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Aligner-mediated cleavage-triggered exponential amplification for sensitive detection of nucleic acids
Talanta 185 (2018) 141–145
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Aligner-mediated cleavage-triggered exponential amplification for sensitive detection of nucleic acids Wanghua Wua, Hongliang Fanb, Xiang Liana, Jianguang Zhoua, Tao Zhanga,
T
⁎
a Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou 310027, PR China b Department of Environmental Medicine, Institute of Hygiene, Zhejiang Academy of Medical Sciences, Hangzhou 310013, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Nucleic acids detection Isothermal exponential amplification DNA cleavage Aligner-mediated cleavage
Exponential amplification reaction (EXPAR), as a simple and high sensitive method, holds great promise in nucleic acids detection. One major challenge in EXPAR is the generation of trigger DNA with a definite 3′-end, which now relies on fingerprinting technology. However, the requirement of different endonucleases for varying target sequences and two head-to-head recognition sites in double stranded DNA, as well as the confinement of trigger DNA's 3′-end to be near/within the recognition site, usually subject EXPAR to compromised universality and/or repeated matching of reaction conditions. Herein, we report a simple and universal method for high sensitive detection of nucleic acids, termed aligner-mediated cleavage-triggered exponential amplification (AMCEA). The aligner-mediated cleavage (AMC) needs only one nicking endonuclease and can make a break at any site of choice in a programmable way. Thus, the 3′-end of target DNA can be easily redefined as required, a key step for initiating the amplification reaction. This capability endows the proposed AMCEA with excellent universality and simplicity. Moreover, it is sensitive and specific, with a detection limit at amol level, a broad dynamic range of 5～6 orders of magnitude and the ability to distinguish single nucleotide mutation. Experiments performed with human serum indicate that AMCEA is compatible with the complex biological sample, and thus has the potentials for practical applications.
1. Introduction Simple, fast and sensitive detection of nucleic acids is important in many fields, such as molecular biology, medical diagnostics and forensic analysis [1–3]. To date, various methods for high sensitive detection of nucleic acids have been developed, such as surface plasmon resonance, electrochemical sensors, molecular imprinted polymer, amplification techniques, and so on [4–9]. In particular, amplification methods have attracted extensive attentions in recent years. Though polymerase chain reaction (PCR) is the most powerful tool and widely used for the quantification of nucleic acids [10], it still has some intrinsic limitations, e.g., the requirement of specific thermal-cycling instrument, considerable power consumption and relatively long reaction time. These issues usually limit its practical utility in point-of-care test. Therefore, in the past two decades, great efforts have been made in developing a variety of isothermal amplification strategies [6], such as loop-mediated amplification (LAMP) [11,12], helicase-dependent amplification (HDA) [13,14], rolling circle amplification (RCA) [15,16], smart amplification process (SMAP) [17], recombinase polymerase amplification (RPA) [18,19] and cross priming amplification (CPA) ⁎
[20]. Nevertheless, most of current isothermal methods employ sophisticated mechanism or extra proteins/enzymes, which greatly increase the difficulty in primer design. Recently, owning to the simple mechanism and high amplification efficiency, the nicking endonuclease (NEase)-based methods, such as strand displacement amplification (SDA) [21,22], exponential amplification reaction (EXPAR) [23] and nicking endonuclease-mediated isothermal amplification (NAMP) [24], have attracted extensive attentions. However, one major limitation of NEase-based methods is the requirement of specific recognition sites in target sequence, which badly restricts their universality. Many efforts, e.g., the adoption of extra bumper primers [25], fingerprinting technique [26], junction probe strategy [27] and beacon-assisted amplification [28], are either unable to address the above issue thoroughly, or likely to worsen the nonspecific background amplification that is already serious in most isothermal methods [29]. For instance, EXPAR is a simple, low-cost and sensitive method, possessing a very high amplification efficiency (106 to 109-fold amplification within minutes). Since its working principle is based on a polymerase-catalyzed extension of trigger DNA (X) along a special template to generate the double-stranded recognition site of
Corresponding author. E-mail address: [email protected] (T. Zhang).
https://doi.org/10.1016/j.talanta.2018.03.067 Received 29 January 2018; Received in revised form 14 March 2018; Accepted 22 March 2018 Available online 26 March 2018 0039-9140/ © 2018 Published by Elsevier B.V.
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Fig. 1. Schematic illustration of AMCEA. Nt. BstNBI firstly binds the recognition site in the stem of DNA aligner (Loading), and is aligned at a specific locus through the hybridization of DA's side-arms with target DNA (Localization), and then makes a cleavage there (Reaction). Subsequently, the cleaved target serves as a trigger to initiate the EXPAR. By simply varying the sequences of aligner's side-arms, a trigger DNA can be generated from any locus of target DNA, endowing this method with excellent universality.
(NaCl) were purchased from Sigma Aldrich (St. Louis, MO). The clinical serum samples of healthy person were obtained from the Hospital of Zhejiang University. All solutions were freshly prepared with DNAgrade water.
