic circuits. Firstly, we constructed a AND-OR logic circuit by modulating the components of the AND and the OR gates. As shown in Figure 4A, the AND gate, which used a hairpin probe including EcoRI cleavage site and EcoRI as two inputs, was cascaded with the OR gate, which consisted of a dumbbell-shaped template and a hairpin probe with MseI cleavage site (MseI was used as the third input of the circuit). When MseI was 7



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present, the primer could be released from the hairpin probe containing the corresponding cleavage site, which was one of the components of the circuit, facilitating RCA reaction and thus generating a “true” signal no matter whether two other inputs were present or not. In the absence of MseI, only when both two other inputs, EcoRI and the hairpin probe with corresponding cleavage site, were present could RCA reaction be conducted and “true” signal be produced. Moreover, using the principle of the NOR and the INHIBIT gates, we constructed a NOR-AND circuit. As shown in Figure 4B, the circuit was composed of a dumbbell-shaped template containing EcoRI and MseI cleavage site and a hairpin probe containing BamHI cleavage site, EcoRI, MseI and BamHI were used as three inputs of the circuit. The presence of BamHI enabled the release of RCA primer, and the presence of either EcoRI or MseI resulted in the cutting of the dumbbell-shaped template with corresponding cleavage sites, inhibiting the following RCA reaction. As shown in Figure 4C and 4D, only when BamHI was present alone could RCA reaction be performed, which resulted in a “true” signal. To further demonstrate the applications of our strategy, we constructed an assay system for endonuclease activity based on the principle of YES gate (Figure 5A). Since endonucleases have been regarded as the promising targets for antimicrobial and antiviral drugs,53,54 our proposed assay system has potential use in the field of drug development. As shown in Figure 5A, a hairpin probe containing an endonuclease cleavage site next to a “hidden” primer and a dumbbell-shaped template were used in the assay system. In the presence of endonuclease, the primer was released after the cleavage of the hairpin probe. The dumbbell-shaped template, dNTPs and DNA polymerase were then added to the system to enable the subsequent RCA reaction, of which the product would be detected by SGI, resulting in a fluorescent signal. In this work, we assayed two endonucleases (EcoRI and MseI) to evaluate the applicability of the

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proposed method. As shown in Figure 5B and 5C, in the absence of specific endonuclease, only small background fluorescence intensity at 524 nm were exhibited; as the concentration of endonucleases increased, the fluorescence intensity at 524 nm increased. The enhancement of fluorescence intensity showed dependence on the concentration of specific endonuclease. As presented in Figure 4D and 4E, a linear correlation was obtained in the concentration ranging from 4 × 10-5 to 2 × 10-3 U/μL for EcoRI and MseI respectively. A detection limit (estimated as 3 times standard deviation of blank florescence signals, “background noise”) of 4 × 10-5 U/μL was achieved for both EcoRI and MseI. We further investigated the specificity of the assay by testing the assay in response to several other randomly selected non-targeted endonucleases, such as BamHI, Acc65I, and SacI, at a concentration of 0.02 U/μL (Figure 5F and 5G). We also used random sequences of dumbbell-shaped DNA templates and hairpin probes as negative controls to test the specificity of the assay system (Figure S1). It was clearly demonstrated that our proposed method could be applied in sensitive detection of endonucleases with high specificity. We further compare our assay system with fluorescent assay based on molecular beacon55 to show the accuracy of our method (Figure S2). CONCLUSION In summary, we applied multi-responsive RCA reactions to construct a set of one-input, two-input, and three-input DNA logic gates/circuits (YES, NOT, AND, OR, NOR, INHIBIT, AND-OR, and NOR-AND) responsive to endonucleases and nucleic acids by finely designing dumbbell-shaped DNA templates and hairpin probes. We have demonstrated the expansibility of logic gates by fabricating more complex three-input logic circuits from two-input gates. This work inspires the integration of various functional sequences such as DNA aptamers, DNAzymes, spacer domains, and endonuclease cleavage sites into RCA components to build 9



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logic devices responsive to biomolecules, which can be potentially used in molecular computing. In addition, our study provides a new insight into the fabrication of smart sensory devices for the multi-channel detection of endonucleases, nucleic acids, and other biomolecules. ASSOCIATED CONTENT Supporting Information. Experimental details, supporting tables, and supporting figures. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Tel and Fax: +86-10-62795290; E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by National Basic Research Program of China (No. 2011CB935704), the National Natural Science Foundation of China (No. 21235004, 21327806), Tsinghua University Initiative Scientific Research Program, and Training Program of Innovation and Entrepreneurship for Undergraduates (No. 201410003046). REFERENCES (1) Fire, A.; Xu, S. Q. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4641-4645. (2) Liu, D. Y.; Daubendiek, S. L.; Zillman, M. A.; Ryan, K.; Kool, E. T. J. Am. Chem. Soc. 1996, 118, 1587-1594.

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(55) Biggins, J. B.; Prudent, J. R.; Marshall, D. J.; Ruppen, M.; Thorson, J. S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13537-13542.

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FIGURES

Figure 1. A, B) Principle of RCA for YES (A) and NOT (B) logic gates. C, D) Fluorescence emission spectra and fluorescence intensity at 524 nm of the different input modes of YES (C) and NOT (D) logic gates: (1) no input, (2) EcoRI.

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Figure 2. Fluorescence emission spectra, fluorescence intensity at 524 nm, schemes and truth tables of AND (A) and OR (B) logic gates. Groups 1-4 in the fluorescent results correspond to the four input combinations in truth tables.

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Figure 3. Fluorescence emission spectra, fluorescence intensity at 524 nm, schemes and truth tables of NOR (A) and INHIBIT (B) logic gates. Groups 1-4 in the fluorescent results correspond to the four input combinations in truth tables.

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Figure 4. A, B) Schemes and truth tables of AND-OR (A) and NOR-AND (B) logic circuits. C, D) Fluorescence emission spectra and fluorescence intensity at 524 nm for AND-OR (C) and NOR-AND (D) logic circuits. Groups 1-8 in the fluorescent results correspond to the eight input combinations in truth tables.

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Figure 5. A) Scheme of fluorescent assay of endonuclease activity based on YES gate. Endonuclease triggers the release of primer, enabling subsequent RCA reaction, which results in a fluorescent signal. B, C) Fluorescence emission spectra for assay of EcoRI (B) and MseI (C) at the following concentrations (10-3 U/μL): 0, 0.08, 0.2, 0.4, 0.8, 2, 4, 8, 20. D, E) Plots of fluorescence intensity at 524 nm vs. concentrations of EcoRI (D) and MseI (E). F, G) Selectivity of fluorescent assays for EcoRI (F) and MseI (G). The concentrations of endonucleases are 0.02 U/μL.

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For TOC only

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