Sensitive Detection of MicroRNAs with Hairpin Probe-Based Circular

Jul 26, 2012 - This hairpin probe-based circular exponential amplification assay exhibits high sensitivity with a detection limit of 3.80 × 10–13 M...
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Sensitive Detection of MicroRNAs with Hairpin Probe-Based Circular Exponential Amplification Assay Guo-lei Wang and Chun-yang Zhang* Single-molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong, 518055, China S Supporting Information *

ABSTRACT: MicroRNAs (miRNAs) play important regulatory roles in a wide range of biological processes, and their aberrant expression is associated with cancer development and a variety of diseases. Here, we develop a simple, sensitive, and specific miRNA assay on the basis of circular exponential amplification in combination with the hairpin probes. The binding of target miRNA with a linear DNA template initiates the first strand displacement amplification (SDA) and generates the universal triggers which are complementary to the 3′ protruding end of a hairpin probe. These universal triggers function not only as the primers to unfold the hairpin probes through an extension reaction, generating distinct fluorescence signals, but also as the amplification templates to initiate the second SDA reaction. Moreover, the second SDA reaction can release new triggers to initiate the above two consecutive SDA reactions, thus constituting a circular exponential amplification which enables the conversion of a small amount of miRNAs to a large number of universal triggers to unfold abundant hairpin probes. This hairpin probe-based circular exponential amplification assay exhibits high sensitivity with a detection limit of 3.80 × 10−13 M and a detection range of 4 orders of magnitude. It can even discriminate single-nucleotide difference between miRNA family members and perform well in real sample analysis. Notably, in this assay, the long-stem hairpin probes are unfolded through an extension reaction rather than through a conventional hybridization reaction controlled by the thermodynamic equilibrium in the case of molecular beacons, making the design of hairpin probes very simple. This hairpin probe-based circular exponential amplification assay holds a great promise for further application in biomedical research and early clinical diagnosis.

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However, these methods either require special chemical21 and enzymatic modification19 of oligonucleotides or rely on the locked nucleic acids (LNAs)20 as the capture probes to circumvent the limitation of small-size miRNAs. Recently, isothermal exponential amplification reaction (EXPAR) is introduced into the profiling of low-abundance miRNA22,23 because of its high sensitivity, low cost, and good tolerance to the inhibitory components in the clinical samples. However, SYBR Green I is usually used as the label in the EXPAR assay, and its preferential binding to GC-rich sequences limits its further applications in multiplex PCR.24 Alternatively, stranddisplacement amplification (SDA) in combination with molecular beacons has been successfully applied for the detection of various nucleic acids with high sensitivity and excellent specificity,25−27 but its sensitivity is mainly dependent on the design of the molecular beacon whose unfolding is determined by the enthalpy and the entropy of both stem and probe−target duplex.28

icroRNAs (miRNAs) are a group of short endogenous noncoding RNAs that can bind to the 3′ untranslated region of mRNAs to regulate the expression of target genes in cell proliferation, differentiation, and tumorigenesis.1−4 Recently, miRNAs have been emerged as new biomarkers for early diagnosis of diseases including cancer.5,6 However, their unique characteristics of small sizes, extremely low abundance in total RNA samples, and high sequence homology among family members have made a great challenge for quantitative analysis.7 Northern blot analysis is usually used for miRNA detection,8−11 but its low sensitivity, labor-intensive steps, and the requirement of a large amount of RNA samples have limited its broad applications. The microarray-based method makes multiple miRNA analysis feasible,12,13 but its low sensitivity and poor specificity are still two unsolved issues.14 The real-time PCRbased method is regarded as a powerful tool for highly sensitive and accurate quantitative analysis,15,16 but its performance is dependent on isolated and highly purified total RNA samples which are difficult to obtain. In addition, the small sizes of miRNAs have restricted the direct application of conventional RT-PCR protocols.17 As the demand for miRNA profiling is rapidly expanding, several new methods, such as solid-phase hybridization assay18−20 and bioluminescence assay,21 have been developed. © 2012 American Chemical Society