NEase, the main challenge to EXPAR is the generation of trigger DNA with a definite 3′-end. This process now relies on the fingerprinting technology [26], which, however, requires different endonucleases for distinct target sequences of interest, and at the same time, two adjacent recognition sites oriented head-to-head in double-stranded DNA. Except for the difficulties in choosing an appropriate endonuclease for a given sequence and the matching of reaction conditions, the trigger DNA can only be produced from double-stranded DNA, and its 3′-end must be within/near the recognition site (Fig. S1), which, to a large extent, restrains the flexibility of EXPAR from detecting the real meaningful fragment of sequences. Our group has recently developed a simple and versatile strategy of using conventional NEases for programmable sequence-specific cleavage of DNA, termed aligner-mediated cleavage (AMC). It is based on a rationally designed hairpin-shaped DNA-aligner (DA), which consists of a recognition site of an NEase in the stem and two side arms complementary to the target sequence. Thus, an NEase can be easily loaded onto DA's stem, localized to a specific locus through the hybridization of the side arms with target DNA, and then makes a cleavage there (Fig. 1). This process needs only one NEase, does not require any special sequence in target DNA and can make a break at any intended site. In this paper, we further demonstrate an aligner-mediated cleavage-triggered exponential amplification (AMCEA). Thanks to the excellent versatility of AMC, the cleavage of target DNA, a key step to generate the trigger DNA, can be easily generated at any locus of target DNA. And the exact 3′ end can be tuned at a single-nucleotide scale by using just one NEase. This avoids the tedious processes of enzyme selection and the repeated optimization of reaction conditions. Thus, the proposed AMCEA is characterized with both excellent simplicity and universality. Moreover, this method is highly sensitive and specific, capable of detecting as less as 1.2 amol DNA and discriminating singlenucleotide mutations.
2.2. General procedure of AMCEA The reaction mixture of AMCEA was prepared separately on ice as part A and part B. Part A contained DA, EXPAR template, dNTPs, Nt. BstNBI and target of interest, and part B consisted of SYBR Green I and Bst 2.0 WarmStart DNA polymerase, both in the final reaction buffer (100 mM NaCl, 50 mM Tris-HCl, pH 8.0, 4 mM MgCl2 and 60 μg mL−1 BSA). Part A was first incubated at 55 °C for 15 min on a Bio-rad CFX96 PCR system, then part B was immediately added. The final reaction mixture, composed of 50 nM DA, 50 nM EXPAR template, 6 U Nt. BstNBI, 0.4 U Bst 2.0 WarmStart DNA polymerase, 0.4 mM dNTPs, 4 mM MgCl2 and 0.5 × SYBR Green I, was kept at 55 °C for 60 min and the real-time fluorescence was monitored at 1-min intervals. 2.3. Gel electrophoresis For gel analysis, the reactions were quenched at 95 °C for 5 min at a certain time (see the black triangles in Fig. 2a and b), then 5 μL of the resultant and 20 × SYBR Green I were loaded onto 12% polyacrylamide gel, and subjected to 120 V constant voltage in 0.5 × TBE buffer (45 mM Tris, 45 mM borate, 1 mM EDTA, pH 8.3) for 50 min at room temperature. The gel was then analyzed on a Maestro Ex IN-VIVO Imaging System (CRI). 2.4. Detection of nucleic acids spiked in human serum In order to demonstrate the utility of AMCEA in complex biological matrix, it was performed with human serum. First, the healthy human serum without any pretreatment was used to dilute target DNA by tenfold in the series. This process generated a concentration gradient from 25 fmol to 2.5 amol. Subsequently, 2.5 μL of spiked serum was subjected to AMCEA mentioned above. The results were compared with those obtained with buffered target DNA.
2. Materials and methods 2.1. Materials and reagents All oligonucleotides of HPLC purity were synthesized from Sangon Biotech Co., Ltd. (Shanghai, China). Detailed sequences of these oligonucleotides are listed in Table S1. Nicking endonuclease Nt. BstNBI (10,000 U mL−1), Bst 2.0 WarmStart DNA polymerase (8000 U mL−1), deoxyribonucleotide triphosphates (dNTPs, 10 mM), Low Molecular Weight DNA Ladder and MgCl2 solution (25 mM) were purchased from New England Biolabs (Ipswich, MA). SYBR Green I (10,000 ×) was purchased from Invitrogen Life Technologies (Carlsbad, CA). DNAgrade water (DNase- and Protease-free) and Tris-hydrochloride buffer (1 M solution, pH 8 .0) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Bovine serum albumin (BSA) and sodium chloride
3. Results and discussion 3.1. Principle of AMCEA The proposed AMCEA is illustrated in Fig. 1. The DNA aligner (DA) consists of two components: a stem-loop structure with a recognition site of Nt. BstNBI in the stem and two side arms complementary to the target sequence. First, a target sequence of interest (TS) hybridizes with DA, and is cleaved by Nt. BstNBI via AMC. Then, the cleaved target 142
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no such a band for either NTC or RS (Lane 2 and 4). A weak band lower than 30 bp in all these four reactions represents DA-1 that is 44 nt long (see Lane 9 for pure DA-1). By contrast, when DA-1 was absent, only TTS initiated EXPAR and resulted in a 30-bp band (Lane 7). TS, as well as RS and NTC, did not generate any resultants. These results clearly indicated the necessity of DA-1, which aligned Nt. BstNBI to cleave target DNA at a specific site to form an active trigger, and thus validated the mechanism of AMCEA shown in Fig. 1a. Note that the POI of RS and NTC were always the same either with or without DA-1, implicating an excellent selectivity of the proposed method. As for the relatively slow reaction rate in the presence of DA-1 when compared with the DAabsent system, the main reason is probably that the binding of Nt. BstNBI with DA-1 notably decreased the amount of free Nt. BstNBI, and thus slowed down the amplification rate. 3.3. Sensitivity of AMCEA To investigate the sensitivity of AMCEA, the real-time fluorescence curves caused by various concentrations of TS were measured. As can be seen in Fig. 3a, the POI values gradually decreased with the increase of TS concentration. Moreover, it showed a linear correlation with the negative logarithm of TS concentration (Fig. 3b). The regression equation is POI = − 36.775 + 4.519 (- lg C) (R2 = 0.992); C represents the concentration of TS. The limit of detection, defined as the concentration corresponding to a signal above three times of standard deviation (3σ), was found as 1.20 amol, and the dynamic range spanned over 5 orders of magnitude, which were comparable to previous EXPAR methods [30–35] (Table S2). The whole detection process of AMCEA took only 30～40 min, shorter than some other methods [31,35].
