Author’s Accepted Manuscript Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate Wei Li, Wei Jiang, Yongshun Ding, Lei Wang www.elsevier.com/locate/bios
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S0956-5663(15)30070-1 http://dx.doi.org/10.1016/j.bios.2015.04.067 BIOS7635
To appear in: Biosensors and Bioelectronic Received date: 13 March 2015 Revised date: 20 April 2015 Accepted date: 21 April 2015 Cite this article as: Wei Li, Wei Jiang, Yongshun Ding and Lei Wang, Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.04.067 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate
Wei Li, a Wei Jiang, b Yongshun Ding, b Lei Wanga, *
a
Key Laboratory of Natural Products Chemical Biology, Ministry of Education, School of Pharmacy, Shandong University, Jinan, 250012, P. R. China.
b
School of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100, P. R. China.
Corresponding author: Tel: +86 531 88380036. Email: [email protected]
Abstract MicroRNAs (miRNAs) play important roles in a variety of biological process and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, displayed an excellent sensitivity. Moreover, the proposed method can accurately identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics. Keywords: toehold-mediated SDR, DNA tetrahedron substrate, miRNA detection, selectivity, sensitivity.
1. Introduction MicroRNAs (miRNAs) are small endogenous noncoding RNAs (18-24 nucleotides (nt)) that act as important regulators of gene expression by mediating mRNA cleavage or translational repression (Pillai et al., 2005; Krol et al., 2010). Such regulatory functions make miRNAs capable of modulating cell proliferation, differentiation and apoptosis (He et al., 2005; Calin et al., 2006). Recently, accumulative evidence indicated that miRNA-expression alteration is directly related to the cancer initiation, oncogenesis, and tumor response to treatments (He et al., 2005; Ventura et al., 2009). Now, miRNAs have been taken as tumor biomarkers in cancer diagnosis and prognosis (Tricoli et al., 2007; Cao et al., 2011). Thus, effective analysis of miRNAs expression in biological samples and clinical specimens is crucial for cancer diagnosis and treatment (Dong et al., 2013; Liu et al., 2014; Liao et al., 2014). Conventional methods, including Northern blotting (Valoczi et al., 2004), quantitative real-time polymerase chain reaction (qRT-PCR) (Chen et al., 2005) and microarrays (Babak et al., 2004), possess some limitations such as low detection sensitivity, tedious sample preparation and imperfect specificity. In the past decade, many new strategies have been developed to improve the reliability of miRNAs analysis (Ge et al., 2014; Liao et al., 2014; Lin et al., 2014; Zhang et al., 2014; Liu et al., 2014; Xi et al., 2014; Dong et al., 2012). Among them, fluorescence method has attracted considerable attention because of its highly sensitivity and easy operation (Zhang et al., 2014; Liu et al., 2014; Xi et al., 2014; Dong et al., 2012). But due to the unique properties of small size, low abundance and highly sequence similarity, the accurate
recognition of miRNAs is still challenging (Dong et al., 2013). Commonly used recognize method in miRNA analysis is direct hybridization that refers to the hybridization between two single-stranded nucleic acids (Xi et al., 2014; Dong et al., 2012; Liu et al., 2014). The recognition probes, either linear or conformational restricted (such as hairpins), when identifying their targets, undergo a direct hybridization reaction driven by thermal fluctuations (Xi et al., 2014; Dong et al., 2012). Although nucleic acid hybridization is a specific recognition event in nature, measurable thermodynamic changes that occur upon hybridization are rather small (Li et al., 2002). Therefore, the ability to accurate identify target miRNA among its family members is limited since they are usually differ by only one or two nucleotides in sequence with the same length (Li et al., 2002; Deng et al., 2014; Liu et al., 2014). Toehold-mediated strand displacement reaction (SDR) is a controllable process that accomplished by a chain migration, in which one strand of DNA in a double-stranded complex is displaced by another DNA strand with the help of a short single-stranded domain (termed “toehold”) (Deng et al., 2014). In this process, the identification of target is obtained by displacement hybridization, which contains two steps: the binding with the toehold domain and the accomplishment of SDR. Differing from the direct hybridization, there is an excess competitor in the probe-target reaction system (Li et al., 2002). The competitor is a single-stranded oligonucleotide that contains the same sequence with the target in the binding region (Wang et al., 2012; Genot et al., 2011; Tang et al., 2013). It can form a stable duplex with another single strand in the absence of the target and can be replaced in the presence of the target. When the target
has a base sequence homologous to the toehold, the branch migration can always occur. Toehold-mediated SDR typically works well because the rate of dissociation from the toehold is much smaller than the rate of strand displacement (Krishnan et al., 2009). Inversely, homologous sequences of the target can barely initiate the strand migration since the dissociation rate caused by mismatch is much quicker than that of strand displacement. Compared to direct hybridization, toehold-mediated recognition mechanism has the ability to enhance the specificity of nucleic-acid recognition and robust sequence discrimination can be achieved by regulating the length and sequence composition of the toehold (Deng et al., 2014; Wang et al., 2012; Zhu et al., 2014). Thus, in this work, we constituted a highly specific and sensitive strategy for miRNA detection based on toehold-mediated SDR and single-molecule counting. single-molecule counting is a new kind of quantitative method, which achieved by counting the target one by one (Walt, 2013). For this approach, the primary issue is the fabrication of substrate, which plays the role of immobilization during detection (Tessler et al., 2009; Peterson et al., 2010). Since DNA tetrahedra can ensure the probes with well controlled density and orientation (Ge et al., 2014; Lin et al., 2014; Pei et al., 2010; Li et al., 2014) and prevent the nonspecific adsorption (Wang et al., 2012), we chose DNA tetrahedron substrate to immobilize the DNA probes. Firstly, functional DNA tetrahedra were specially assembled with a hairpin, which has an overhanging toehold domain at one of the terminal. Then, the tetrahedral were immobilized on the glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA prehybidized with the toehold and triggered a SDR,
which accomplished by the unfolding of the hairpin. While, the homologous sequences of the target miRNA can barely initiate the strand migration. Finally, a biotinylation ssDNA was used as reporter probe (bio-RP) and hybridized with the new emerging single strands. Upon the addition of streptavidin coated quantum dots (QDs), fluorescence images can be achieved and the numbers of dots were counted one by one for quantification. As a proof of concept, let-7a, which was associated with carcinoma of the lungs, was selected as a model target sequence. By introducing the toehold-mediated SDR, the proposed method exhibited an admirable specific to its family members (let-7b, let-7c, and let-7i). The detection limit of this method was 5 fM, which was attributed to the low background DNA tetrahedron substrate and single-molecule counting. Moreover, miRNA analysis in total RNA samples from human lung tissues were also performed, indicating that our strategy was reliable and had a great potential application in biomedical research and early clinical diagnostics.
