High Specific and Ultrasensitive Isothermal Detection of MicroRNA by

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High Specific and Ultrasensitive Isothermal Detection of MicroRNA by padlock probe-based Exponential Rolling Circle Amplification Haiyun Liu, Lu Li, Lili Duan, Xu Wang, Yanxia Xie, Lili Tong, Qian Wang, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac401715k • Publication Date (Web): 16 Jul 2013 Downloaded from http://pubs.acs.org on July 21, 2013

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Analytical Chemistry

High Specific and Ultrasensitive Isothermal Detection of MicroRNA by padlock probe-based Exponential Rolling Circle Amplification Haiyun Liu‡, Lu Li‡, Lili Duan, Xu Wang, Yanxia Xie, Lili Tong, Qian Wang and Bo Tang,* College of Chemistry, Chemical Engineering and Materials Science, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan, Shandong Province, 250014, China

ACS Paragon Plus Environment

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ABSTRACT: In this paper, a padlock probe-based exponential rolling circle amplification (PERCA) assay is developed for high specific and sensitive detection of microRNA (miRNA). The padlock probe is composed of a hybridization sequence to miRNA and a nicking site for niking endonuclease. Using the miRNA as template, specific ligation to padlock probe and linear rolling circle reaction (LRCA) are achieved under isothermal conditions. After multiple nicking reactions, many copies of short DNA products are successively produced and then used as trigger in next circle amplification. Thus, a small amount of miRNAs are converted to a large number of triggers to initiate rolling circle amplification reaction and circular exponential signal amplification is achieved. This padlock probe-based exponential rolling circle amplification assay exhibits a remarkable sensitivity of 0.24 zmol using optimized sequences of padlock probe. The target-dependent circularization of padlock probe and the ligation reaction could improve the specificity effectively, making single–nucleotide difference be discriminated between miRNA family members. The miRNA analysis in human lung cells was performed with this method. The result indicates this highly sensitive P-ERCA strategy will become a promising miRNA quantification method in early clinical diagnostics. KEYWORDS: MicroRNA, exponential rolling circle amplification, padlock probe, isothermal detection, fluorescence


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MicroRNAs (miRNAs) are short endogenous noncoding RNA about 18-24 nucleotieds (nt), that play important roles in normal and pathologic processes. They regulate gene activity and act to promote or repress cell proliferation, migration, and apoptosis.1-5 Recent studies have found that some miRNAs have altered expression in cancer cells, aberrant expression of miRNAs are associated with cancer initiation, tumor stage, and tumor response to treatments.5, 6 Nowdays, miRNAs have been regarded as biomarkers and therapeutic targets in cancer treatment.1, 4, 7-11 So, effective detection for miRNAs is crucial for better understanding their roles in cancer cells and further validating their function in biomedical research and clinical diagnosis. However, it is difficult to analyze the miRNAs because of their unique characteristics, including their small size, sequence homology among family members, and low abundance in total RNA samples.12 So, strategies for specific, especially sensitive quantitive detection of miRNAs are in urgent need. Recently, many different methods have been used to profile miRNAs expression. Northern blot is widely used to visualize specific miRNA,13-16 but it requires large amounts of sample and the sensitivity is not satisfied.12 Microarray technology offers a way to analyze a little volume and multiple samples simultaneously. Nevertheless, its sensitivity and specificity should also be improved.17-22 RT-qPCR has been proposed for miRNA analysis and the sensitivity of miRNA detection can reach to single molecule level.23 However, it requires precise control of temperature cycling for successful amplification and the short length of miRNAs make their experimental design very sophisticated.23-26 Except these standard methods for miRNA detection mentioned above, various new strategies have been developed to improve the detection sensitivity and adaptability, such as nanopartical-based assay,27-30 bioluminescence-based assay,31 modified invader assay,32 ribozyme-based assay,33 sequencing-based assay,34 EXPAR


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assay,35 strand displacement assay,36 hairpin-based amplification.37 Among these methods, rolling-circle amplification (RCA) has become increasingly popular in the miRNA detection due to its simplicity, specificity, and high sensitivity.38-40 After the RCA-based miRNA detection was firstly reported,38 several novel strategies have been developed to improve the specificity and sensitivity of this method by introducing a second primer,39 a dumbbell-shaped DNA probe,40, 41 DNAzyme,42 or encoded gel microparticles.43 Recently, a primer generation-rolling circle amplification (PG-RCA) has been reported.44,