Fig. 2. Real-time fluorescence curves caused by different sequences (2.5 fmol) a) with and b) without DA-1. NTC represents the no-target control. c) Gel image of the products of AMCEA in the presence (Lane 1–4) or absence (Lane 5–8) of DA-1. Lane 9, 2.5 fmol pure DA-1.
sequence (CTS) leaves DA because the shortened hybridization length is unable to maintain a stable Y-shaped structure. At this point, the 3′ end of target DNA has been definitely redefined, and thus can serve as a trigger to initiate the amplification reaction. Namely, the 3′ end of CTS (X′) transiently hybridizes with EXPAR template that contains the recognition sequence of NtBstNBI in the middle and two fragments X on each side, and is extended along the template by Bst polymerase to form a complete double-stranded recognition site. As a result, Nt. BstNBI binds the newly formed recognition site and makes a nick on the extension product, followed by a second polymerase-catalyzed extension to displace the downstream strand (X′). Cycling above nicking and extension steps will generate more and more X′, and every X′ can initiate more reactions, leading to an exponential amplification. Due to the excellent versatility of AMC, the cleavage of target DNA, a key step to generate a trigger for initiating EXPAR, can be readily achieved at any site of choice by simply varying the sequences of DA's side arms. This process utilizes just one NEase and does not rely on any special sequence in target DNA. Therefore, the proposed AMCEA is very simple and highly universal.
3.4. Specificity of AMCEA To further evaluate the specificity of AMCEA, six single-base mismatched target DNA (TSX, X = 1～6, Table S1) were examined. Fig. 3c displays the positions of mismatches, all near the cleavage site of AMC. As shown in Fig. 3d, all mismatched DNA resulted in higher POI values when compared with that of perfectly matched target TS. In particular, the POI values of TS2～TS4 were much larger than those of other TSX and very close to NTC, indicating an excellent capability to discriminate single-base mismatch of AMCEA. This can be explained by the “dualeffects” imposed on both cleavage and extension processes, i.e., the mismatches near the cleavage site would cause imperfect hybridization between TSX and DA-1, and thus decrease the efficiency of AMC; in the meanwhile, the mismatches, especially the ones that are close to the 3′end of CTSX, would notably slow down the extension rate. As for TS1, TS5 and TS6, the POI values are just a little bit larger than that of TS. This is because their mutations are either relatively far away from the cleavage/extension site (TS1) or do not exist in the cleaved TSX (TS5 and TS6). These results reconfirmed the explanation about the “dual-effects”. Moreover, since the exact 3′ end of CTS can be continuously tuned at a single-nucleotide scale via AMC, it is convenient and flexible to identify mismatches at any site of target DNA, showing a good potential for single nucleotide polymorphism (SNP) analysis [36,37].
3.2. Feasibility of AMCEA To demonstrate the feasibility of AMCEA, three artificial sequences, i.e., a target sequence (TS), a truncated target sequence (TTS, to mimic the cleavage product of TS via AMC) that can directly initiate EXPAR, and a random sequence (RS) as a non-target control, were firstly examined by using both fluorescence and gel analysis. As shown in Fig. 2a, the fluorescence increase in all cases displayed a sigmoid curve, on which the time corresponding to the maximum slope was termed as point of inflection (POI). It was found that the POI value caused by TS in the presence of DNA aligner (DA-1) was similar to that of TTS, and much lower than those of both RS and NTC (H2O as no-target control). By contrast, in the absence of DA-1 (Fig. 2b), the POI value of TS was almost the same with those of RS and NTC, and much higher than that of TTS. Gel analysis further confirmed these results. As shown in Fig. 2c, a bright band of ~30 bp (Lane 1) was observed for TS in the presence of DA-1. It corresponded to the duplex composed of EXPAR template (Temp-1) and its antisense strand, which was well consistent with the result of original EXPAR initiated by TTS (Lane 3). Moreover, there was
3.5. Universality of AMCEA In order to demonstrate the universality of AMCEA, two more sequences unique for SARS N gene (SARS-N) [38] and Bacillus anthracis protective antigen gene (BA-PA) [39] were also examined. In both cases, the real-time fluorescence showed a wide linear dynamic range of 5～6 orders of magnitude, with the limit of detection of 0.22 amol for SARSN and 0.68 amol for BA-PA, respectively (Fig. 4a and b). These results are comparable to or even better than that of the TS, indicating a good universality of AMCEA. We ascribed it to the excellent versatility of AMC. Since the cleavage of target DNA (trigger generation) can be achieved at any site of choice, AMCEA can virtually be used to detect 143
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Fig. 3. Sensitivity and specificity of AMCEA. a) Real-time fluorescence curves caused by series of TS concentration. b) The linear relationship between POI values and the negative logarithm of TS concentration. c) Schematic illustration of the positions of mismatches (lowercase letters) and the sites where the cleavage/extension occurs (red triangle). d) Real-time fluorescence curves caused by matched (TS) and single-nucleotide mismatched sequences (TSx) (2.5 fmol).
