Homogeneous and Label-Free Detection of MicroRNAs Using

Jun 6, 2014 - ... analyzed the bands of triggers X and Y using the ImageJ software (Figure 2B). .... 21325523), the Award for the Hundred Talent Progr...
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Homogeneous and Label-Free Detection of MicroRNAs Using Bifunctional Strand Displacement Amplification-Mediated Hyperbranched Rolling Circle Amplification Li-rong Zhang,† Guichi Zhu,† and Chun-yang Zhang* Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China ABSTRACT: MicroRNAs (miRNAs) are an emerging class of biomarkers and therapeutic targets for various diseases including cancers. Here, we develop a homogeneous and label-free method for sensitive detection of let-7a miRNA based on bifunctional strand displacement amplification (SDA)-mediated hyperbranched rolling circle amplification (HRCA). The binding of target miRNA with the linear template initiates the bifunctional SDA reaction, generating two different kinds of triggers which can hybridize with the linear template to initiate new rounds of SDA reaction for the production of more and more triggers. In the meantime, the released two different kinds of triggers can function as the first and the second primers, respectively, to initiate the HRCA reaction whose products can be simply monitored by a standard fluorometer with SYBR Green I as the fluorescent indicator. The proposed method exhibits high sensitivity with a detection limit of as low as 1.8 × 10−13 M and a large dynamic range of 5 orders of magnitude from 0.1 pM to 10 nM, and it can even discriminate the single-base difference among the miRNA family members. Moreover, this method can be used to analyze the total RNA samples from the human lung tissues and might be further applied for sensitive detection of various proteins, small molecules, and metal ions in combination with specific aptamers.

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and multiple primer design increases the experimental cost and complexity. To improve the detection sensitivity and simplicity, a variety of new methods has been developed, such as molecular beacons,9 electrochemical sensor,10 nanoparticle-based assay,11 and the surface-enhanced Raman scattering method.12 However, the involvement of costly fluorescent-labeled probes,9 the immobilization/separation steps,10 the time-consuming synthesis of nanoparticles,11 and the fluctuation of Raman spectra12 limit their practical applications. Therefore, the development of a simple and sensitive method for the miRNA assay still remains a great challenge. Herein, we develop a homogeneous and label-free method for sensitive detection of let-7a miRNA using bifunctional strand displacement amplification (SDA)-mediated hyperbranched rolling circle amplification (HRCA). HRCA is an isothermally exponential amplification strategy through a turn-by-turn cascade of multiple hybridization, primer extension, and strand displacement13 and has been employed for sensitive detection of DNA methylation,14 viral RNA,15 and proteins.16 However, the involvement of two different kinds of extra primers might complicate the experimental procedures and increase the risk of nonspecific amplification. In this research, we design a bifunctional SDA reaction to simultaneously generate two different kinds of triggers,

icroRNAs (miRNAs) comprise a large family of small noncoding RNA molecules and play an important role in many biological pathways, such as cell proliferation, differentiation, and apoptosis.1 Most of miRNAs are derived from long primary transcripts that undergo the cleavage processing of precursor miRNAs (pre-miRNAs) by the Dicer enzyme.2 Recently, more and more evidence indicates that the aberrant expression of miRNAs in human tissues and blood samples is associated with the cancer initiation, the tumor stage, and the response of tumors to treatments.3 Moreover, miRNAs might be used as the potential therapeutic targets for the discovery of new drugs.4 miRNAs have simpler structure and less complex postsynthetic processing than proteins and DNAs and can be used as promising biomarkers for cancer diagnosis and prognosis.5 Consequently, the accurate and quantitative analysis of miRNA expression has become imperative for the biological research, early clinical diagnosis, and the screening of new anticancer drugs. Several unique characteristics of miRNAs, such as small size, sequence similarity among the miRNA family members, and low abundance in total RNA samples, make their accurate analysis a great challenge.6 The Northern blotting is the widely used method for the miRNA assay,7 but it suffers from poor sensitivity and large sample consumption. The quantitative real-time polymerase chain reaction (qRT-PCR) is another conventional method with the advantage of high sensitivity and practicality,8 but the requirement of precise temperature control © 2014 American Chemical Society