2. EXPERIMENTAL SECTION 2.1. Materials and Reagents QDs-streptavidin (605 nm) conjugates were obtained from Invitrogen Co. (Eugene, OK, USA). Tris (>99.8%) was purchased from Amresco Inc. (Solon, OH). Tween-20, 3-glycidyloxypropyltrimethoxysilane (GOPS) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Microscope cover glasses (22 mm × 22 mm) were purchased from Cole-Parmer (Illinois, USA). Diethyl pyrocarbonate (DEPC) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). MirVana TM
miRNA Isolation Kit, DNA oligonucleotide stands and RNAs were purchased from Invitrogen Trading Co., Ltd. (Shanghai, China) (the sequences were showed in Table S1). Human lung cells (A549) were obtained from Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences. Other chemicals (analytical grade) were obtained from standard reagent suppliers. The physiological buffer saline (PBS) consisted of 0.15 M NaCl, 2.4 mM NaH2PO4, and 7.6 mM Na2HPO4 (pH 7.4). PBST buffer consisted of PBS and 0.05 % Tween-20 (pH 7.4). TE buffer consisted of 10 mM Tris and 1.0 mM Na2EDTA (pH 8.0). TM buffer consisted of 20 mM Tris and 50 mM MgCl2 (pH 8.0). TAE-Mg buffer consisted of 40 mM Tris, 20 mM Acetic Acid, 2 mM EDTANa2, and 12.5 mM (CH3COO)2Mg. Tris-HCl buffer consisted of 20 mM Tris, 5 mM MgCl 2, 137 mM NaCl, and 5 mM KCl (pH 7.4). To create and maintain an RNase-free environment, the solutions were prepared with 0.1% diethyl pyrocarbonate(DEPC)-treated Milli-Q water and autoclaved. The tips and tubes were also treated with 0.1 % DEPC-H2O. 2.2. Apparatus EFM imaging were performed with an Olympus IX81 fluorescence microscope (Tokyo, Japan) equipped with a high-numerical-aperture 60 × (1.45 NA) oil-immersion objective lens, a mercury lamp source, a mirror unit consisting of a 470-490 nm excitation filter (BP470-490), a 505 nm dichromatic mirror (DM 505), a 510-550 nm emission filter (IF 580), and a 16-bit thermoelectrically cooled electron multiplying charge coupled device (EMCCD) (Cascade 512 B, Tucson, AZ, USA). Imaging acquisition was performed using the MetaMorph software (Universal
Imaging, Downingtown, PA, USA). All captured images were then further processed with the Analyze Particles function in the public-domain image-processing software ImageJ to determine the number of single fluorescence particles counting. BioPhotometer Plus was purchased from Eppendorf (Hamburg, Germany). Tensionmeter-K121 processor was purchased from Krüss Company (Germany). 2.3. Assembly and Electrophoresis Analysis of DNA Tetrahedron Four DNA strands (A-HP-6, B-NH2, C-NH2 and D-NH2) were diluted in TM buffer, yielding a final concentration of 50 μM. Two microliters of each strand was mixed with 92 μL TM buffer, and the resulting mixture was heated to 95 °C for 2 min and then cooled in ice bath. The final concentration of the DNA Tetrahedron is 1μM. In order to verify the assembly of DNA Tetrahedron, a polyacrylamide gel electrophoresis (native-PAGE) experiment was performed. Tetrahedra were run on 9.6% native-PAGE gel in TAE-Mg buffer with a constant current of 30 mA at 15 °C. 2.4. The Construction and Characterization of DNA Tetrahedron Decorated Substrates The DNA tetrahedron decorated substrate was constructed on a glass slide. The epoxy-functionalized glass slides were prepared according to the reported protocol (Dai et al., 2014; Ding et al., 2014; Wang et al., 2012). Fifty microliters of the functional DNA tetrahedron were added to the epoxy-functionalized glass slide and allowed to react overnight at 37 °C. Then, the substrate was rinsed three times with PBS buffer. To characterize the fabrication of DNA tetrahedron decorated substrates, a contact
angle experiment was performed on Tensionmeter-K121 processor. The concentration of DNA tetrahedron added to the glass slides was 1 μM. The substrates were immersed into and retracted from ultra-pure water medium. Furthermore, to calculate the surface density, the concentration of DNA tetrahedron before and after incubation was measured using BioPhotometer Plus. 2.5. Single-Molecule Detection of MiRNA The freshly prepared substrate was incubated with 50 μL miRNA solutions of various concentrations (1.0 pM, 0.8 pM, 0.5 pM, 0.3 pM, 0.1 pM, 0.05 pM and 0.005 pM) at 37 °C. After washing thrice with PBS buffer, 50 μL bio-RP (0.1 nM) was added onto the substrate and incubated at 37 °C for 2 h. Following the washing step, the substrate was incubated with 50 μL QDs (dissolved into 0.1 nM with PBS buffer) at 37 °C for 2 h. Then the superfluous QDs were removed and the substrate was rinsed three times with PBST and PBS buffer. Before fluorescence imaging with epi-fluorescence microscopy, 50 μL PBS buffer were added. 2.6. Fluorescence Imaging Fluorescence imaging was performed with an Olympus IX81 fluorescence microscope equipped with a 16-bit thermoelectrically cooled EMCCD, a mercury lamp, a mirror unit consisting of a 470 to 490 nm excitation filter (BP470-490) and a >580 nm emission filter (IF580). The images corresponding to different locations were obtained by manually moving the XY sample stage. And the images were analyzed by MetaMorph software. 2.7. Preparation of Total RNA
The Human lung cells (A549) were cultured in cell culture dishes in a humidified atmosphere at 37 °C with 5% CO2 and 95% air. Cells were grown in RPMI 1640 supplemented with 15% FBS, 100 μg/mL streptomycin and 100 μg/mL penicillin. Total RNA of human lung cells was extracted with the MirVanaTM miRNA Isolation Kit according to the manufacturer’s procedures. The concentration of total RNA was measured by BioPhotometer Plus at 260 nm. Then the total RNA was diluted with Tris-HCl buffer and analyzed with the proposed miRNA method.