Received: May 8, 2012 Accepted: July 26, 2012 Published: July 26, 2012 7037

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DNA polymerase, ThermoPol buffer, and DEPC-treated water. Part A was first incubated at 95 °C for 3 min to denature the template and the miRNA target. Then, part A was cooled to 55 °C, followed by addition of part B. The reaction of part A with part B was performed at 55 °C for 30 min to generate the universal triggers. After the reaction finished, part C was added immediately to the reaction solution. The circular exponential amplification reaction was performed at 52 °C in a total volume of 10 μL of solution containing 0.2 μM template, 250 μM dNTPs, 0.8 U/ μL RNase inhibitor, 0.8 U/μL Nt.BstNBI nicking enzyme, 0.1 U/μL Vent (exo-) DNA polymerase, 0.05 μM hairpin probe, 0.5× Nt.BstNBI buffer (25 mM Tris−HCl, pH 7.9, 5 mM MgCl2, 50 mM NaCl, 0.5 mM dithiothreitol), and 1× ThermoPol buffer (20 mM Tris−HCl, pH 8.8, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X100). The real-time fluorescence was monitored at intervals of 30 s with a Roche LightCycler 480 II Real-Time PCR System (Rotkreuz, Switzerland). Gel Electrophoresis. Products of the first SDA reaction were analyzed by 12% nondenaturating polyacrylamide gel electrophoresis (PAGE) in 1× TBE buffer (9 mM Tris−HCl, pH 7.9, 9 mM boric acid, 0.2 mM EDTA) at a 110 V constant voltage for 40 min at room temperature. The gel was stained by SYBR Green I and scanned by a Kodak Image Station 4000MM (Rochester, NY).

Herein, we introduce a hairpin probe-based circular exponential amplification assay for sensitive detection of miRNAs. Unlike the conventional molecular beacon whose unfolding requires the hybridization of molecular beacon with a perfect-matched target at a certain temperature, in this assay, the hairpin probe with a long stem is unfolded through an extension reaction with the universal trigger as the primer, making the design of hairpin probe very simple and the subsequent experiments very easy. Moreover, miRNA targettriggering circular exponential amplification can unfold abundant hairpin probes and induce significant enhancement of fluorescence signals, endowing this hairpin probe-based circular exponential amplification assay with excellent specificity and high sensitivity.

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EXPERIMENTAL SECTION Materials. The HPLC-purified hairpin probes, DNA oligonucleotides, and miRNAs were synthesized by TaKaRa Bio. Inc. (Dalian, China). Sequences of the oligonucleotides were designed with the help of Primer Premier 5 (PREMIER Biosoft Int., Palo Alto, CA) and mfold software (Table 1). Table 1. Sequences of Hairpin Probe, Template, and miRNAsa note hairpin probe template let-7a let-7b let-7c

sequence (5′-3′)

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FAM-ACT ATA CAA CCT ACT ACC TTT CAG ACT CAC GTA GTA GGT TGT ATA Gt(BHQ1)G TTT GTC ATC GCA GTG TTC CTC A-NH2 GTC ATC GCA GTG TTC CTC AAC AGA CTC AAA CTA TAC AAC CTA CTA CCT CAA-NH2 UGA GGU AGU AGG UUG UAU AGU U UGA GGU AGU AGG UUG UGU GGU U UGA GGU AGU AGG UUG UAU GGU U

RESULTS AND DISCUSSION Principle of Hairpin Probe-Based Circular Exponential Amplification Assay. The strategy for miRNA detection is illustrated in Scheme 1. The linear template consists of three domains: a miRNA-binding domain (I), a recognition domain (II) for Nt.BstNBI, and an amplification domain (III). The hairpin probe has a stem-loop structure with a fluorophore (FAM) at the 5′ end and a quencher (BHQ1) across from FAM on the opposite strand of the stem. In addition, the hairpin probe is designed to have the same recognition domain (II) as the linear template and a special 3′ protruding end (IV) as the binding domain for the universal trigger released from the first SDA reaction (Table 1). In the presence of target miRNA, the specific templatemiRNA binding leads to a part duplex at domain I, making the target miRNA be extended along the template to form a complete duplex in the presence of Vent (exo-) DNA polymerase and dNTPs.22 Subsequently, the nicking enzyme recognizes the duplex site at domain II and cleaves the upper DNA strand at a site four bases downstream, generating a new replication site for polymerase. As a result of stranddisplacement activity of Vent (exo-) DNA polymerase, a large number of universal triggers are generated through the repeated extension, cleavage, and the release of short oligonucleotides. The released universal triggers might function as the primers to anneal with the domain IV of the hairpin probe and subsequently initiate a new extension reaction. This elongation unfolds the hairpin probe, making spatial separation of the fluorophore from the quencher and consequently the restoration of FAM fluorescence. In the meantime, the resultant DNA duplex with a special recognition site can be cleaved by Nt.BstNBI and then extended by Vent (exo-) DNA polymerase, recreating an amplification template for the second SDA reaction which enables the further release of new triggers. It should be noted that these new triggers have the same sequence as the target miRNA except for three bases (two at