Received: May 5, 2014 Accepted: June 6, 2014 Published: June 6, 2014 6703

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(exo-) DNA polymerase, the nicking endonuclease of Nb.BsmI, the deoxynucleotide mixture (dNTPs), and the corresponding buffers were purchased from New England Biolabs, Inc. (Ipswich, MA, USA). The RNase inhibitor and RNase-free water were obtained from TaKaRa Bio. Inc. (Dalian, China). SYBR Green I and SYBR gold were purchased from Life Technologies (Carlsbad, CA, USA). The paraffin-embedding lung tissue samples were obtained from the Department of Pathology, Affiliated Hospital of Guangdong Medical College (Zhanjiang, China). Bifunctional SDA-Mediated HRCA Reaction. All oligonucleotides were dissolved in RNase-free water before use. The target miRNA and the linear template were first denatured at 80 °C for 3 min, followed by cooling to room temperature for annealing. The bifunctional SDA-mediated HRCA reaction was performed in 10 μL of reaction solution containing 25 nM linear template, 5 nM circlular template, 400 μM dNTPs, 0.4 U Vent (exo-) DNA polymerase, 6 U Nb.BsmI, 20 mM Tris-HCl buffer (pH 8.8), 10 mM (NH4)2SO4, 10 mM KCl, 6 mM MgSO4, 0.1% Triton X-100, and the target miRNAs at 62 °C for 2 h. The reaction was terminated by incubation at 80 °C for 20 min. Measurement of Fluorescence Spectra. The 10 μL of amplification products was diluted to a final volume of 50 μL with water and then mixed with 0.5× SYBR Green I. The fluorescence spectra were measured by a Hitachi F-4500 fluorometer (Tokyo, Japan) at the excitation wavelength of 495 nm. The fluorescence intensity at 526 nm was used for data analysis. Gel Electrophoresis. The amplification products of bifunctional SDA reaction were analyzed by 10% nondenaturating polyacrylamide gel electrophoresis (PAGE) in 0.5× TBE buffer (22.5 mM Tris-boric acid, 5 mM EDTA, pH 8.0) at a 120 V constant voltage at room temperature for 50 min. The amplification products of bifunctional SDA-mediated HRCA reaction were analyzed by 2% agarose gel electrophoresis in 1× TAE buffer

which can function as the primers to initiate the HRCA reaction without the addition of any extra primers. In addition, this bifunctional SDA-mediated HRCA reaction can be completed in one tube without the involvement of any separation steps. The proposed method allows for sensitive detection of target miRNA with a detection limit of as low as 1.8 × 10−13 M and a large dynamic range of 5 orders of magnitude from 0.1 pM to 10 nM, and it can even discriminate single-base difference among the miRNA family members. Moreover, this method can be used to analyze the total RNA samples from the human lung tissues.

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EXPERIMENTAL SECTION Materials. All oligonucleotides (Table 1) were HPLC purified and obtained from TaKaRa Bio. Inc. (Dalian, China). The Vent Table 1. Sequences of the Templates, the Synthesized Triggers, and the miRNAsa note linear template circular template synthesized trigger X synthesized trigger Y let-7a let-7b let-7c

sequence (5′−3′) CAA CTA TAC AAC CTA CTA CCG AAT GCA GAC ATA GAG ACT TAG AAT GCA ACT ATA CAA CCT ACT ACC TCA-P P-CAA CTA TAC AAC CTA CTA CCG AAT GAA TAT GAA CAC ATT CTA AGT CTC TAT GTC TGG CAA CAG TGT CAT TCG GTA GTA GGT TGT ATA GTT G CAT TCT AAG TCT CTA TGT CTG UGA GGU AGU AGG UUG UAU AGU U UGA GGU AGU AGG UUG UGU GGU U UGA GGU AGU AGG UUG UAU GGU U

a

The underlined regions in the linear template represent the recognition site of Nb.BsmI. The bases in let-7b and let-7c that differ from those in let-7a are marked in bold. There are two base mismatches between let-7a and let-7b and one base mismatch between let-7a and let-7c.