45

PG-RCA is a process including a cascade

reaction of linear rolling circle amplification and nicking reactions. In contrast with conventional linear rolling circle amplification, the amplification was in an exponential mode. The remarkable sensitivity may offer great possibility for low-abundance miRNA detection. But due to the high sequence homology among miRNA family members and small size, there’s still a great challenge for specific detection of miRNA, especially for discrimination of single-nucleotide difference within the same and short length. Herein, we developed a padlock probe-based exponential rolling circle amplification (PERCA) assay for high specific and ultrasensitive detection of miRNA. After a padlock probe was designed reasonable, specific ligation and circulation with the miRNA as template was achieved under isothermal conditions. The ligation reaction and the target-dependent circularization of padlock probe could improve the specificity of the miRNA assay effectively. The exponential amplification allowed quantification of miRNA with a remarkable sensitivity of 0.24 zmol. The miRNA analysis in human lung cells was also performed, indicating this P-ERCA strategy will become a specific and ultrasensitive miRNA quantification method in medical research and early clinical diagnostics.


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EXPERIMENTAL SECTION Materials and Apparatus. Oligonucleotides were purchased from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). The miRNA, diethyprocarbonated (DEPC)-treated deionized water, deoxynucleotides (dNTPs), TE buffer, PBS and ribonuclease inhibitor were obtained from TaKaRa Biotechnology Co., Ltd. (Dalian, China). T4 RNA ligase 2, T4 DNA ligase, Phi29 DNA polymerase and Nb.BbvCI were purchased from New England Biolabs (Ipswich, MA). SYBR Green I was purchased from Xiamen Zeesan Biotechnology Co., Ltd. (Xiamen, China). Fetal bovine serum was purchased from Gibco (Carlsbad, CA). RPMI 1640 and MirVana miRNA Isolation Kit was purchased from Life Technologies (Carlsbad, CA). Before use, the oligonucleotides and miRNA were diluted to appropriate concentration with diethprocarbonated (DEPC)-treated water, then separated into 20 µL centrifugal tubes. All of the oligonucleotides and miRNA were purified by HPLC. DEPCtreated deionized water was used in all experiments. The sequences of the oligonucleotiedes and miRNAs were listed in Table 1. Isothermal amplification reaction was performed on a Veriti 96 Well Thermal Cycler (Applied Biosystems, CA). Gel electrophoresis was conducted using DYCZ-24DN Electrophoresis Cell (LIUYI, Beijing, China) and GelDoc-It Imaging Systems (UVP, Cambridge, UK). The fluorescent spectra was measured using Cary Eclipse Fluorescence Spectrophotometer (Varian, CA).


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Table 1. Sequences of oligonucleotides and miRNAsa

a

Name

Sequence (5′-3′)

Padlock probe I

5′-phosphate-CTA CTA CCT CAT TTG CAT TTC AGT TTA CCT CAG CGC ATT TCG CAA TTT TAA CTA TAC ACC-3′

Padlock probe II

5′-phosphate-CTA CTA CCT CAC CTC AGC AAC TAT ACA ACC TAC TAC CTC ACC TCA GCA ACT ATA CAA CCT ACT ACC TCA CCT CAG CAA CTA TAC AAC-3′

let-7a

5′-UGA GGU AGU AGG UUG UAU AGU U-3′

let-7b

5′-UGA GGU AGU AGG UUG UGU GGU U-3′

let-7c

5′-UGA GGU AGU AGG UUG UAU GGU U-3′

miR-21

5′-UAG CUU AUC AGA CUG AUG UUG A-3′

The bold characters indicate recognition sequences of Nb.BbvCI; The underlined bases are the

different bases between let-7b, let-7c and let-7a. Ligation Reactions. The ligation reaction was carried out with 10 µL reaction mixture containing 1 × ligation buffer [40 mM Tris-HCl, 10 mM MgCl2, 10 mM Dithiothreitol (DTT), 500 mM ATP (pH 7.8)], 2 U of T4 RNA ligase 2, 2 µL of the padlock probe and 2 µL of miRNA. Before adding T4 RNA ligase 2 and ligation buffer, the oligonucleotide mixture was denatured at 65 ºC for 3 min, and cooled slowly to room temperature over a 10 min period. After annealing, T4 RNA ligase 2 and ligation buffer were added to the mixture and incubated at 37 ºC for 2 h. RCA Reactions. The amplification reaction was conducted at 30 ºC in 20 µL reaction mixture containing 10 µL ligation reaction products, 20 mM Tris–HCl buffer (pH 8.8), 10 mM