Fig. 4. Real-time fluorescence curves caused by series of a) SARS-N, b) BA-PA and c) TS spiked in human serum concentrations. Inset: the linear relationship between POI values and the negative logarithm of target concentration.
any sequence of interest. More importantly, only one NEase is needed in the whole process, which will greatly simplify the probe design and avoid the repeated optimization of reaction conditions.
3.6. Utility in complex biological matrix To evaluate the utility of AMCEA in complex biological matrix, it was carried out in human serum. To this end, different concentration of 144
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TS was spiked into serum, and then subjected to AMCEA under the identical conditions mentioned above. As shown in Fig. 4c, the realtime fluorescence curves were similar to that obtained by TS in buffer. Likewise, a linear correlation between the POI values and the negative logarithm of TS concentration (-lgC) was found. Its dynamic range also spans 5 orders of magnitude (from 25 fmol to 2.5 amol), with a regression equation of POI = − 14.668 + 2 (- lgC). The limit of detection was calculated as 1.37 amol using the 3σ method, which is nearly the same with the result of standard assay in buffer. Only a minor difference was caused by human serum, i.e., the shortened reaction time, which did not affect the quantitative ability of this method. Therefore, the proposed AMCEA has a good compatibility with real biological samples.
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4. Conclusions In summary, a highly universal, sensitive and selective amplification method, AMCEA, for the detection of nucleic acids has been developed. This method integrates the programmable cleavage of target DNA (AMC) with the NEase-based isothermal amplification (EXPAR). Since AMC uses only one NEase (also the same with EXPAR), and more importantly can make a break at any site of target sequence, it is easy to obtain a trigger DNA with a definite 3′-end as required to initiate EXPAR. As far as we know, the proposed AMCEA has the best versatility and simplicity among EXPAR methods. Moreover, it is sensitive and specific, with the capability of detecting amol-level DNA and distinguishing single nucleotide mutation through convenient and flexible probe design. It also functions well in the presence of complex biological matrix, holding good potentials for practical application. Acknowledgments This work was supported by the National Natural Science Foundation of China (21275129), National Key Foundation for Exploring Scientific Instruments (2013YQ470781), the Fundamental Research Funds for the Central Universities (2016QNA5008), Medical and Health Technology Project of Zhejiang Province (2015KYA061), and the Autonomous Research Project (ICT1601) of the State Key Laboratory of Industrial Control Technology. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2018.03.067. References [1] X. Chen, Y. Ba, L. Ma, X. Cai, Y. Yin, K. Wang, J. Guo, Y. Zhang, J. Chen, X. Guo, Q. Li, X. Li, et al., Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases, Cell Res. 18 (2008) 997–1006. [2] L. Uttley, B.L. Whiteman, H.B. Woods, S. Harnan, S.T. Philips, I.A. Cree, Building the evidence base of blood-based biomarkers for early detection of cancer: a rapid systematic mapping review, EBioMedicine 10 (2016) 164–173. [3] P. Yáñez-Sedeño, L. Agüí, R. Villalonga, J.M. Pingarrón, Biosensors in forensic analysis. A review, Anal. Chim. Acta 823 (2014) 1–19. [4] C. Zhang, D. Xing, Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends, Nucleic Acids Res. 35 (2007) 4223–4237. [5] H. Sipova, J. Homola, Surface plasmon resonance sensing of nucleic acids: a review, Anal. Chim. Acta 773 (2013) 9–23. [6] Y. Zhao, F. Chen, Q. Li, L. Wang, C. Fan, Isothermal amplification of nucleic acids, Chem. Rev. 115 (2015) 12491–12545. [7] P. Zhang, J. Zhang, C. Wang, C. Liu, H. Wang, Z. Li, Highly sensitive and specific multiplexed microRNA quantification using size-coded ligation chain reaction, Anal. Chem. 86 (2014) 1076–1082. [8] Y. Guo, K. Yang, J. Sun, J. Wu, H. Ju, A pH-responsive colorimetric strategy for DNA detection by acetylcholinesterase catalyzed hydrolysis and cascade amplification, Biosens. Bioelectron. 94 (2017) 651–656. [9] V. Ratautaite, S.N. Topkaya, L. Mikoliunaite, M. Ozsoz, Y. Oztekin, A. Ramanaviciene, A. Ramanavicius, Molecularly imprinted polypyrrole for DNA determination, Electroanalysis 25 (2013) 1169–1177. [10] S.A. Bustin, Quantification of mRNA using real-time reverse transcription PCR (RT-
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Aligner-mediated cleavage-triggered exponential amplification for sensitive detection of nucleic acids Wanghua Wua, Hongliang Fanb, Xiang Liana, Jianguang Zhoua, Tao Zhanga,
T
⁎
a Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou 310027, PR China b Department of Environmental Medicine, Institute of Hygiene, Zhejiang Academy of Medical Sciences, Hangzhou 310013, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Nucleic acids detection Isothermal exponential amplification DNA cleavage Aligner-mediated cleavage
