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. 2020 Dec 8;5(50):32738–32743. doi: 10.1021/acsomega.0c05141

Dual-Signal Amplification Strategy for Sensitive MicroRNA Detection Based on Rolling Circle Amplification and Enzymatic Repairing Amplification

Fubing Xiao †, Jie Liu †, Qinghui Guo ‡, Zhibo Du §, Hong Li †, Chunlong Sun ∥, Wenfang Du †,∥,*
PMCID: PMC7758957  PMID: 33376911

Abstract

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MicroRNAs (miRNAs) play crucial regulatory roles as post-transcriptional regulators for gene expression and serve as promising biomarkers for diagnosis and prognosis of diseases. Herein, a dual-signal amplification method has been developed for sensitive and selective detection of miRNA based on rolling circle amplification (RCA) and enzymatic repairing amplification (ERA) with low nonspecific background. This strategy designs a padlock probe that can be cyclized in the presence of target miRNA to initiate the RCA reaction, after which the TaqMan probes that are complementary to the RCA products can be cyclically cleaved to produce obvious fluorescence signals with the help of endonuclease IV (Endo IV). Attributed to the dual-signal amplification procedure and the high fidelity of Endo IV, the RCA–ERA method allows quantitative detection of miR-21 in a dynamic range from 2 pM to 5 nM with a low background signal. Moreover, it has the ability to discriminate single-base difference between miRNAs and shows good performance for miRNA detection in complex biological samples. The results demonstrate that the RCA–ERA assay holds a great promise for miRNA-based diagnostics.

1. Introduction

MicroRNAs (miRNAs), approximately 19–25 nucleotides long, are a series of endogenous, noncoding RNA molecules.1−3 Because they play crucial roles as post-transcriptional regulators for gene expression and are involved in several biological processes, aberrant expression of miRNAs is closely associated with various diseases, including cancer.4−6 Therefore, miRNAs have been increasingly treated as promising biomarkers for classification, diagnosis, and prognosis of diseases.7−9 However, detection of miRNAs is always challenged by their inherent characteristics, including short lengths, similarities of the sequences, susceptibility to degradation, and low abundance.10−12 Hence, establishing sensitive and selective strategies for miRNA detection is urgently needed, especially for miRNA-based diagnosis.

The most traditional methods for miRNA detection are Northern blotting13,14 and microarrays.15,16 However, there are some drawbacks such as low sensitivity, labor consumption, and requirement of a large number of samples which limit their wide use. In recent years, several nucleic acid amplification methods have been developed to improve the sensitivity, such as duplex-specific nuclease signal amplification,17 quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR),18 and rolling circle amplification (RCA).19,20 RCA, an isothermal DNA replication technique, could generate extremely long DNA strands with tandem repeats, which hold great potential for various applications such as biosensing, drug delivery, and clinical diagnosis. Jonstrup et al. first reported an RCA-based method for miRNA detection by designing a padlock probe which can be cyclized and replicated in the presence of target miRNA.21 Cheng et al. developed a simple and sensitive miRNA detection system by using target-primed and branched rolling-circle amplification by introduction of a second primer.22 To achieve desired specificity and sensitivity, several novel mechanisms were incorporated into the RCA for miRNA detection, such as cyclic enzymatic amplification method,23 strand displacement amplification,24 and catalytic hairpin assembly.25 However, with ever increasing needs for point-of-care applications, nucleic acid amplification strategies with low nonspecific background remain a great challenge.

Enzymatic repairing amplification (ERA) reaction is a mechanism of cyclic cleavage of TaqMan probes mediated by endonuclease IV (Endo IV) with a low background signal. Hence, it may be a good idea to establish a miRNA detection method by combining RCA with ERA. Motivated by this hypothesis, here we develop a dual-signal amplification approach for highly sensitive and specific miRNA analysis based on the ERA reaction after RCA. The RCA–ERA reaction includes two steps: (1) padlock probes are circularized with miRNA as a template in the presence of T4 RNA ligase 2 and RCA reaction utilizing miRNA as a primer with the assistance of phi29 DNA polymerase and (2) cyclic cleavage of TaqMan probes to produce fluorescence signals in the presence of Endo IV. By this way, target miRNA can be quantitatively detected with high sensitivity and selectivity.