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(NH4)2SO4, 10 mM KCl, 6 mM MgSO4, 400 µM each dNTP, 0.1% Triton X-100, 0.4 U Phi29 DNA polymerase and 1 U Nb.BbvCI. Gel Electrophoresis analysis. P–ERCA product was analyzed by 16% Urea denaturing polyacrylamide gel electrophoresis (PAGE). The gel was carried in 1× electrophoresis Trisborate-EDTA (TBE) at 100 V for 10 min, and stained with SYBR Green I for 15 min. The imaging of gel was performed using UVP GelDoc-It Imaging Systems. Measurement of Fluorescent Spectra. The 6 µL P-ERCA amplification product was mixed with 4 µL 20 × SYBR Green I and diluted to final volume of 600 µL with 10 mM PBS (pH 7.4). The fluorescent spectra were measured using a Cary Eclipse Fluorescence Spectrophotometer. The excitation wavelength was 480 nm, and the spectra was recorded between 520 nm and 650 nm. The fluorescence emission maximum was at 528 nm. Cell Lysis and RNA preparation. The human lung cells (A549) were cultured according to instructions of American Type Culture Collection. Cells were grown in RPMI 1640 (Hyclone, penicillin 100 U/mL, streptomycin 100 µg/mL) plus 10% fetal bovine serum (FBS, Gibco) and maintained at 37 ºC in a humidified atmosphere of 5% CO2 and 95% air. The cells were collected and centrifuged at 3000 rpm for 5 min in culture medium, washed once with PBS buffer, then spun down at 3000 rpm for 5 min. The cell pellets were suspended in 600 µL lysis solution. Total RNA was extracted from human lung cells using mirVana miRNA Isolation Kit according to the manufacturer’s procedures. The RNA concentration was determined to be 3.9 µg/mL from the UV vis absorption at 260 nm. The sample of let-7a in these cells was diluted, then analyzed with the proposed miRNA assays. RESULTS AND DISCUSSION


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Scheme 1. Scheme for miRNA detection with the padlock probe-based exponential rolling circle amplification (P-ERCA) reaction.a

a

The reaction involves three principal steps: (1) padlock probe is ligated specifically and

circularized with the miRNA as template in the presence of T4 RNA ligate 2; (2) A long concatenated sequence copy of the padlock probe is synthesized by Phi29 DNA polymerase; (3) Multiple padlock probes can be hybridized to DNA product, nicking endonuclease recognizes the niking sites and cleaves sequences, multiple triggers are produced and initiate a new reaction cycle. Through multiple reaction cycles, an exponential amplification for a small amount miRNA is achieved. Principle of P-ERCA assay. The strategy for miRNA detection on the basis of the padlock probe-based exponential rolling circle amplification (P-ERCA) is shown in Scheme 1. The padlock probe is composed of a hybridization sequence to miRNA (black) and a nicking site for


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niking endonuclease (red). The 5′- and 3′-terminal of the padlock probe are designed to be complementary to miRNA target. The padlock probe can be ligated specifically and circularized with the miRNA as template in the presence of T4 RNA ligate 2. Once miRNA and padlock probe form a complex, Phi29 DNA polymerase synthesizes a long concatenated sequence copy of the padlock probe through linear rolling circle amplification. Next, multiple padlock probes can be hybridized to multiple sites of the LRCA DNA product and nicking endonuclease recognizes the sites and cleaves sequences of double strand formation, producing multiple short DNA products as new triggers to initiate multiple reaction cycles until some of the reaction components, most likely dNTP substrates, are depleted. Hence, LRCA and cleavage can be repeated continuously in cycles, conventional LRCA is converted to an exponential amplification and high sensitive assay for miRNA can be achieved. The P-ERCA reaction was further confirmed by measurement of fluorescent spectra and gel electrophoresis. As shown in Figure 1, the fluorescence intensity gradually increases in the presence of let-7a (curve b), indicating that let-7a initiates the P-ERCA reaction and produces a large number of DNA products. On the contrary, the fluorescence intensity is faint and unchanged in the control reaction without let-7a (curve a), indicating that no reaction occurs. To verify the P-ERCA pathway of cleavage by nicking endonuclease and production of multiple DNA products, denaturing polyacrylamide gel electrophoresis was also performed. The P-ERCA after triggering by the let-7a target was conducted and productions were analyzed by 16% denaturing polyacrylamide gel electrophoresis. Lane 1 in the inset of Figure 1 shows the amplification products by P-ERCA in the presence of let-7a, the wide bands of lane 1 could be attributed to the multiple components of the cleavage. This is probably because the LRCA product from padlock probe II has nicking sites every 22 bases and even the ladder products


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could look like smear.44 When the reaction was conducted in the absence of let-7a, there is no product observed (Lane 0 in the inset of Figure 1), indicating no reaction occurs also.