3. Results and Discussion 3.1. Rationale of the Proposed Method In this work, a single-molecule quantitative method has been developed for specific and sensitive detection of miRNA. The rationale of this method is shown in Scheme 1. Firstly, we have designed a DNA tetrahedron with a toehold and hairpin at one vertex, while the other three vertexes were labeled with amino group. The DNA tetrahedron was immobilized on substrate through the reaction between amino and epoxy and it has a capture hairpin probe that consists of a 6-nt external toehold, 12-bp stem and 6-nt loop. Next, the target miRNA prehybridized with the behold domain and then triggered a strand displacement reaction that accomplished by the unfolding of the hairpin. Subsequently, the bio-RP were introduced and hybridized with the new emerging single strands. Upon the addition of QDs, fluorescence images were obtained through epi-fluorescence microscopy. Finally, the numbers of the bright dots in those images were counted one by one for quantification.
Scheme 1 is here.
As described above, the bright dots detected in the proposed method was attributed to the accomplishment of the toehold-mediated SDR. To demonstrate the feasibility of this strategy, typical fluorescence images were displayed in Fig. S1. In the presence of let-7a, a large number of fluorescence dots were observed. Inversely, in the absence of let-7a, almost no fluorescence dot was found after incubated with QDs. 3.2. Self-assembly and Characterization of DNA Tetrahedron The self-assembly of DNA tetrahedron was first reported by Goodman et al. through a simple and quick method (Goodman et al., 2004). Four single-stranded DNA were assembled into a tetrahedron and the tetrahedron consisted of six double-stranded edges connected to each other by two unpaired nucleotides. In the proposed method, there was a hairpin probe at one vertex of the DNA tetrahedron and the hairpin has a toehold domain at one of the terminal. Other three vertexes were labeled with amino group in order to immobilize the DNA tetrahedron on the glass substrate. To demonstrate the self-assembly of DNA tetrahedron, the functional DNA tetrahedron was analyzed using polyacryamide gel electrophoresis. As shown in Fig. 1, DNA tetrahedra moved more slowly than the single strand DNA and any other combinations lacking one or more strands, which correspond to previous reports (Ding et al., 2014; Wang et al., 2012; Goodman et al., 2004; Goodman et al., 2005).
This confirmed the successful assembling of the DNA tetrahedron nanostructure.
Fig. 1 is here.
3.3. Characterization of DNA Tetrahedron Decorated Substrates To immobilize the DNA tetrahrdra onto the substrate, the glass substrates were cleaned via a substrate pretreatment described in the literature and silanized of substrate surfaces with epoxy groups (Wang et al., 2012; Jiang et al., 2010). The amino groups at the vertexes of the DNA tetrahedron can react with the epoxy groups on the substrate. To further demonstrate the immobilization of DNA tetrahedra on the substrate, the contact angle experiments of several different group terminal substrates were applied. As shown in Fig. S2, the bare glass substrates had a contact angle value (CAV) about 43.2°, indicating a moderate hydrophilic surface. The activated treatment created a layer of hydroxyl groups on the bare glass surface and obtained a CAV of 17.8°. Since the hydroxyl group was highly polar group, the hydroxyl-terminated substrate was more hydrophilic than the bare glass. After the silanization of the substrate surface, the epoxy group, a hydrophobic group, was decorated on the glass, which makes the CAV rise to almost 60°. When the DNA tetrahedra were immobilized onto the epoxy-terminal substrates, the CAV was down to 30° because of the hydrophilic of the DNA tetrahedra, which indicated that the DNA tetrahedra were successfully immobilized on the substrates. The DNA tetrahedron decorated substrate used here was to restrain the
nonspecific adsorption of QDs. Our group had studied the nonspecific adsorption of QDs on different substrates, which indicated the DNA tetrahedra can prevent the nonspecific adsorption (Wang et al., 2012). When the concentration of DNA tetrahedra added to the substrate was 1 μM, the negative-control experiment has barely fluorescence spots. The ability to restrain the nonspecific adsorption of QDs may because of the DNA tetrahedron layer separates QDs from the glass slide, preventing substrate-induced reactions. Moreover, the electrostatic repulsive force between QDs and DTDS makes it hard for the adsorption of QDs to occur. The DNA tetrahedron carries high negative charges as well as the QDs that decorated with streptavidin (the isoelectric point is 6.0). The surface density was calculated by UV spectrophotometry and we acquired 9.2×1012 tetrahedra/cm2 (more details in the supporting information). 3.4. Optimization of Reaction Conditions The kinetic rate of SDR can be controlled by adjusting the length and sequence composition of the toehold. It was reported that the toehold length of 6 nt was the smallest for initiating strand migration at the maximum rate in solution (Wang et al., 2012). However, the influence of toehold length on the kinetic of DNA tetrahedron decorated substrate has not been reported yet. Though design the sequences of DNA tetrahedron makes the effective toehold length vary from 5 nt to 8 nt, respectively. To assay miRNA with sensitive and selective, the reaction time between miRNA and capture probe was optimized. As shown in Fig. 2, when the toehold has a 5 nt length, it could not reach an equilibrium in 2 h. While the toehold with 6 nt had an
equilibrium at 2 h, and the toehold with 7 nt and 8 nt both had an equilibrium at 1.5 h. If the reaction time was longer, the selective of this method may decrease because the similar sequences can also initiate the SDR. Thus 2 h was chosen as the best time for toehold with 5 nt and 6 nt and 1.5 h for toehold with 7 nt and 8 nt.