Scheme 1. Schematic Illustration of miRNA Assay Based on the Bifunctional SDA-Mediated HRCA

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Figure 1. (A) Real-time fluorescence monitoring of bifunctional SDA reaction in the presence of 1 nM let-7a miRNA with SYBR Green I as the fluorescent indicator. (B) The amplification products of bifunctional SDA analyzed by 10% nondenaturating PAGE. Lane M is the DNA ladder marker. Lanes 1 and 2 represent the products in the absence and in the presence of 1 nM let-7a miRNA, respectively. Lanes 3 and 4 represent the synthesized trigger X (25 nt) and the synthesized trigger Y (21 nt), respectively. (C) Fluorescence measurement of SDA-mediated HRCA reaction in the presence of 1 nM let-7a and circular template (red line), 1 nM let-7a without circular template (blue line), and circular template without let-7a miRNA (black line). The SDA-mediated HRCA reaction time is 2 h. (D) Amplification products of SDA-mediated HRCA reaction analyzed by 2% agarose gel electrophoresis. Lane M is the DNA ladder marker. Lanes 1, 2, and 3 represent the products in the presence of circular template without let-7a miRNA, 1 nM let-7a without circular template, and 1 nM let-7a with circular template, respectively.

triggers. In the meantime, the released triggers X and Y can function as the first and the second primers, respectively, to initiate the HRCA reaction in the presence of circular template, generating larger numbers of double-stranded DNA (dsDNA) fragments with different length, which can be simply detected by using SYBR Green I as the fluorescent indicator.17 While in the absence of target miRNA, neither the SDA nor HRCA reaction can be initiated and no fluorescence enhancement is observed. To evaluate the amplification products of bifunctional SDA, we monitored the real-time fluorescence of bifunctional SDA reaction with SYBR Green I as the fluorescent indicator in the absence of circular template. As shown in Figure 1A, in the presence of let-7a miRNA, the fluorescence intensity increases rapidly with the reaction time and reaches a plateau at 35 min (Figure 1A, red line), indicating that the let-7a miRNA can initiate the bifunctional SDA reaction. In contrast, no fluorescence enhancement is observed in the absence of let-7a miRNA (Figure 1A, black line). The above results are further confirmed by the 10% nondenaturating PAGE analysis. As shown in Figure 1B, in the absence of let-7a miRNA, only the band of single-stranded linear template is observed, but no band of the generated triggers is observed (Figure 1B, lane 1), indicating no occurrence of bifunctional SDA reaction. However, in the presence of let-7a miRNA, a small amount of target can be converted to large numbers of triggers X and Y through bifunctional SDA reaction, and the bands of dsDNA template,

(40 mM Tris-acetic acid, 2 mM EDTA, pH 8.0) at a 120 V constant voltage at room temperature for 25 min. The gels were stained by SYBR gold and analyzed by a Kodak Image station 4000MM (Carestream Health, Rochester, NY, USA). The bands of nondenaturating PAGE were analyzed by the ImageJ software for quantitative analysis.

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RESULTS AND DISCUSSION Principle of miRNA Assay. The principle of the miRNA assay is illustrated in Scheme 1. The linear template contains three main regions of X*-Y*-X*. The sequence of X* is complementary to the target miRNA and the generated trigger X, and the sequence of Y* is complementary to the generated trigger Y. It should be noted that there are two recognition sites of Nb.BsmI between X* and Y* in the sequence of X*-Y*-X*. When the target miRNA is added to the reaction solution, it will specifically bind to the X* region of linear template, and the 3′ end of miRNA can function as a primer to initiate an extension reaction with the catalysis of Vent (exo-) polymerase and dNTPs, producing a DNA duplex. Then, the nicking enzyme Nb.BsmI cleaves the two recognition sites of DNA duplex to initiate a new extension cycle, generating abundant triggers X and Y. The released triggers X and Y can specifically hybridize with the X* and Y* regions of linear template to start new rounds of SDA reaction, resulting in an exponential amplification through the repeated extension, cleavage, and the release of 6705

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Figure 2. (A) Variance of the amplification products with the concentration of linear template in the presence of 1 nM let-7a miRNA. The concentration of linear template is 10 nM (lane 1), 25 nM (lane 2), 50 nM (lane 3), 100 nM (lane 4), and 150 nM (lane 5), respectively. The SDA reaction time is 35 min. (B) Variance of the products of triggers X and Y with the concentration of linear template. (C) Variance of the amplification products with the reaction temperature in the presence of 1 nM let-7a miRNA. The reaction temperature is 57 °C (lane 1), 60 °C (lane 2), 62 °C (lane 3), 64 °C (lane 4), and 65 °C (lane 5), respectively. The SDA reaction time is 35 min. (D) Variance of the products of triggers X and Y with the reaction temperature. Error bars show the standard deviation of three experiments.