Figure 1. Fluorescence intensity of amplification products by P-ERCA in the absence (a) and presence (b) of let-7a. Inset: Denaturing polyacrylamide gel electrophoresis (16%) of amplification products by the P-ERCA reaction in the absence (a) and presence (b) of let-7a. The products were stained with SYBR Green I. The marker was indicated by M. The reaction solution contained 3 nM padlock probe and 18 zmol let-7a. The ligation reactions were performed at 37 ºC for 2 h and the amplification reactions were performed at 30 ºC for 6 h. Specificity of the Assay. It is a great challenge to carry out the miRNA assay with high specificity due to the high sequence homology among family members and small size of miRNA. For example, members of the let-7 miRNA family differ by only one or two nucleotides in sequence with same length (only 22 bases). In this assay, a padlock probe was introduced to improve the selectivity. In the padlock probe, two target-complementary segments are present at opposite ends of a linear DNA probe molecule. Upon hybridization to the target miRNA, the ends of padlock probe are brought in contact and the probe is circularized by enzymatic ligation of the ends, trapping the probe molecule at the site of hybridization. Due to the strict requirement


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for coincident hybridization to two target segments, target miRNA was recognized with very high specificity; even single-nucleotide variants of sequences could be distinguished using the padlock probe.46-50 Here, a circular probe was used to be compared with the padlock probe in the specificity evaluation of P-ERCA. Using the circular probe perfectly complementary to let-7a, fluorescence intensity produced by let-7a is only 1.6-fold of that produced by let-7c. When a padlock probe was used, the fluorescent signal produced by let-7a is approximately 6.2-fold higher than that of let-7c (Figure 2A), revealing padlock probe has higher specificity to discriminate a single-nucleotide difference between let-7a and let-7c compared with a circular probe. At the same time, the molecular dynamics simulation had been carried out to study the specificity of the two probes and the corresponding DNA-RNA binding free energy was shown in Figure 2B. It is clearly seen that the binding free energy of let-7a was only 1.1-fold of that let7c in circular probe, while the ratio of binding free energy of let-7a to the let-7c was about 1.5fold in padlock probe. This was consistent with the experimentally observed. The results illustrates that ligation reaction allows efficient, specific assay for discriminating singlenucleotide differences between miRNAs. The specificity of P-ERCA is also affected by the ligase for sealing the termini of padlock probe. Both T4 DNA ligase and T4 RNA ligase 2 are generally used as an efficient catalyst of RNA ligation in RNA/DNA hybrids. The let-7a and let7c were detected using T4 DNA ligase and T4 RNA ligase 2 respectively. The results shown in Figure 2C indicated though the fluorescent intensity produced by T4 RNA ligase 2 was lower than that of T4 DNA ligase, T4 RNA ligase 2 exhibited higher specificity than T4 DNA ligase in this assay, and the result was consistent with that reported by other literatures.39 After the padlock probe and T4 RNA ligase 2 were used, the specificity of the proposed P-ERCA reaction was evaluated. Three members of let-7 family (let-7a, let-7b, and let-7c), with only one- or two-


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nucleotides differences and miR-21 are chosen as the detection model. As shown in Figure 2D, fluorescence signal produced by let-7a which is perfectly complementary to the padlock probe reaches 219, which is 39-fold more than that of mir-21, 12-fold more than that of let-7b and 6.2fold more than that of let-7c, even though there is only a single-nucleotide difference between let-7a and let-7c, suggesting the high specificity of our strategy for miRNA detection.