2. Results and Discussion

2.1. Principle of the RCA–ERA Assay

The principle of the RCA–ERA technology is illustrated in Scheme 1. A padlock probe is designed to be complementary to the target miRNA at the 5′- and 3′-terminal and has a sequence similar to the TaqMan probe. In the presence of target miRNA, miRNA can act as a template to ligate and cyclize the padlock probe to generate a circular DNA with the help of T4 RNA ligase 2. Subsequently, the RCA reaction is initiated with miRNA as the primer and the circular DNA as the template by the addition of phi29 DNA polymerase. Such an RCA reaction is able to synthesize a long linear single-stranded DNA (ssDNA) with a large number of repeating sequences that are complementary to the TaqMan probe. The TaqMan probe with a tetrahydrofuran abasic site mimic flanked by nucleotides modified with a fluorophore (FAM) and a quencher (TAMRA) can anneal on the repeating sequences of the linear ssDNA product. The tetrahydrofuran abasic site mimic can be cleaved specifically by Endo IV which leads to the dissociation of the fluorophore and the quencher, and then, each repeating sequence is liberated to hybridize with another TaqMan probe to activate the cyclic annealing and cleavage of the TaqMan probe. As a result, a distinct fluorescence signal can be recorded after the RCA–ERA dual-signal amplification process. However, when the target miRNA is absent, the circular DNA template cannot form, precluding the RCA and ERA reactions and thus merely maintaining a low fluorescence. On the basis of this principle, the target can be quantitatively determined by measuring the fluorescence signal after the RCA–ERA reaction. Because of the combination of RCA and Endo IV-mediated signal amplification, the proposed assay provides a highly sensitive platform for miRNA detection with low background.

Scheme 1. Schematic Illustration of the RCA–ERA Strategy for miRNA Detection.

Scheme 1

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2.2. Feasibility of the RCA–ERA Assay

To prove the feasibility of the RCA–ERA method for the miRNA assay, the fluorescence spectra in the reactions were monitored using microRNA-21 (miR-21) as a model target. MiR-21 has been reported to be related with varying human cancers, so quantitative detection of miR-21 is significant for early cancer diagnosis. It can be seen from Figure 1A that the reaction system displayed a high fluorescence intensity in the presence of miR-21 (curve a), demonstrating a successful RCA–ERA reaction. In contrast, the fluorescence intensity was faint in a control experiment without miR-21 (curve b). In control reactions without padlock probe (curve c) or Endo IV (curve d), the fluorescence intensities were almost the same as that of the blank (without miR-21), indicating that both padlock probe and Endo IV were essential for the RCA–ERA reaction process. Taken together, these results revealed that the fluorescence signal change was an indicator for miR-21. To further verify the feasibility of this approach, the RCA products were analyzed using agarose gel electrophoresis (Figure 1B). Lane 3 shows the circular DNA after ligation with miR-21 as a template. Moreover, the long ssDNA products were obtained in the presence of miR-21 (lane 6), while no RCA products were observed in the absence of miR-21 (lane 4) or T4 RNA ligase 2 (lane 5). Thus, these findings confirmed that the RCA–ERA approach can be exploited for miRNA detection.

Figure 1.

Figure 1

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(A) Fluorescence emission spectra of the miRNA analysis under different conditions. (a) Padlock probe + T4 RNA ligase 2 + miR-21 (500 pM) + phi29 DNA polymerase + dNTPs + TaqMan probe + Endo IV, (b) control with no miR-21, (c) control with no padlock probe, and (d) control with no Endo IV. (B) Gel electrophoresis image for RCA products. Lane M: DNA marker (10–300 bp); Lane 1: 2 μM miR-21; Lane 2: 1 μM padlock probe; Lane 3: 1 μM padlock probe + 1 μM miR-21 + 2 U T4 RNA ligase 2; Lane 4: 100 nM padlock probe + 2 U T4 RNA ligase 2 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs. Lane 5: 100 nM padlock probe + 10 nM miR-21 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs; Lane 6: 100 nM padlock probe + 10 nM miR-21 + 2 U T4 RNA ligase 2 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs.