trigger X, and trigger Y are observed distinctly (Figure 1B, lane 2). To evaluate the amplification products of bifunctional SDA-mediated HRCA reaction, we performed both the fluorescence measurement (Figure 1C) and the agarose gel electrophoresis analysis (Figure 1D). In the absence of let-7a miRNA, neither bifunctional SDA nor HRCA reactions can be initiated even in the presence of circular template, resulting in no observable fluorescence signal (Figure 1C, black line). While in the presence of let-7a miRNA and circular template, a high fluorescence signal with the characteristic emission peak of 526 nm is observed (Figure 1C, red line), indicating that large amounts of amplification products are generated by bifunctional SDA-mediated HRCA. Notably, in the presence of let-7a miRNA but without circular template, the bifunctional SDA reaction still can be initiated, but the HRCA reaction is unable to be activated, resulting in a relatively low fluorescence signal (Figure 1C, blue line). These results are further confirmed by 2% agarose gel electrophoresis (Figure 1D). As shown in Figure 1D, the distinct bands with different molecular weight are observed only in the presence of both let-7a miRNA and circular template (Figure 1D, lane 3), but no band is observed in the control group without let-7a miRNA (Figure 1D, lane 1). While in the presence of let-7a miRNA but without circular

template, only the band of dsDNA template from SDA amplification is observed (Figure 1D, lane 2). Optimization of Experimental Condition. The concentration of linear template is one of the crucial factors which influence the amplification efficiency of SDA reaction.18 We investigated the effect of linear template concentration upon SDA amplification using the 10% nondenaturating PAGE (Figure 2A) and quantitatively analyzed the bands of triggers X and Y using the ImageJ software (Figure 2B). As shown in Figure 2B, the products of triggers X and Y increase with the increasing concentration of linear template from 10 nM to 25 nM, followed by the decrease beyond the concentration of 25 nM. Thus, 25 nM linear template is used in the subsequent research. Notably, the results in Figure 2A,B can be well explained by the dependence of SDA amplification efficiency upon the hybridization of template with the target.19 The lowconcentration linear template is unable to hybridize with the available targets and adversely affect the amplification efficiency, resulting in the decrease of both dsDNA templates and the triggers (Figure 2A,B). The high-concentration linear template might lead to a high amplification efficiency and the generation of abundant triggers, but the released triggers can subsequently hybridize with the excess templates at the X* region and Y* region (Scheme 1), resulting in the increase of dsDNA templates and the decrease of triggers (Figure 2A,B). 6706

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The reaction temperature might influence the enzyme activity and the hybridization efficiency of nucleic acids and is a key factor in the isothermal amplification.9 We investigated the effect of reaction temperature upon SDA amplification using the 10% nondenaturating PAGE (Figure 2C) and quantitatively analyzed the bands of triggers X and Y using the ImageJ software (Figure 2D). As shown in Figure 2D, the products of triggers X and Y increase with the increase of reaction temperature from 57 to 62 °C, followed by the decrease beyond the temperature of 62 °C. Thus, 62 °C is used as the reaction temperature of SDA amplification in the subsequent research. We further investigated the effect of circular template concentration upon the amplification efficiency of HRCA reaction. Although the high amplification efficiency can be obtained at high-concentration circular template, the background signal might increase correspondingly.20 To optimize the concentration of circular template, we examined the variance of the (F − F0)/F0 value with the concentration of circular template, where F and F0 are the fluorescence intensity at 526 nm in the presence and in the absence of target miRNAs, respectively. As shown in Figure 3, the

Figure 4. (A) Variance of fluorescence intensity with the concentration of let-7a miRNA. (B) The fluorescence intensity shows a log−linear correlation with the concentration of let-7a miRNA in the range from 0.1 pM to 10 nM. Error bars show the standard deviation of three experiments.

triggers; (2) The generated triggers can further act as the primers to initiate an exponential HRCA reaction. Specificity of miRNA Assay. Since there is only one base mismatch between let-7a and let-7c and two base mismatches between let-7a and let-7b, it is a great challenge to discriminate these miRNA family members with high sequence homology. To investigate the detection specificity, we further measured these let-7 family members under the optimal condition. As shown in Figure 5, the fluorescence intensity of let-7a is 8.8-fold higher than that of let-7b and 3.7-fold higher than that of let-7c, suggesting the high specificity of the proposed method for miRNA assay. Real Sample Analysis. To investigate the feasibility of the proposed method for real sample analysis, we used the miRNeasy FFPE kit (Qiagen, Germany) to measure the total RNA sample extracted from the human lung tissues according to the manufacturer’s procedure. The concentration of total RNA was determined by measuring the absorbance at 260 nm with a spectrophotometer. The extracted total RNA sample was diluted to 2 ng/μL with RNase-free water, and 1 μL of total RNA sample (2 ng in total) was added to 10 μL of reaction solution for measurement. As shown in Figure 6, the fluorescence intensity obtained from 2 ng of total RNA sample (Figure 6, blue line) is much higher than that from the control sample (Figure 6, black line). The amount of let-7a miRNA in the total RNA sample is calculated to be 11.12 amol (i.e., 3.35 × 109 copies/μg), which is consistent with the reported results (∼109 copies/μg).22,23 To further confirm the accuracy of the proposed method in real sample analysis, a spiked sample