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Figure 2. (A) Comparison of the fluorescence intensity produced by let-7a and let-7c when a circular probe or a padlock probe was used respectively; (B) Comparison of the binding free energy of let-7a and let-7c with circular probe and padlock probe; (C) Comparison of the fluorescence intensity produced by let-7a and let-7c when T4 DNA ligase or T4 RNA ligase 2 was used respectively; (D) Comparison of the fluorescence intensity produced by let-7a, let-7b, let-7c and miR-21 using a padlock probe perfectly complementary to let-7a, where F and F0 are fluorescence intensities of amplification products by P-ERCA in the presence and absence of let7a, respectively. The reaction solution contained 3 fM padlock probe and 18 zmol let-7a. The ligation reactions were performed at 37 ºC for 2 h and P-ERCA reactions were performed at 30 ºC for 6 h. Error bars were estimated from three replicate measurements.


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Optimization of Padlock Probe Sequences. The padlock probe used to ligate miRNA and initiate multiple reaction cycles carries two types of sequences for signal amplification: a hybridization sequence to the miRNA and a nicking site. Precise design for sequences is important to the amplification efficiency. Here, two padlock probes (padlock probes I and padlock probes II) were designed and amplification efficiency was compared. Padlock probe I used in the reaction contains only one complementary segment to miRNA and one nicking site for Nb.BbvCI, while padlock probe II contains three complementary segments and three nicking sites (Table 1). P-ERCA reaction for let-7a detection was achieved using the two probes, results were shown in Figure 3. The fluorescence intensity produced by padlock probe II was 3.2-fold of that produced by padlock probes I. The increased complementary segments and nicking sites could increase the efficiency of LRCA and nicking reaction in each reaction cycle, producing more short DNA products as triggers and leading to multiple signal amplification.

Figure 3. Comparison of amplification efficiency with padlock probe I and II, where F and F0 are fluorescence intensities of amplification products by P-ERCA in the presence and absence of let-7a, respectively. The reactions were conducted with 3 fM padlock probe and 18 zmol let-7a. The ligation reactions were performed at 37 ºC for 2 h and amplification reactions were performed at 30 ºC for 6 h. Error bars were estimated from three replicate measurements.


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Optimization of P-ERCA Reaction Conditions. Several detection conditions such as the concentrations of dNTP substrates, Phi29 DNA polymerase and nicking enzyme Nb.BbvCI, the time and temperature of ligation and amplification were further optimized to improve the detection sensitivity. The final fluorescence was measured in the presence of 18 zmol let-7a. To investigate the influence of the concentration of dNTP, 100 µM, 200 µM, 400 µM, 600 µM and 800 µM each dNTP was used. As shown in Figure 4A, as the concentration of dNTP increased, the fluorescence intensity increased gradually until 400 µM dNTP was used. The concentration of Phi29 DNA polymerase is another factor which affects the amplification reaction. When different concentrations of Phi29 DNA polymerase were used, the fluorescence signals were analyzed and dates were showed in Figure 4B, the produced fluorescence signals increased with increasing concentration of Phi29 DNA polymerase until the concentration reached to 0.4 U. The effect of the amount of Nb.BbvCI on the assay was assessed also. The dates were shown in Figure 4C. Though the amount of nicking enzyme was changed from 0.2 U to 2 U, there was little change in the fluorescence signals. In order to obtain optimal detection sensitivity, 400 µM each dNTP, 0.4 U Phi29 DNA polymerase and 1 U Nb.BbvCI were selected, respectively. The changes of fluorescence intensity with the time of ligation and amplification were also investigated (Figure 4D), 2 hours and 6 hours were selected as optimal ligation time and amplification time for miRNA detection. Several temperatures of ligation (35 ºC, 37 ºC and 39 ºC) and exponential rolling circle amplification (30 ºC and 37 ºC) were compared to obtain higher detection sensitivity (Figure 4E). Finally, 37 ºC and 30 ºC were considered to be optimal ligation temperature and amplification temperature respectively.


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Figure 4. The relationship between the fluorescence intensity and the concentration of dNTP (A), Phi29 DNA polymerase (B), Nb.BbvCI (C), the time of ligation and amplification (D) and the temperature of ligation and amplification (E), where F and F0 are fluorescence intensities of amplification products by P-ERCA in the presence and absence of let-7a, respectively. The concentration of let-7a was 18 zmol. The error bar represents the standard deviation of three measurements. Sensitivity of the Assay. Under the optimized concentrations, the let-7a was detected to evaluate the sensitivity of the proposed strategy. The fluorescent intensities of products by P– ERCA reaction with different amount of let-7a were measured (Figure 5), and there was