Fig. 2 is here.
After affirm the reaction time, the selective of the method was investigated. To achieve the best sensing performance, the toehold length vary from 5 nt to 8 nt all be investigated with target miRNA (Let-7a) and its similar family members (let-7b, Let-7c, and Let-7i). Obviously, in Fig. 3, the toehold length with 6 nt, 7 nt, and 8 nt all had an excellent specificity towards the similar sequences. However, through calculated the ratio of the dots numbers produced by let-7a and its similar sequences (as shown in table S2), the toehold length with 6 nt had a best specificity. These results may be attributed to that for both the target miRNA and its similar family members, the reaction rate of SDR was increased with the increasing of toehold length. Hence, the toehold length with 6 nt was selected in the following experiments.
Fig. 3 is here.
3.5. Specificity of the Assay Due to the high sequence similarity among family members, it is a great
challenge to carry out the miRNAs assay with high specificity. For example, members of the let-7a family differ by only one or two nucleotides. To investigate the specificity of this method, we measured these let-7 family members under the optical condition. As shown in Fig. 3, the toehold with 6 nt showed perfect selectivity to the target miRNA, dots numbers produced by let-7a is 13.1-, 12.3- and 20.9-fold of that produced by let-7b, let-7c and let-7i, respectively, much higher than the reported methods (Liao et al., 2014; Zhang et al., 2014; Liu et al., 2014). These results indicated that the proposed strategy exhibited sufficient capability of distinguishing single-base difference among the miRNA family members by introducing the toehold-mediated SDR. 3.6. Sensitivity of the Assay Under the optimal conditions, we further measured the target miRNA at various concentrations. As shown in Fig. 4, the number of fluorescence dots was increased with the increasing concentration of let-7a. There was a good linear relationship between the number of dots and the concentration of let-7a over a range from 5 fM to 1 pM. The linear regression equation was Y=26.863+1041.898C with a correlation coefficient of 0.998, where Y and C were the number of fluorescence dots and the concentration of let-7a (pM), respectively. Compared with other reported methods, this proposed strategy demonstrates excellent sensitivity (Table S3). The enhancement of sensitivity might be attributed to the lower background of DNA tetrahedron substrates and the higher fluorescence quantum yield of QDs.
Fig. 4 is here.
3.7. Detection efficiency of the DNA tetrahedron probes In order to evaluate the detection efficiency of the proposed method, we calculated the number of fluorescence dots in theory according to one equation (more detail in the supporting information). The detection efficiency was the ratio of detected number and the theoretical number. When the concentration of target miRNA was 1 pM, the detection efficiency was 13%. This corresponds with previous reports (Wang et al., 2012) and is much higher than that in the direct immobilization method (5%) (Li et al., 2011). The improvement is mainly benefit from the introduction of DNA tetrahedra, which have made the probes more accessible. 3.8. Real Sample Assay To investigate the capability of this proposed method in real sample analysis, we perform the miRNA assay in total RNA sample extracted from the human lung tissues. The quantity of let-7a in human lung cells was measured by the standard addition method using synthetic let-7a as the standard. The total RNA sample was diluted to 2 ng/μL with Tris-HCl buffer, and 1μL of total RNA sample were spiked in standard solutions with synthetic let-7a at concentration of 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 pM, respectively. Then, the toehold-mediated SDR and fluorescence imaging were performed under the optimal conditions. The results are shown as Fig. 5, the amount of let-7a in the human lung total RNA is calculated at 3.50×109 copies/μg, which is consistent with previous reports (Zhang et al., 2014; Liu et al., 2014; Cheng et al.,
2009).
Fig. 5 is here.
4. Conclusion In summary, we have developed a selective and sensitive method for miRNA on the basis of toehold-mediated SDR and DNA tetrahedron substrate. To our knowledge, it was the first time to employ the toehold-mediated SDR into single-molecule quantitative method. First, after the introduction of DNA tetrahedron substrate, a low nonspecific adsorption was obtained. Under the optical conditions, the detection limit of this method was 5 fM without any amplification strategy. Second, by combining the toehold-mediated SDR, this method revealed an admirable specificity and could accurate recognize the target miRNA among its family members. Third, miRNA analysis in real samples was performed, indicating that our strategy was reliable and had a great potential application in biomedical research, early clinical diagnostics and oncotherapy.
Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant nos. 21175081, 21175082, 21375078 and 21475077).
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Figure Captions
Scheme 1. Schematic illustration of quantitative detection of single miRNA based on toehold-mediated strand displacement reaction.
Fig. 1 Electrophoretic analysis of DNA tetrahedron, 1-5 line stand for the
electrophoretic
image
of
sequence
A-HP-6+B-NH2+C-NH2+D-NH2,
B-NH2+C-NH2+D-NH2, B-NH2+C-NH2, B-NH2, and marker (20 bp).
Fig. 2 Effect of reaction time between miRNA and capture probe with different lengths of toehold. The error bars showed the standard deviation of three replicate determinations.
Fig. 3 Specificity of the proposed method to target miRNA (let-7a) and its family members (let-7b, let-7c and let-7i). The error bars are standard deviations of three
repetitive measurements.
Fig. 4 Linear relationship between the numbers of fluorescence dots and the concentration of target miRNA. The error bars represents the standard deviation of three repetitive measurements.
Fig. 5 The determination of let-7a in total RNA sample through standard addition method. The error bars represent the standard deviation of three repetitive measurements.
highlights
A toehold-mediated SDR-based strategy for miRNA analysis was proposed.
It was the first time to employ the toehold-mediated SDR into single-molecule counting method.
A high specificity and sensitivity for miRNA detection was achieved.