Exponential amplification reaction (EXPAR), as a simple and high sensitive method, holds great promise in nucleic acids detection. One major challenge in EXPAR is the generation of trigger DNA with a definite 3′-end, which now relies on fingerprinting technology. However, the requirement of different endonucleases for varying target sequences and two head-to-head recognition sites in double stranded DNA, as well as the confinement of trigger DNA's 3′-end to be near/within the recognition site, usually subject EXPAR to compromised universality and/or repeated matching of reaction conditions. Herein, we report a simple and universal method for high sensitive detection of nucleic acids, termed aligner-mediated cleavage-triggered exponential amplification (AMCEA). The aligner-mediated cleavage (AMC) needs only one nicking endonuclease and can make a break at any site of choice in a programmable way. Thus, the 3′-end of target DNA can be easily redefined as required, a key step for initiating the amplification reaction. This capability endows the proposed AMCEA with excellent universality and simplicity. Moreover, it is sensitive and specific, with a detection limit at amol level, a broad dynamic range of 5～6 orders of magnitude and the ability to distinguish single nucleotide mutation. Experiments performed with human serum indicate that AMCEA is compatible with the complex biological sample, and thus has the potentials for practical applications.
1. Introduction Simple, fast and sensitive detection of nucleic acids is important in many fields, such as molecular biology, medical diagnostics and forensic analysis [1–3]. To date, various methods for high sensitive detection of nucleic acids have been developed, such as surface plasmon resonance, electrochemical sensors, molecular imprinted polymer, amplification techniques, and so on [4–9]. In particular, amplification methods have attracted extensive attentions in recent years. Though polymerase chain reaction (PCR) is the most powerful tool and widely used for the quantification of nucleic acids [10], it still has some intrinsic limitations, e.g., the requirement of specific thermal-cycling instrument, considerable power consumption and relatively long reaction time. These issues usually limit its practical utility in point-of-care test. Therefore, in the past two decades, great efforts have been made in developing a variety of isothermal amplification strategies [6], such as loop-mediated amplification (LAMP) [11,12], helicase-dependent amplification (HDA) [13,14], rolling circle amplification (RCA) [15,16], smart amplification process (SMAP) [17], recombinase polymerase amplification (RPA) [18,19] and cross priming amplification (CPA) ⁎
[20]. Nevertheless, most of current isothermal methods employ sophisticated mechanism or extra proteins/enzymes, which greatly increase the difficulty in primer design. Recently, owning to the simple mechanism and high amplification efficiency, the nicking endonuclease (NEase)-based methods, such as strand displacement amplification (SDA) [21,22], exponential amplification reaction (EXPAR) [23] and nicking endonuclease-mediated isothermal amplification (NAMP) [24], have attracted extensive attentions. However, one major limitation of NEase-based methods is the requirement of specific recognition sites in target sequence, which badly restricts their universality. Many efforts, e.g., the adoption of extra bumper primers [25], fingerprinting technique [26], junction probe strategy [27] and beacon-assisted amplification [28], are either unable to address the above issue thoroughly, or likely to worsen the nonspecific background amplification that is already serious in most isothermal methods [29]. For instance, EXPAR is a simple, low-cost and sensitive method, possessing a very high amplification efficiency (106 to 109-fold amplification within minutes). Since its working principle is based on a polymerase-catalyzed extension of trigger DNA (X) along a special template to generate the double-stranded recognition site of
Corresponding author. E-mail address: [email protected] (T. Zhang).
https://doi.org/10.1016/j.talanta.2018.03.067 Received 29 January 2018; Received in revised form 14 March 2018; Accepted 22 March 2018 Available online 26 March 2018 0039-9140/ © 2018 Published by Elsevier B.V.
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Fig. 1. Schematic illustration of AMCEA. Nt. BstNBI firstly binds the recognition site in the stem of DNA aligner (Loading), and is aligned at a specific locus through the hybridization of DA's side-arms with target DNA (Localization), and then makes a cleavage there (Reaction). Subsequently, the cleaved target serves as a trigger to initiate the EXPAR. By simply varying the sequences of aligner's side-arms, a trigger DNA can be generated from any locus of target DNA, endowing this method with excellent universality.
(NaCl) were purchased from Sigma Aldrich (St. Louis, MO). The clinical serum samples of healthy person were obtained from the Hospital of Zhejiang University. All solutions were freshly prepared with DNAgrade water.