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remarkable increase with the increased amount of let-7a. A good linearity was obtained in 3 orders of magnitude from 0.3 zmol to 18 zmol miRNA. The correlation equation was F-F0 = 28.26 + 10.91 A (F and F0 represent fluorescence intensities of amplification products by PERCA in the presence and absence of let-7a; A represents the amount of let-7a, zmol) with a correlation coefficient r = 0.9919. A relative standard deviation (RSD) of 4.2% for 11 repetitive measurements of 6.0 zmol let-7a was obtained, providing a good reproducibility of this miRNA assay. The detection limit was estimated to be 0.24 zmol (3σ, n = 11). The proposed assay has achieved one of the most sensitive approaches for miRNA detection compared other reported methods (Table 2). The low detection limit allows ultrasensitive accurate quantitation of miRNA at low concentration, which is of great significance in the early diagnosis of diseases.

Figure 5. The relationship between the fluorescent response and the amount of target miRNA (let-7a). The reactions were conducted with 3 fM padlock probe and miRNA from 0.3 zmol to 18 zmol. Table 2. The comparison for existing miRNA amplification methods Methods

Detection limit Dynamic range References


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Branched RCA

6 amol

3

39

CC-SDR

136 zmol

3

36

D-RCA

20 zmol

8

40

Encoded gel microparticles-RCA 15 zmol

6

43

P-ERCA

0.24 zmol

3

This work

EXPAR

0.1 zmol

10

35

Stem-loop RT-PCR

7 copies

7

23

Real Sample Assay. P–ERCA was further applied to quantify the amount of let-7a in total RNA sample that was extracted from human lung cell. The concentration of let-7a in human lung cells was determined by standard addition method using synthetic let-7a as the standard. The human lung total RNA (3.9 µg/mL) extracted from human lung cells was diluted to 0.78 ng/mL with RNase free TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Aliquots of the diluted RNA sample (1 µL) were spiked with standard solutions containing synthetic let-7a at concentrations of 0 zmol, 0.6 zmol, 1.8 zmol, 3.0 zmol, 4.2 zmol, 6 zmol, 9 zmol, 12 zmol and 18 zmol, respectively. Then P-ERCA and fluorescence detection were performed under the same conditions as described in the experimental section. The results were showed in Figure S1. It was calculated the content of let-7a in human lung total RNA sample was 5.05×109 copies/µg, which were in good agreement with those obtained in previous studies.39, 41. CONCLUSIONS In summary, a high specific and ultrasensitive isothermal detection of label-free miRNA is achieved based on P-ERCA reaction. This reaction is initiated by the target miRNA and catalyzed by DNA products generated and accumulated during the reaction. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does


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not require exogenous primers, which often cause primer dimerization or non-specific amplification. This experiment is a simple. With the exponential amplification, fluorescence signals can be sensitively detected with a remarkable sensitivity of 0.24 zmol. Moreover, using the reasonable designed padlock probe and T4 RNA ligase 2 in the specific ligation, high specificity was achieved, even single–nucleotide difference can be discriminated. In addition, the proposed strategy has successfully achieved the detection of let-7a in total RNA sample extracted from human lung cells. The results indicate that the proposed P-ERCA strategy holds a great potential for further application in biomedical research and early clinical diagnostics. ASSOCIATED CONTENT Supporting Information. Process of molecular dynamics simulation, calculations of binding free energies and Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected]. Tel: (86)531 86180010. Fax: (86)531 86180017. Author Contributions ‡These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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This work was supported by 973 Program (2013CB933800), National Natural Science Foundation of China (21035003, 21227005, 21205074, 31200545), Specialized Research Fund for the Doctoral Program of Higher Education of China (20113704130001), Program for Changjiang Scholars and Innovative Research Team in University, the Shandong Distinguished Middle-Aged and Young Scientist Encourage and Reward Foundation (BS2012SW022) and Key Project of Chinese Ministry of Education (212102)


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For Table of Contents only

Padlock probe-based exponential rolling circle amplification (P-ERCA) is developed to detect miRNA with high specificity and sensitivity. Specific ligation reaction and target-dependent circularization of padlock probe improve the specificity of the assay effectively. Exponential amplification allows quantification of miRNA with a remarkable sensitivity of 0.24 zmol. The potential applications of this method in bioanalysis and clinical diagnostics have been demonstrated by miRNA analysis in human lung cells.


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High Specific and Ultrasensitive Isothermal Detection of MicroRNA by

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