MiRNA analysis in total RNA samples from human lung tissues was performed.
PII: DOI: Reference:
S0956-5663(15)30070-1 http://dx.doi.org/10.1016/j.bios.2015.04.067 BIOS7635
To appear in: Biosensors and Bioelectronic Received date: 13 March 2015 Revised date: 20 April 2015 Accepted date: 21 April 2015 Cite this article as: Wei Li, Wei Jiang, Yongshun Ding and Lei Wang, Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.04.067 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate
Wei Li, a Wei Jiang, b Yongshun Ding, b Lei Wanga, *
a
Key Laboratory of Natural Products Chemical Biology, Ministry of Education, School of Pharmacy, Shandong University, Jinan, 250012, P. R. China.
b
School of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100, P. R. China.
Corresponding author: Tel: +86 531 88380036. Email: [email protected]
Abstract MicroRNAs (miRNAs) play important roles in a variety of biological process and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, displayed an excellent sensitivity. Moreover, the proposed method can accurately identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics. Keywords: toehold-mediated SDR, DNA tetrahedron substrate, miRNA detection, selectivity, sensitivity.
1. Introduction MicroRNAs (miRNAs) are small endogenous noncoding RNAs (18-24 nucleotides (nt)) that act as important regulators of gene expression by mediating mRNA cleavage or translational repression (Pillai et al., 2005; Krol et al., 2010). Such regulatory functions make miRNAs capable of modulating cell proliferation, differentiation and apoptosis (He et al., 2005; Calin et al., 2006). Recently, accumulative evidence indicated that miRNA-expression alteration is directly related to the cancer initiation, oncogenesis, and tumor response to treatments (He et al., 2005; Ventura et al., 2009). Now, miRNAs have been taken as tumor biomarkers in cancer diagnosis and prognosis (Tricoli et al., 2007; Cao et al., 2011). Thus, effective analysis of miRNAs expression in biological samples and clinical specimens is crucial for cancer diagnosis and treatment (Dong et al., 2013; Liu et al., 2014; Liao et al., 2014). Conventional methods, including Northern blotting (Valoczi et al., 2004), quantitative real-time polymerase chain reaction (qRT-PCR) (Chen et al., 2005) and microarrays (Babak et al., 2004), possess some limitations such as low detection sensitivity, tedious sample preparation and imperfect specificity. In the past decade, many new strategies have been developed to improve the reliability of miRNAs analysis (Ge et al., 2014; Liao et al., 2014; Lin et al., 2014; Zhang et al., 2014; Liu et al., 2014; Xi et al., 2014; Dong et al., 2012). Among them, fluorescence method has attracted considerable attention because of its highly sensitivity and easy operation (Zhang et al., 2014; Liu et al., 2014; Xi et al., 2014; Dong et al., 2012). But due to the unique properties of small size, low abundance and highly sequence similarity, the accurate
recognition of miRNAs is still challenging (Dong et al., 2013). Commonly used recognize method in miRNA analysis is direct hybridization that refers to the hybridization between two single-stranded nucleic acids (Xi et al., 2014; Dong et al., 2012; Liu et al., 2014). The recognition probes, either linear or conformational restricted (such as hairpins), when identifying their targets, undergo a direct hybridization reaction driven by thermal fluctuations (Xi et al., 2014; Dong et al., 2012). Although nucleic acid hybridization is a specific recognition event in nature, measurable thermodynamic changes that occur upon hybridization are rather small (Li et al., 2002). Therefore, the ability to accurate identify target miRNA among its family members is limited since they are usually differ by only one or two nucleotides in sequence with the same length (Li et al., 2002; Deng et al., 2014; Liu et al., 2014). Toehold-mediated strand displacement reaction (SDR) is a controllable process that accomplished by a chain migration, in which one strand of DNA in a double-stranded complex is displaced by another DNA strand with the help of a short single-stranded domain (termed “toehold”) (Deng et al., 2014). In this process, the identification of target is obtained by displacement hybridization, which contains two steps: the binding with the toehold domain and the accomplishment of SDR. Differing from the direct hybridization, there is an excess competitor in the probe-target reaction system (Li et al., 2002). The competitor is a single-stranded oligonucleotide that contains the same sequence with the target in the binding region (Wang et al., 2012; Genot et al., 2011; Tang et al., 2013). It can form a stable duplex with another single strand in the absence of the target and can be replaced in the presence of the target. When the target
has a base sequence homologous to the toehold, the branch migration can always occur. Toehold-mediated SDR typically works well because the rate of dissociation from the toehold is much smaller than the rate of strand displacement (Krishnan et al., 2009). Inversely, homologous sequences of the target can barely initiate the strand migration since the dissociation rate caused by mismatch is much quicker than that of strand displacement. Compared to direct hybridization, toehold-mediated recognition mechanism has the ability to enhance the specificity of nucleic-acid recognition and robust sequence discrimination can be achieved by regulating the length and sequence composition of the toehold (Deng et al., 2014; Wang et al., 2012; Zhu et al., 2014). Thus, in this work, we constituted a highly specific and sensitive strategy for miRNA detection based on toehold-mediated SDR and single-molecule counting. single-molecule counting is a new kind of quantitative method, which achieved by counting the target one by one (Walt, 2013). For this approach, the primary issue is the fabrication of substrate, which plays the role of immobilization during detection (Tessler et al., 2009; Peterson et al., 2010). Since DNA tetrahedra can ensure the probes with well controlled density and orientation (Ge et al., 2014; Lin et al., 2014; Pei et al., 2010; Li et al., 2014) and prevent the nonspecific adsorption (Wang et al., 2012), we chose DNA tetrahedron substrate to immobilize the DNA probes. Firstly, functional DNA tetrahedra were specially assembled with a hairpin, which has an overhanging toehold domain at one of the terminal. Then, the tetrahedral were immobilized on the glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA prehybidized with the toehold and triggered a SDR,
which accomplished by the unfolding of the hairpin. While, the homologous sequences of the target miRNA can barely initiate the strand migration. Finally, a biotinylation ssDNA was used as reporter probe (bio-RP) and hybridized with the new emerging single strands. Upon the addition of streptavidin coated quantum dots (QDs), fluorescence images can be achieved and the numbers of dots were counted one by one for quantification. As a proof of concept, let-7a, which was associated with carcinoma of the lungs, was selected as a model target sequence. By introducing the toehold-mediated SDR, the proposed method exhibited an admirable specific to its family members (let-7b, let-7c, and let-7i). The detection limit of this method was 5 fM, which was attributed to the low background DNA tetrahedron substrate and single-molecule counting. Moreover, miRNA analysis in total RNA samples from human lung tissues were also performed, indicating that our strategy was reliable and had a great potential application in biomedical research and early clinical diagnostics.