a The italicized region of the hairpin probe identifies the stem sequence, and the underlined regions in the hairpin probe and the template identify the recognition site of Nt.BstNBI, respectively. The lowercase letter of t in the middle of the hairpin probe and the 5′ end of the hairpin probe are labeled with Black Hole Quencher 1 (BHQ1) and fluorescein (FAM), respectively. The bases in let-7b and let-7c that differ from those in let-7a are marked in boldface.

Before use, the hairpin probes were diluted to 5 μM in a buffer containing 1.5 mM MgCl2 and 10 mM Tris−HCl, pH 8.0. The hairpin probes were incubated at 95 °C for 5 min and then slowly cooled to room temperature over 30 min to make the probe perfectly fold into a hairpin structure. The vent (exo-) DNA polymerase and nicking endonuclease Nt.BstNBI were obtained from New England Biolabs (Ipswich, MA). The deoxynucleotide solution mixture (dNTPs), RNase inhibitor, and diethyl pyrocarbonate (DEPC)-treated water were purchased from TaKaRa Bio. Inc. (Dalian, China). SYBR Green I (20 μg/μL) was obtained from Xiamen Biovision Biotech Co., Ltd. (Xiamen, China). The human brain total RNA was obtained from Ambion (Austin, TX). DEPC-treated deionized water was used in all experiments. Hairpin Probe-Based Circular Exponential Amplification Assay. The reaction mixtures for amplification reaction were prepared separately on ice as part A, part B, and part C. Part A consisted of the linear template, dNTPs, Nt.BstNBI buffer, and miRNA target. Part B consisted of RNase inhibitor, Nt.BstNBI nicking enzyme, Vent (exo-) DNA polymerase, ThermoPol buffer, and DEPC-treated water. Part C consisted of the hairpin probe, Nt.BstNBI nicking enzyme, Vent (exo-) 7038

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Scheme 1. Scheme for miRNA Detection with Hairpin Probe-Based Circular Exponential Amplification Assaya

a

This assay involves four principal steps: (1) miRNA-template binding and the following extension reaction produce a double-stranded DNA/RNA duplex in the presence of polymerase; (2) strand displacement amplification generates abundant universal triggers in the presence of nicking enzyme and polymerase; (3) universal trigger-hairpin probe binding and the following extension reaction unfold the hairpin probe and restore FAM fluorescence; (4) a second strand displacement amplification generates abundant new triggers which can bind to the linear template to initiate a new reaction cycle. Through the reaction cycle, the two consecutive SDA reactions in tandem constitute a circular exponential amplification which enables the conversion of a small amount of miRNAs to a large number of universal triggers to unfold abundant hairpin probes.

the 5′ end and one at the 3′ end) less than the target miRNA and the replacement of uridines and ribonucleotides with thymines and deoxyribonucleotides, respectively. Therefore, these new triggers can hybridize with the linear template, and their elongation launches the first SDA to yield more universal triggers which can initiate the second SDA. As a result, two consecutive SDA reactions in tandem constitute a circular chain reaction which leads to an exponential growth of the triggers, generating circular exponential amplification (EXPAR). Notably, in this circular EXPAR, the long-stem hairpin probe is unfolded by an extension reaction rather than by conventional hybridization reaction between the loop region of molecular beacon and single-stranded DNA/RNA.25,26 Thus, this hairpin probe can be simply designed without the requirement of thermodynamic equilibrium associated with the enthalpy and the entropy of both stem and probe−target duplexes. To monitor the first SDA reaction in real time, SYBR Green I is used as the fluorescent dye. As shown in Figure 1A, the fluorescence intensity gradually increases in the presence of let7a miRNA (curve b in Figure 1A), indicating that let-7a miRNA initiates the first SDA reaction and produces a large number of universal triggers (lane b in the inset of Figure 1A). On the contrary, in the control reaction without let-7a miRNA, the fluorescence intensity remains unchanged (curve a in Figure