NEase, the main challenge to EXPAR is the generation of trigger DNA with a definite 3′-end. This process now relies on the fingerprinting technology [26], which, however, requires different endonucleases for distinct target sequences of interest, and at the same time, two adjacent recognition sites oriented head-to-head in double-stranded DNA. Except for the difficulties in choosing an appropriate endonuclease for a given sequence and the matching of reaction conditions, the trigger DNA can only be produced from double-stranded DNA, and its 3′-end must be within/near the recognition site (Fig. S1), which, to a large extent, restrains the flexibility of EXPAR from detecting the real meaningful fragment of sequences. Our group has recently developed a simple and versatile strategy of using conventional NEases for programmable sequence-specific cleavage of DNA, termed aligner-mediated cleavage (AMC). It is based on a rationally designed hairpin-shaped DNA-aligner (DA), which consists of a recognition site of an NEase in the stem and two side arms complementary to the target sequence. Thus, an NEase can be easily loaded onto DA's stem, localized to a specific locus through the hybridization of the side arms with target DNA, and then makes a cleavage there (Fig. 1). This process needs only one NEase, does not require any special sequence in target DNA and can make a break at any intended site. In this paper, we further demonstrate an aligner-mediated cleavage-triggered exponential amplification (AMCEA). Thanks to the excellent versatility of AMC, the cleavage of target DNA, a key step to generate the trigger DNA, can be easily generated at any locus of target DNA. And the exact 3′ end can be tuned at a single-nucleotide scale by using just one NEase. This avoids the tedious processes of enzyme selection and the repeated optimization of reaction conditions. Thus, the proposed AMCEA is characterized with both excellent simplicity and universality. Moreover, this method is highly sensitive and specific, capable of detecting as less as 1.2 amol DNA and discriminating singlenucleotide mutations.
2.2. General procedure of AMCEA The reaction mixture of AMCEA was prepared separately on ice as part A and part B. Part A contained DA, EXPAR template, dNTPs, Nt. BstNBI and target of interest, and part B consisted of SYBR Green I and Bst 2.0 WarmStart DNA polymerase, both in the final reaction buffer (100 mM NaCl, 50 mM Tris-HCl, pH 8.0, 4 mM MgCl2 and 60 μg mL−1 BSA). Part A was first incubated at 55 °C for 15 min on a Bio-rad CFX96 PCR system, then part B was immediately added. The final reaction mixture, composed of 50 nM DA, 50 nM EXPAR template, 6 U Nt. BstNBI, 0.4 U Bst 2.0 WarmStart DNA polymerase, 0.4 mM dNTPs, 4 mM MgCl2 and 0.5 × SYBR Green I, was kept at 55 °C for 60 min and the real-time fluorescence was monitored at 1-min intervals. 2.3. Gel electrophoresis For gel analysis, the reactions were quenched at 95 °C for 5 min at a certain time (see the black triangles in Fig. 2a and b), then 5 μL of the resultant and 20 × SYBR Green I were loaded onto 12% polyacrylamide gel, and subjected to 120 V constant voltage in 0.5 × TBE buffer (45 mM Tris, 45 mM borate, 1 mM EDTA, pH 8.3) for 50 min at room temperature. The gel was then analyzed on a Maestro Ex IN-VIVO Imaging System (CRI). 2.4. Detection of nucleic acids spiked in human serum In order to demonstrate the utility of AMCEA in complex biological matrix, it was performed with human serum. First, the healthy human serum without any pretreatment was used to dilute target DNA by tenfold in the series. This process generated a concentration gradient from 25 fmol to 2.5 amol. Subsequently, 2.5 μL of spiked serum was subjected to AMCEA mentioned above. The results were compared with those obtained with buffered target DNA.
2. Materials and methods 2.1. Materials and reagents All oligonucleotides of HPLC purity were synthesized from Sangon Biotech Co., Ltd. (Shanghai, China). Detailed sequences of these oligonucleotides are listed in Table S1. Nicking endonuclease Nt. BstNBI (10,000 U mL−1), Bst 2.0 WarmStart DNA polymerase (8000 U mL−1), deoxyribonucleotide triphosphates (dNTPs, 10 mM), Low Molecular Weight DNA Ladder and MgCl2 solution (25 mM) were purchased from New England Biolabs (Ipswich, MA). SYBR Green I (10,000 ×) was purchased from Invitrogen Life Technologies (Carlsbad, CA). DNAgrade water (DNase- and Protease-free) and Tris-hydrochloride buffer (1 M solution, pH 8 .0) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Bovine serum albumin (BSA) and sodium chloride
3. Results and discussion 3.1. Principle of AMCEA The proposed AMCEA is illustrated in Fig. 1. The DNA aligner (DA) consists of two components: a stem-loop structure with a recognition site of Nt. BstNBI in the stem and two side arms complementary to the target sequence. First, a target sequence of interest (TS) hybridizes with DA, and is cleaved by Nt. BstNBI via AMC. Then, the cleaved target 142
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no such a band for either NTC or RS (Lane 2 and 4). A weak band lower than 30 bp in all these four reactions represents DA-1 that is 44 nt long (see Lane 9 for pure DA-1). By contrast, when DA-1 was absent, only TTS initiated EXPAR and resulted in a 30-bp band (Lane 7). TS, as well as RS and NTC, did not generate any resultants. These results clearly indicated the necessity of DA-1, which aligned Nt. BstNBI to cleave target DNA at a specific site to form an active trigger, and thus validated the mechanism of AMCEA shown in Fig. 1a. Note that the POI of RS and NTC were always the same either with or without DA-1, implicating an excellent selectivity of the proposed method. As for the relatively slow reaction rate in the presence of DA-1 when compared with the DAabsent system, the main reason is probably that the binding of Nt. BstNBI with DA-1 notably decreased the amount of free Nt. BstNBI, and thus slowed down the amplification rate. 3.3. Sensitivity of AMCEA To investigate the sensitivity of AMCEA, the real-time fluorescence curves caused by various concentrations of TS were measured. As can be seen in Fig. 3a, the POI values gradually decreased with the increase of TS concentration. Moreover, it showed a linear correlation with the negative logarithm of TS concentration (Fig. 3b). The regression equation is POI = − 36.775 + 4.519 (- lg C) (R2 = 0.992); C represents the concentration of TS. The limit of detection, defined as the concentration corresponding to a signal above three times of standard deviation (3σ), was found as 1.20 amol, and the dynamic range spanned over 5 orders of magnitude, which were comparable to previous EXPAR methods [30–35] (Table S2). The whole detection process of AMCEA took only 30～40 min, shorter than some other methods [31,35].