2. EXPERIMENTAL SECTION 2.1. Materials and Reagents QDs-streptavidin (605 nm) conjugates were obtained from Invitrogen Co. (Eugene, OK, USA). Tris (>99.8%) was purchased from Amresco Inc. (Solon, OH). Tween-20, 3-glycidyloxypropyltrimethoxysilane (GOPS) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Microscope cover glasses (22 mm × 22 mm) were purchased from Cole-Parmer (Illinois, USA). Diethyl pyrocarbonate (DEPC) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). MirVana TM
miRNA Isolation Kit, DNA oligonucleotide stands and RNAs were purchased from Invitrogen Trading Co., Ltd. (Shanghai, China) (the sequences were showed in Table S1). Human lung cells (A549) were obtained from Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences. Other chemicals (analytical grade) were obtained from standard reagent suppliers. The physiological buffer saline (PBS) consisted of 0.15 M NaCl, 2.4 mM NaH2PO4, and 7.6 mM Na2HPO4 (pH 7.4). PBST buffer consisted of PBS and 0.05 % Tween-20 (pH 7.4). TE buffer consisted of 10 mM Tris and 1.0 mM Na2EDTA (pH 8.0). TM buffer consisted of 20 mM Tris and 50 mM MgCl2 (pH 8.0). TAE-Mg buffer consisted of 40 mM Tris, 20 mM Acetic Acid, 2 mM EDTANa2, and 12.5 mM (CH3COO)2Mg. Tris-HCl buffer consisted of 20 mM Tris, 5 mM MgCl 2, 137 mM NaCl, and 5 mM KCl (pH 7.4). To create and maintain an RNase-free environment, the solutions were prepared with 0.1% diethyl pyrocarbonate(DEPC)-treated Milli-Q water and autoclaved. The tips and tubes were also treated with 0.1 % DEPC-H2O. 2.2. Apparatus EFM imaging were performed with an Olympus IX81 fluorescence microscope (Tokyo, Japan) equipped with a high-numerical-aperture 60 × (1.45 NA) oil-immersion objective lens, a mercury lamp source, a mirror unit consisting of a 470-490 nm excitation filter (BP470-490), a 505 nm dichromatic mirror (DM 505), a 510-550 nm emission filter (IF 580), and a 16-bit thermoelectrically cooled electron multiplying charge coupled device (EMCCD) (Cascade 512 B, Tucson, AZ, USA). Imaging acquisition was performed using the MetaMorph software (Universal
Imaging, Downingtown, PA, USA). All captured images were then further processed with the Analyze Particles function in the public-domain image-processing software ImageJ to determine the number of single fluorescence particles counting. BioPhotometer Plus was purchased from Eppendorf (Hamburg, Germany). Tensionmeter-K121 processor was purchased from Krüss Company (Germany). 2.3. Assembly and Electrophoresis Analysis of DNA Tetrahedron Four DNA strands (A-HP-6, B-NH2, C-NH2 and D-NH2) were diluted in TM buffer, yielding a final concentration of 50 μM. Two microliters of each strand was mixed with 92 μL TM buffer, and the resulting mixture was heated to 95 °C for 2 min and then cooled in ice bath. The final concentration of the DNA Tetrahedron is 1μM. In order to verify the assembly of DNA Tetrahedron, a polyacrylamide gel electrophoresis (native-PAGE) experiment was performed. Tetrahedra were run on 9.6% native-PAGE gel in TAE-Mg buffer with a constant current of 30 mA at 15 °C. 2.4. The Construction and Characterization of DNA Tetrahedron Decorated Substrates The DNA tetrahedron decorated substrate was constructed on a glass slide. The epoxy-functionalized glass slides were prepared according to the reported protocol (Dai et al., 2014; Ding et al., 2014; Wang et al., 2012). Fifty microliters of the functional DNA tetrahedron were added to the epoxy-functionalized glass slide and allowed to react overnight at 37 °C. Then, the substrate was rinsed three times with PBS buffer. To characterize the fabrication of DNA tetrahedron decorated substrates, a contact
angle experiment was performed on Tensionmeter-K121 processor. The concentration of DNA tetrahedron added to the glass slides was 1 μM. The substrates were immersed into and retracted from ultra-pure water medium. Furthermore, to calculate the surface density, the concentration of DNA tetrahedron before and after incubation was measured using BioPhotometer Plus. 2.5. Single-Molecule Detection of MiRNA The freshly prepared substrate was incubated with 50 μL miRNA solutions of various concentrations (1.0 pM, 0.8 pM, 0.5 pM, 0.3 pM, 0.1 pM, 0.05 pM and 0.005 pM) at 37 °C. After washing thrice with PBS buffer, 50 μL bio-RP (0.1 nM) was added onto the substrate and incubated at 37 °C for 2 h. Following the washing step, the substrate was incubated with 50 μL QDs (dissolved into 0.1 nM with PBS buffer) at 37 °C for 2 h. Then the superfluous QDs were removed and the substrate was rinsed three times with PBST and PBS buffer. Before fluorescence imaging with epi-fluorescence microscopy, 50 μL PBS buffer were added. 2.6. Fluorescence Imaging Fluorescence imaging was performed with an Olympus IX81 fluorescence microscope equipped with a 16-bit thermoelectrically cooled EMCCD, a mercury lamp, a mirror unit consisting of a 470 to 490 nm excitation filter (BP470-490) and a >580 nm emission filter (IF580). The images corresponding to different locations were obtained by manually moving the XY sample stage. And the images were analyzed by MetaMorph software. 2.7. Preparation of Total RNA
The Human lung cells (A549) were cultured in cell culture dishes in a humidified atmosphere at 37 °C with 5% CO2 and 95% air. Cells were grown in RPMI 1640 supplemented with 15% FBS, 100 μg/mL streptomycin and 100 μg/mL penicillin. Total RNA of human lung cells was extracted with the MirVanaTM miRNA Isolation Kit according to the manufacturer’s procedures. The concentration of total RNA was measured by BioPhotometer Plus at 260 nm. Then the total RNA was diluted with Tris-HCl buffer and analyzed with the proposed miRNA method.