Figure 3. Variance of the (F − F0)/F0 value with the concentration of circular template. The concentration of let-7a miRNA is 0.1 nM. Error bars show the standard deviation of three experiments.

(F − F0)/F0 value increases with the increasing concentration of circular template from 0.2 to 5 nM, followed by the decrease beyond the concentration of 5 nM. Therefore, 5 nM circular template is used in the subsequent research. Sensitivity of miRNA Assay. We further measured let-7a miRNA at various concentrations under the optimal condition. As shown in Figure 4A, the fluorescence intensity increases with the increase of let-7a miRNA concentration from 0 to 10 nM. In logarithmic scales, the fluorescence intensity has a linear correlation with the concentration of let-7a miRNA over a range from 0.1 pM to 10 nM (Figure 4B). The regression equation is F = 369.5 + 407.8 log10 C with a correlation coefficient of 0.969, where F and C are the fluorescence intensity and the concentration of let-7a miRNA (pM), respectively. The detection limit is calculated to be 1.8 × 10−13 M (or 1.8 amol) based on the average signal of blank plus three times of the standard deviation. Notably, the sensitivity of the proposed method has improved by as much as 5 orders of magnitude as compared with that of the silver nanorod-based surface-enhanced Raman scattering method (28 nM)12 and 3 orders of magnitude as compared with that of Q-STAR probe-based rolling circle amplification assay (0.2 nM).21 The improved sensitivity of the current method can be attributed to the two-stage amplification: (1) The bifunctional SDA reaction enables the conversion of low-abundant miRNA to numerous 6707

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SDA allows for the simultaneous generation of two different kinds of triggers, which can function as two primers for the HRCA reaction. As a result, this miRNA assay can be simply performed in one tube with neither the addition of any extra primers nor the involvement of any separation steps. Taking advantage of the high amplification efficiency of bifunctional SDA-mediated HRCA reaction, the proposed method can sensitively measure let-7a miRNA with a detection limit of 1.8 × 10−13 M and a large dynamic range of 5 orders of magnitude from 0.1 pM to 10 nM, and it can even discriminate the single-base difference among the miRNA family members. Importantly, the proposed method can be used to analyze the total RNA sample from the human lung tissues for early clinical diagnosis and might be further applied for sensitive detection of various proteins, small molecules, and metal ions in combination with specific aptamers.

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AUTHOR INFORMATION

Corresponding Author

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

L.R.Z. and G.Z. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 21325523), the Award for the Hundred Talent Program of the Chinese Academy of Sciences, and the Fund for Shenzhen Engineering Laboratory of Singlemolecule Detection and Instrument Development (Grant No. (2012) 433).

Figure 5. Specificity of miRNA assay. (A) Fluorescence spectra in response to let-7a (red), let-7b (green), let-7c (blue), and the control samples (black). (B) Variance of fluorescence intensity in response to let-7a, let-7b, let-7c, and the control samples. The concentration of each let-7a, let-7b, and let-7c is 0.1 nM. Error bars show the standard deviation of three experiments.

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Figure 6. Variance of fluorescence intensity in response to the mixture of 10 amol synthesized let-7a and 2 ng of total RNA sample (red line), 2 ng of total RNA sample (blue line), and the control sample (black line).

was prepared by the addition of 10 amol synthesized let-7a into 2 ng of total RNA (Figure 6, red line). The amount of let-7a miRNA in the spiked sample was measured to be 20.21 amol and with an acceptable recovery of 90.9%. These results demonstrate that the proposed method holds a great promise for real sample analysis with great accuracy and reliability.

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CONCLUSIONS In summary, we have developed a homogeneous and label-free method for sensitive detection of let-7a miRNA using a bifunctional SDA-mediated HRCA reaction. The bifunctional 6708

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