1A), indicating that no SDA reaction occurs and no universal trigger is produced (lane a in the inset of Figure 1A). After the first SDA reaction, a solution containing hairpin probes, Vent (exo-) DNA polymerase, and nicking enzyme is added to initiate the second SDA and the subsequent circular EXPAR. The variance of fluorescence intensity with time is monitored in real time. As shown in Figure 1B, the fluorescence intensity increases in a sigmoidal fashion in the presence of let7a miRNA (curve d in Figure 1B), suggesting that the universal triggers generated in the first SDA can efficiently initiate the second SDA and the subsequent circular EXPAR, which can unfold abundant hairpin probes and induce the enhancement of fluorescence signals. It should be noted that the control experiment without let-7a miRNA shows nonspecific background amplification as well (curve c in Figure 1B); however, it takes a long time to reach a plateau. The point of inflection (POI), which is defined as the time corresponding to the maximum slope in the sigmoidal curve,22,23,29,30 is used for quantitative analysis of miRNA in this assay. Since the nonspecific background amplification exhibits an extremely high POI value (curve c in Figure 1B), it does not interfere with the specific amplification of target miRNAs and the subsequent quantitative analysis.22,30 7039

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appropriate temperature of 52 °C is chosen in the following assay. Detection of let-7a miRNA. To investigate the capability of the proposed method for miRNA detection, let-7a with different concentrations is analyzed under the optimum condition. Figure 2A shows the real-time fluorescence curves

Figure 1. (A) Real-time fluorescence monitoring of the first SDA reaction with SYBR Green I as the fluorescent indicator. The fluorescence curves are obtained in the absence (a) and in the presence (b) of 5 nM let-7a, respectively. Inset: polyacrylamide gel electrophoresis of the amplification products from the first SDA reaction. Lane m is the DNA ladder marker. Lanes a and b represent the amplification products in the absence (a) and in the presence (b) of 5 nM let-7a, respectively. (B) Real-time fluorescence monitoring of the circular EXPAR with the hairpin probe as the fluorescent indicator after the first SDA reaction. The fluorescence curves are obtained in the absence (c) and in the presence (d) of 5 nM let-7a, respectively.

Figure 2. (A) Real-time fluorescence curves in response to different concentrations of let-7a. (B) Variance of the POI values as a function of let-7a concentration. Inset: the POI value is log−linear correlation with let-7a concentration in the range from 1 pM to 10 nM. Error bars show the standard deviation of five experiments.

Optimization of Hairpin Probe Concentration. The effect of hairpin probe concentration on the hairpin probebased circular EXPAR is investigated by comparing the fluorescence intensity produced by 5 nM let-7a with that by the blank without let-7a (see the Supporting Information, Figure S-1). With the increase in the concentration of hairpin probes, the fluorescence intensity increases in the presence of let-7a, but the fluorescence intensity increases in the blank without let-7a as well. When 0.05 μM hairpin probe is used, the interval of the POI between let-7a and the blank reaches the maximum value. Therefore, 0.05 μM is chosen as the optimum concentration of hairpin probes in the following assay. Optimization of Amplification Temperature. The amplification temperature has a crucial effect on the reactivity of enzyme and the hybridization efficiency of nucleic acids. The effect of amplification temperature on the hairpin probe-based circular EXPAR is investigated by comparing the fluorescence intensity produced by 5 nM let-7a with that by the blank without let-7a (see the Supporting Information, Figure S-2). As the amplification temperature increases, the initial time at which the fluorescence signals are detectable becomes short in the presence of let-7a, but the initial time becomes relatively short as well in the blank without let-7a, leading to a small interval of the POI between let-7a and the blank at a high temperature. In contrast, a low temperature leads to a large interval of the POI between let-7a and the blank, but the circular EXPAR at a low temperature will take a long time, which is against the rapid miRNA assay. Therefore, an