2.3. Optimizations of the Reaction Conditions

In order to achieve the optimum assay performance, we first optimized the reaction conditions including the length of the complementary sequence of the TaqMan probe (the design of the padlock probe), the time of the RCA reaction, and the concentrations of the padlock probe and TaqMan probe. To obtain an optimal fluorescence recovery efficiency of the TaqMan probe, six complementary sequences (cTaq-1, cTaq-2, cTaq-3, cTaq-4, cTaq-5, and cTaq-6) with 14–18 bases were investigated. The maximum fluorescence recovery efficiencies (F′/F0′ – 1) (where F0 and F′ are the fluorescence intensities of the TaqMan probe in the absence and presence of complementary sequence, respectively) of the TaqMan probe was obtained using cTaq-4 (Figure S1, Supporting Information). Hence, the padlock probe was designed to include the complementary sequence of cTaq-4. Although the high-amplification efficiency can be obtained at high padlock probe concentration, the background signal might increase correspondingly. As displayed in Figure S2 (Supporting Information), the highest fluorescence intensity ratio (F/F0 – 1, where F and F0 are the fluorescence intensities in the presence and absence of miR-21, respectively) was gained when the concentration of the padlock probe was 10 nM. Hence, the 10 nM padlock probe was selected and used in the subsequent experiments. The fluorescence intensity ratio increased gradually with increasing reaction time and reached a plateau after 4 h, which was chosen as the optimum RCA reaction time (Figure S3, Supporting Information). Moreover, the concentration of the TaqMan probe might influence the amplification efficiency of the ERA reaction and the sensitivity of the proposed method, so the effect of the concentration of the TaqMan probe was also studied. The F/F0 – 1 values first increased and then decreased with increasing concentrations of the TaqMan probe (Figure S4, Supporting Information). Therefore, the concentration of the TaqMan probe was fixed at 0.2 μM.

2.4. Sensitivity of the Assay

Under the optimal conditions, miR-21 with various concentrations was detected to estimate the sensitivity of the developed strategy. As shown in Figure 2A, as the miR-21 concentrations increased, a gradual increase of the fluorescence intensities with the fluorescent peak at 525 nm was clearly observed. Figure 2B depicts the relationship between the changes in the fluorescence intensity ratio (F/F0 – 1) and the amount of target miR-21 ranging from 2 pM to 5 nM. The F/F0 – 1 values were found to present a linear correlation with the logarithmic concentrations of miR-21 in the range from 100 pM to 5 nM (inset of Figure 2B), with a calibration equation of F/F0 – 1 = 132.7145 + 12.7740 lg(CmiR-21/M) (R2 = 0.9937), where F and F0 are the fluorescence intensities in the presence and absence of miR-21, respectively. The limit of detection (LOD) was estimated to be 0.2 pM according to the 3σ rule, which was comparable to some previously reported fluorescence assays for miRNA.26−28 Moreover, the analytical performance of the proposed method was also compared to other reported methods (Table S2, Supporting Information). The high sensitivity and the low background might attribute to the dual-signal amplification procedure and the high fidelity of Endo IV in specifically cleaving the TaqMan probe in dsDNA. Furthermore, three repetitive assays of miR-21 at 2 pM, 200 pM, and 5 nM were carried out, and the relative standard deviations of 6.2, 4.1, and 3.0% were obtained, respectively, implying a good reproducibility of this strategy.

Figure 2.

Figure 2

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(A) Fluorescence emission spectra of the miRNA detection strategy at different concentrations of miR-21. (B) Fluorescence ratio (F/F0 – 1) vs logarithmic miR-21 concentrations. Inset: linear relationship between F/F0 – 1 and the logarithmic concentration of miR-21. Error bars are standard deviations of three repetitive experiments.