3. Results and Discussion 3.1. Rationale of the Proposed Method In this work, a single-molecule quantitative method has been developed for specific and sensitive detection of miRNA. The rationale of this method is shown in Scheme 1. Firstly, we have designed a DNA tetrahedron with a toehold and hairpin at one vertex, while the other three vertexes were labeled with amino group. The DNA tetrahedron was immobilized on substrate through the reaction between amino and epoxy and it has a capture hairpin probe that consists of a 6-nt external toehold, 12-bp stem and 6-nt loop. Next, the target miRNA prehybridized with the behold domain and then triggered a strand displacement reaction that accomplished by the unfolding of the hairpin. Subsequently, the bio-RP were introduced and hybridized with the new emerging single strands. Upon the addition of QDs, fluorescence images were obtained through epi-fluorescence microscopy. Finally, the numbers of the bright dots in those images were counted one by one for quantification.
Scheme 1 is here.
As described above, the bright dots detected in the proposed method was attributed to the accomplishment of the toehold-mediated SDR. To demonstrate the feasibility of this strategy, typical fluorescence images were displayed in Fig. S1. In the presence of let-7a, a large number of fluorescence dots were observed. Inversely, in the absence of let-7a, almost no fluorescence dot was found after incubated with QDs. 3.2. Self-assembly and Characterization of DNA Tetrahedron The self-assembly of DNA tetrahedron was first reported by Goodman et al. through a simple and quick method (Goodman et al., 2004). Four single-stranded DNA were assembled into a tetrahedron and the tetrahedron consisted of six double-stranded edges connected to each other by two unpaired nucleotides. In the proposed method, there was a hairpin probe at one vertex of the DNA tetrahedron and the hairpin has a toehold domain at one of the terminal. Other three vertexes were labeled with amino group in order to immobilize the DNA tetrahedron on the glass substrate. To demonstrate the self-assembly of DNA tetrahedron, the functional DNA tetrahedron was analyzed using polyacryamide gel electrophoresis. As shown in Fig. 1, DNA tetrahedra moved more slowly than the single strand DNA and any other combinations lacking one or more strands, which correspond to previous reports (Ding et al., 2014; Wang et al., 2012; Goodman et al., 2004; Goodman et al., 2005).
This confirmed the successful assembling of the DNA tetrahedron nanostructure.
Fig. 1 is here.
3.3. Characterization of DNA Tetrahedron Decorated Substrates To immobilize the DNA tetrahrdra onto the substrate, the glass substrates were cleaned via a substrate pretreatment described in the literature and silanized of substrate surfaces with epoxy groups (Wang et al., 2012; Jiang et al., 2010). The amino groups at the vertexes of the DNA tetrahedron can react with the epoxy groups on the substrate. To further demonstrate the immobilization of DNA tetrahedra on the substrate, the contact angle experiments of several different group terminal substrates were applied. As shown in Fig. S2, the bare glass substrates had a contact angle value (CAV) about 43.2°, indicating a moderate hydrophilic surface. The activated treatment created a layer of hydroxyl groups on the bare glass surface and obtained a CAV of 17.8°. Since the hydroxyl group was highly polar group, the hydroxyl-terminated substrate was more hydrophilic than the bare glass. After the silanization of the substrate surface, the epoxy group, a hydrophobic group, was decorated on the glass, which makes the CAV rise to almost 60°. When the DNA tetrahedra were immobilized onto the epoxy-terminal substrates, the CAV was down to 30° because of the hydrophilic of the DNA tetrahedra, which indicated that the DNA tetrahedra were successfully immobilized on the substrates. The DNA tetrahedron decorated substrate used here was to restrain the
nonspecific adsorption of QDs. Our group had studied the nonspecific adsorption of QDs on different substrates, which indicated the DNA tetrahedra can prevent the nonspecific adsorption (Wang et al., 2012). When the concentration of DNA tetrahedra added to the substrate was 1 μM, the negative-control experiment has barely fluorescence spots. The ability to restrain the nonspecific adsorption of QDs may because of the DNA tetrahedron layer separates QDs from the glass slide, preventing substrate-induced reactions. Moreover, the electrostatic repulsive force between QDs and DTDS makes it hard for the adsorption of QDs to occur. The DNA tetrahedron carries high negative charges as well as the QDs that decorated with streptavidin (the isoelectric point is 6.0). The surface density was calculated by UV spectrophotometry and we acquired 9.2×1012 tetrahedra/cm2 (more details in the supporting information). 3.4. Optimization of Reaction Conditions The kinetic rate of SDR can be controlled by adjusting the length and sequence composition of the toehold. It was reported that the toehold length of 6 nt was the smallest for initiating strand migration at the maximum rate in solution (Wang et al., 2012). However, the influence of toehold length on the kinetic of DNA tetrahedron decorated substrate has not been reported yet. Though design the sequences of DNA tetrahedron makes the effective toehold length vary from 5 nt to 8 nt, respectively. To assay miRNA with sensitive and selective, the reaction time between miRNA and capture probe was optimized. As shown in Fig. 2, when the toehold has a 5 nt length, it could not reach an equilibrium in 2 h. While the toehold with 6 nt had an
equilibrium at 2 h, and the toehold with 7 nt and 8 nt both had an equilibrium at 1.5 h. If the reaction time was longer, the selective of this method may decrease because the similar sequences can also initiate the SDR. Thus 2 h was chosen as the best time for toehold with 5 nt and 6 nt and 1.5 h for toehold with 7 nt and 8 nt.