in response to different concentrations of let-7a, and the POI value decreases with the increase in let-7a concentration (Figure 2B). In logarithmic scales, the POI value exhibits a linear correlation with let-7a concentration over a range of 4 orders of magnitude from 1 pM to 10 nM (inset of Figure 2B). The regression equation is Y = −117.72−16.42 log10 C with a correlation coefficient of 0.9961, where Y and C are the POI value and the miRNA concentration (M). The detection limit, derived from the difference between the average POI value of the blank and three times the standard deviation of POI value of the blank, is estimated to be 3.80 × 10−13 M. Notably, the sensitivity of this method is 3 orders of magnitude higher than that of molecular beacon-based methods,31 2 orders of magnitude higher than that of the Q-STAR probe-based rolling circle amplification assay,32 and more than 1 order of magnitude higher than that of bimolecular beacon-based amplification assay as well.33 The improved sensitivity of this method can be attributed to the following two factors: (1) the extremely high amplification efficiency of circular EXPAR which enables the conversion of a small amount of miRNAs to a large number of universal triggers, and (2) the involvement of a new mechanism to unfold the hairpin probe through universal trigger-initiated extension reaction. Detection Specificity. A great challenge of the miRNA assay is to distinguish the miRNA family members with high 7040

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sample analysis, 20 amol let-7a is spiked into 200 ng of total RNA for the assay, and the amount of let-7a in the spiked sample is estimated to be 33.95 amol (RSD = 2.6%, n = 5) with a recovery ratio of 115.1%, suggesting that the proposed method is capable of sensitive miRNA assay in real samples.

similarity. To investigate the specificity of the proposed method, three members of let-7 family (let-7a, let-7b, and let7c), with only one- or two-nucleotide differences out of 22 nucleotides between them, are selected as the detection model. As let-7a is perfectly complementary to the amplification template, its real-time fluorescence signal can be distinguished well from those produced by let-7b and let-7c (inset of Figure 3). The ΔPOI, defined as the difference in the POI value

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CONCLUSIONS In summary, we have developed a highly selective and sensitive method for miRNA assay on the basis of circular EXPAR in combination with the hairpin probes. In this assay, the longstem hairpin probes are unfolded through an extension reaction with the universal triggers as the primers rather than through a conventional hybridization reaction controlled by the thermodynamic equilibrium in the case of molecular beacons, making the design of hairpin probes very simple. In addition, miRNA target-triggering circular EXPAR can unfold abundant hairpin probes and induce significant enhancement of fluorescence signals. This method has significant advantages of excellent specificity with the capability of discriminating single-nucleotide difference between miRNA family members, high sensitivity with a detection limit of 3.80 × 10−13 M and a detection range of 4 orders of magnitude, and good performance in real sample analysis. More importantly, this hairpin probe-based circular exponential amplification assay holds a great potential for further application in biomedical research and early clinical diagnosis.

Figure 3. Detection specificity of the hairpin probe-based circular EXPAR evaluated by the ΔPOI of let-7a, let-7b, and let-7c. The ΔPOI of the blank is defined as 0. The concentration of each let-7a, let-7b, and let-7c is 100 pM. Error bars show the standard deviation of five experiments. Inset: typical real-time fluorescence curves in response to let-7a (red line), let-7b (green line), let-7c (purple line), and the blank (black line).

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ASSOCIATED CONTENT

S Supporting Information *

between the particular sample and the blank, is calculated to evaluate the detection specificity. As shown in Figure 3, the ΔPOI of let-7a reaches 43.5, which is 13-fold more than that of let-7b and 7-fold more than that of let-7c as well, even though there is only a single base difference near the 3′ end of the miRNA between let-7a and let-7c (Table 1), suggesting the high specificity of this miRNA assay. Real Sample Analysis. To demonstrate the capability of the proposed method in real sample analysis, we perform the miRNA assay using human brain total RNA. The RNA sample is diluted to 200 ng/μL with DEPC-treated water, and 1 μL of the total RNA sample (200 ng in total) is used for the measurement. As shown in Figure 4, the real-time fluorescence signal produced by 200 ng of total RNA can be distinguished well from that produced by the blank. According to the simultaneously constructed calibration curve (see the Supporting Information, Figure S-3), the amount of let-7a in the total RNA sample is estimated to be 10.93 amol per 200 ng (RSD = 3.4%, n = 5). For further evaluation of its performance in real

Supplementary Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 755 86392211. Fax: +86 755 86392299. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program 973 (Grant Nos. 2011CB933600 and 2010CB732600), National Natural Science Foundation of China (Grant No. 21075129), Guangdong Innovation Research Team Fund for Low-cost Healthcare Technologies, Natural Science Foundation of Shenzhen City (Grant Nos. JC201005270327A and CXB201005250029A), Fund for Shenzhen Engineering Laboratory of Single-molecule Detection and Instrument Development (Grant No. (2012) 433), and Award for the Hundred Talent Program of the Chinese Academy of Science.

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