Fig. 2. Real-time fluorescence curves caused by different sequences (2.5 fmol) a) with and b) without DA-1. NTC represents the no-target control. c) Gel image of the products of AMCEA in the presence (Lane 1–4) or absence (Lane 5–8) of DA-1. Lane 9, 2.5 fmol pure DA-1.
sequence (CTS) leaves DA because the shortened hybridization length is unable to maintain a stable Y-shaped structure. At this point, the 3′ end of target DNA has been definitely redefined, and thus can serve as a trigger to initiate the amplification reaction. Namely, the 3′ end of CTS (X′) transiently hybridizes with EXPAR template that contains the recognition sequence of NtBstNBI in the middle and two fragments X on each side, and is extended along the template by Bst polymerase to form a complete double-stranded recognition site. As a result, Nt. BstNBI binds the newly formed recognition site and makes a nick on the extension product, followed by a second polymerase-catalyzed extension to displace the downstream strand (X′). Cycling above nicking and extension steps will generate more and more X′, and every X′ can initiate more reactions, leading to an exponential amplification. Due to the excellent versatility of AMC, the cleavage of target DNA, a key step to generate a trigger for initiating EXPAR, can be readily achieved at any site of choice by simply varying the sequences of DA's side arms. This process utilizes just one NEase and does not rely on any special sequence in target DNA. Therefore, the proposed AMCEA is very simple and highly universal.
3.4. Specificity of AMCEA To further evaluate the specificity of AMCEA, six single-base mismatched target DNA (TSX, X = 1～6, Table S1) were examined. Fig. 3c displays the positions of mismatches, all near the cleavage site of AMC. As shown in Fig. 3d, all mismatched DNA resulted in higher POI values when compared with that of perfectly matched target TS. In particular, the POI values of TS2～TS4 were much larger than those of other TSX and very close to NTC, indicating an excellent capability to discriminate single-base mismatch of AMCEA. This can be explained by the “dualeffects” imposed on both cleavage and extension processes, i.e., the mismatches near the cleavage site would cause imperfect hybridization between TSX and DA-1, and thus decrease the efficiency of AMC; in the meanwhile, the mismatches, especially the ones that are close to the 3′end of CTSX, would notably slow down the extension rate. As for TS1, TS5 and TS6, the POI values are just a little bit larger than that of TS. This is because their mutations are either relatively far away from the cleavage/extension site (TS1) or do not exist in the cleaved TSX (TS5 and TS6). These results reconfirmed the explanation about the “dual-effects”. Moreover, since the exact 3′ end of CTS can be continuously tuned at a single-nucleotide scale via AMC, it is convenient and flexible to identify mismatches at any site of target DNA, showing a good potential for single nucleotide polymorphism (SNP) analysis [36,37].
3.2. Feasibility of AMCEA To demonstrate the feasibility of AMCEA, three artificial sequences, i.e., a target sequence (TS), a truncated target sequence (TTS, to mimic the cleavage product of TS via AMC) that can directly initiate EXPAR, and a random sequence (RS) as a non-target control, were firstly examined by using both fluorescence and gel analysis. As shown in Fig. 2a, the fluorescence increase in all cases displayed a sigmoid curve, on which the time corresponding to the maximum slope was termed as point of inflection (POI). It was found that the POI value caused by TS in the presence of DNA aligner (DA-1) was similar to that of TTS, and much lower than those of both RS and NTC (H2O as no-target control). By contrast, in the absence of DA-1 (Fig. 2b), the POI value of TS was almost the same with those of RS and NTC, and much higher than that of TTS. Gel analysis further confirmed these results. As shown in Fig. 2c, a bright band of ~30 bp (Lane 1) was observed for TS in the presence of DA-1. It corresponded to the duplex composed of EXPAR template (Temp-1) and its antisense strand, which was well consistent with the result of original EXPAR initiated by TTS (Lane 3). Moreover, there was
3.5. Universality of AMCEA In order to demonstrate the universality of AMCEA, two more sequences unique for SARS N gene (SARS-N) [38] and Bacillus anthracis protective antigen gene (BA-PA) [39] were also examined. In both cases, the real-time fluorescence showed a wide linear dynamic range of 5～6 orders of magnitude, with the limit of detection of 0.22 amol for SARSN and 0.68 amol for BA-PA, respectively (Fig. 4a and b). These results are comparable to or even better than that of the TS, indicating a good universality of AMCEA. We ascribed it to the excellent versatility of AMC. Since the cleavage of target DNA (trigger generation) can be achieved at any site of choice, AMCEA can virtually be used to detect 143
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Fig. 3. Sensitivity and specificity of AMCEA. a) Real-time fluorescence curves caused by series of TS concentration. b) The linear relationship between POI values and the negative logarithm of TS concentration. c) Schematic illustration of the positions of mismatches (lowercase letters) and the sites where the cleavage/extension occurs (red triangle). d) Real-time fluorescence curves caused by matched (TS) and single-nucleotide mismatched sequences (TSx) (2.5 fmol).