Fig. 2 is here.
After affirm the reaction time, the selective of the method was investigated. To achieve the best sensing performance, the toehold length vary from 5 nt to 8 nt all be investigated with target miRNA (Let-7a) and its similar family members (let-7b, Let-7c, and Let-7i). Obviously, in Fig. 3, the toehold length with 6 nt, 7 nt, and 8 nt all had an excellent specificity towards the similar sequences. However, through calculated the ratio of the dots numbers produced by let-7a and its similar sequences (as shown in table S2), the toehold length with 6 nt had a best specificity. These results may be attributed to that for both the target miRNA and its similar family members, the reaction rate of SDR was increased with the increasing of toehold length. Hence, the toehold length with 6 nt was selected in the following experiments.
Fig. 3 is here.
3.5. Specificity of the Assay Due to the high sequence similarity among family members, it is a great
challenge to carry out the miRNAs assay with high specificity. For example, members of the let-7a family differ by only one or two nucleotides. To investigate the specificity of this method, we measured these let-7 family members under the optical condition. As shown in Fig. 3, the toehold with 6 nt showed perfect selectivity to the target miRNA, dots numbers produced by let-7a is 13.1-, 12.3- and 20.9-fold of that produced by let-7b, let-7c and let-7i, respectively, much higher than the reported methods (Liao et al., 2014; Zhang et al., 2014; Liu et al., 2014). These results indicated that the proposed strategy exhibited sufficient capability of distinguishing single-base difference among the miRNA family members by introducing the toehold-mediated SDR. 3.6. Sensitivity of the Assay Under the optimal conditions, we further measured the target miRNA at various concentrations. As shown in Fig. 4, the number of fluorescence dots was increased with the increasing concentration of let-7a. There was a good linear relationship between the number of dots and the concentration of let-7a over a range from 5 fM to 1 pM. The linear regression equation was Y=26.863+1041.898C with a correlation coefficient of 0.998, where Y and C were the number of fluorescence dots and the concentration of let-7a (pM), respectively. Compared with other reported methods, this proposed strategy demonstrates excellent sensitivity (Table S3). The enhancement of sensitivity might be attributed to the lower background of DNA tetrahedron substrates and the higher fluorescence quantum yield of QDs.
Fig. 4 is here.
3.7. Detection efficiency of the DNA tetrahedron probes In order to evaluate the detection efficiency of the proposed method, we calculated the number of fluorescence dots in theory according to one equation (more detail in the supporting information). The detection efficiency was the ratio of detected number and the theoretical number. When the concentration of target miRNA was 1 pM, the detection efficiency was 13%. This corresponds with previous reports (Wang et al., 2012) and is much higher than that in the direct immobilization method (5%) (Li et al., 2011). The improvement is mainly benefit from the introduction of DNA tetrahedra, which have made the probes more accessible. 3.8. Real Sample Assay To investigate the capability of this proposed method in real sample analysis, we perform the miRNA assay in total RNA sample extracted from the human lung tissues. The quantity of let-7a in human lung cells was measured by the standard addition method using synthetic let-7a as the standard. The total RNA sample was diluted to 2 ng/μL with Tris-HCl buffer, and 1μL of total RNA sample were spiked in standard solutions with synthetic let-7a at concentration of 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 pM, respectively. Then, the toehold-mediated SDR and fluorescence imaging were performed under the optimal conditions. The results are shown as Fig. 5, the amount of let-7a in the human lung total RNA is calculated at 3.50×109 copies/μg, which is consistent with previous reports (Zhang et al., 2014; Liu et al., 2014; Cheng et al.,
2009).
Fig. 5 is here.
4. Conclusion In summary, we have developed a selective and sensitive method for miRNA on the basis of toehold-mediated SDR and DNA tetrahedron substrate. To our knowledge, it was the first time to employ the toehold-mediated SDR into single-molecule quantitative method. First, after the introduction of DNA tetrahedron substrate, a low nonspecific adsorption was obtained. Under the optical conditions, the detection limit of this method was 5 fM without any amplification strategy. Second, by combining the toehold-mediated SDR, this method revealed an admirable specificity and could accurate recognize the target miRNA among its family members. Third, miRNA analysis in real samples was performed, indicating that our strategy was reliable and had a great potential application in biomedical research, early clinical diagnostics and oncotherapy.
Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant nos. 21175081, 21175082, 21375078 and 21475077).
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Figure Captions
Scheme 1. Schematic illustration of quantitative detection of single miRNA based on toehold-mediated strand displacement reaction.
Fig. 1 Electrophoretic analysis of DNA tetrahedron, 1-5 line stand for the
electrophoretic
image
of
sequence
A-HP-6+B-NH2+C-NH2+D-NH2,
B-NH2+C-NH2+D-NH2, B-NH2+C-NH2, B-NH2, and marker (20 bp).
Fig. 2 Effect of reaction time between miRNA and capture probe with different lengths of toehold. The error bars showed the standard deviation of three replicate determinations.
Fig. 3 Specificity of the proposed method to target miRNA (let-7a) and its family members (let-7b, let-7c and let-7i). The error bars are standard deviations of three
repetitive measurements.
Fig. 4 Linear relationship between the numbers of fluorescence dots and the concentration of target miRNA. The error bars represents the standard deviation of three repetitive measurements.
Fig. 5 The determination of let-7a in total RNA sample through standard addition method. The error bars represent the standard deviation of three repetitive measurements.
highlights
A toehold-mediated SDR-based strategy for miRNA analysis was proposed.
It was the first time to employ the toehold-mediated SDR into single-molecule counting method.
A high specificity and sensitivity for miRNA detection was achieved.
MiRNA analysis in total RNA samples from human lung tissues was performed.