Fig. 4. Real-time fluorescence curves caused by series of a) SARS-N, b) BA-PA and c) TS spiked in human serum concentrations. Inset: the linear relationship between POI values and the negative logarithm of target concentration.
any sequence of interest. More importantly, only one NEase is needed in the whole process, which will greatly simplify the probe design and avoid the repeated optimization of reaction conditions.
3.6. Utility in complex biological matrix To evaluate the utility of AMCEA in complex biological matrix, it was carried out in human serum. To this end, different concentration of 144
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TS was spiked into serum, and then subjected to AMCEA under the identical conditions mentioned above. As shown in Fig. 4c, the realtime fluorescence curves were similar to that obtained by TS in buffer. Likewise, a linear correlation between the POI values and the negative logarithm of TS concentration (-lgC) was found. Its dynamic range also spans 5 orders of magnitude (from 25 fmol to 2.5 amol), with a regression equation of POI = − 14.668 + 2 (- lgC). The limit of detection was calculated as 1.37 amol using the 3σ method, which is nearly the same with the result of standard assay in buffer. Only a minor difference was caused by human serum, i.e., the shortened reaction time, which did not affect the quantitative ability of this method. Therefore, the proposed AMCEA has a good compatibility with real biological samples.
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4. Conclusions In summary, a highly universal, sensitive and selective amplification method, AMCEA, for the detection of nucleic acids has been developed. This method integrates the programmable cleavage of target DNA (AMC) with the NEase-based isothermal amplification (EXPAR). Since AMC uses only one NEase (also the same with EXPAR), and more importantly can make a break at any site of target sequence, it is easy to obtain a trigger DNA with a definite 3′-end as required to initiate EXPAR. As far as we know, the proposed AMCEA has the best versatility and simplicity among EXPAR methods. Moreover, it is sensitive and specific, with the capability of detecting amol-level DNA and distinguishing single nucleotide mutation through convenient and flexible probe design. It also functions well in the presence of complex biological matrix, holding good potentials for practical application. Acknowledgments This work was supported by the National Natural Science Foundation of China (21275129), National Key Foundation for Exploring Scientific Instruments (2013YQ470781), the Fundamental Research Funds for the Central Universities (2016QNA5008), Medical and Health Technology Project of Zhejiang Province (2015KYA061), and the Autonomous Research Project (ICT1601) of the State Key Laboratory of Industrial Control Technology. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2018.03.067. References [1] X. Chen, Y. Ba, L. Ma, X. Cai, Y. Yin, K. Wang, J. Guo, Y. Zhang, J. Chen, X. Guo, Q. Li, X. Li, et al., Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases, Cell Res. 18 (2008) 997–1006. [2] L. Uttley, B.L. Whiteman, H.B. Woods, S. Harnan, S.T. Philips, I.A. Cree, Building the evidence base of blood-based biomarkers for early detection of cancer: a rapid systematic mapping review, EBioMedicine 10 (2016) 164–173. [3] P. Yáñez-Sedeño, L. Agüí, R. Villalonga, J.M. Pingarrón, Biosensors in forensic analysis. A review, Anal. Chim. Acta 823 (2014) 1–19. [4] C. Zhang, D. Xing, Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends, Nucleic Acids Res. 35 (2007) 4223–4237. [5] H. Sipova, J. Homola, Surface plasmon resonance sensing of nucleic acids: a review, Anal. Chim. Acta 773 (2013) 9–23. [6] Y. Zhao, F. Chen, Q. Li, L. Wang, C. Fan, Isothermal amplification of nucleic acids, Chem. Rev. 115 (2015) 12491–12545. [7] P. Zhang, J. Zhang, C. Wang, C. Liu, H. Wang, Z. Li, Highly sensitive and specific multiplexed microRNA quantification using size-coded ligation chain reaction, Anal. Chem. 86 (2014) 1076–1082. [8] Y. Guo, K. Yang, J. Sun, J. Wu, H. Ju, A pH-responsive colorimetric strategy for DNA detection by acetylcholinesterase catalyzed hydrolysis and cascade amplification, Biosens. Bioelectron. 94 (2017) 651–656. [9] V. Ratautaite, S.N. Topkaya, L. Mikoliunaite, M. Ozsoz, Y. Oztekin, A. Ramanaviciene, A. Ramanavicius, Molecularly imprinted polypyrrole for DNA determination, Electroanalysis 25 (2013) 1169–1177. [10] S.A. Bustin, Quantification of mRNA using real-time reverse transcription PCR (RT-
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