2.5. Selectivity of the Proposed Method

The selectivity of the proposed miRNA detection method was tested by measuring the fluorescence signal response of the system for miR-141, miR-143, two-base mismatched miR-21 (2M-21), and single-base mismatched miR-21 (SM-21). As depicted in Figure 3, the fluorescence intensity ratios of miR-141 and miR-143 exhibited nearly negligible change compared with the blank. The F/F0 – 1 value obtained for the mixture of miR-21, miR-141, and miR-143 was about the same as that with only miR-21. Moreover, it is noticed that the F/F0 – 1 values for SM-21 and 2M-21 were much smaller than that for miR-21. These data demonstrated that the developed method was highly selective to the target miRNA.

Figure 3.

Figure 3

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Selectivity of the RCA–ERA assay for miRNA detection. Bars represent the fluorescence intensity ratio (F/F0 – 1) in the presence of different miRNAs with the same concentration of 200 pM.

2.6. Real Sample Assay

To examine the capability of the proposed approach for miRNA analysis in real biological samples, total RNA extracts from three human cancer cell lines, including MCF-10A, Hela, and MCF-7, were selected for further performance test of this strategy. Total RNA was extracted from each cell line by employing the UNlQ-10 column Trizol total RNA purification kit (Sangon Biotech Co., Ltd., Shanghai, China). From the data of qRT-PCR (Table S3, Supporting Information), the expression level of miR-21 in MCF-10A and Hela cell lines was estimated to be 0.281883- and 0.480277-fold of that in the MCF-7 cell line, indicating the up-regulation of miR-21 in the MCF-7 cell line as compared with that of MCF-10A and Hela cell lines. According to the data shown in Figure 4, the relative expression levels of miR-21 in the cell lysates determined by our method were consistent with those obtained by the standard method qRT-PCR. In addition, it can be observed that the expression levels of miR-21 were various in different cell lines, which was in good agreement with previous data.29 These results proved that the developed technology has great potential for miRNA analysis in clinical diagnosis.

Figure 4.

Figure 4

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Detection of the relative expression levels of miR-21 in different cell lysates, as measured by using our method and qRT-PCR method. Error bars are the standard deviation of three repetitive experiments.

3. Conclusions

In conclusion, we have established a dual-signal amplification strategy to quantify miRNA by coupling RCA and ERA reactions. The assay is based on target miRNA-triggered RCA followed by an Endo IV-mediated ERA reaction for the detection of the RCA products. Compared with other RCA-based amplification methods, the RCA-ERA assay exhibited low background and comparable amplification accuracy. The RCA-ERA method was shown to allow quantitative detection of miR-21 in a dynamic range from 2 pM to 5 nM with a detection limit down to 0.2 pM. Moreover, this assay also showed excellent specificity and had the capability to discriminate single-base mismatch. When used for detecting complex biological samples, the RCA–ERA assay achieved good performance for miR-21 detection in total RNA extracts from three human cell lines. Therefore, the developed strategy offered a potential detection platform for genetic analysis and clinical diagnosis.

4. Experimental Section

4.1. Materials

T4 RNA ligase 2, phi29 DNA polymerase, and endonuclease IV (Endo IV) were purchased from New England Biolabs (Ipswich, MA, USA). HPLC-purified miRNAs, RNase inhibitor, and RNase-free water were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The DNA probes, 5× TBE buffer (225 mM Tris–boric acid, 50 mM EDTA, pH 8.0), and dNTP mixture were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). GeneRuler Ultra Low Range DNA Ladder was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). RPMI 1640 medium, penicillin, 15% heat-inactivated fetal bovine serum, and streptomycin were obtained from Thermo Scientific HyClone (MA, USA). Sequences of DNA probes and miRNAs are given in Table S1, Supporting Information. All other reagents were of analytical grade and used without additional purification. RNase-free water was used in all experiments.

4.2. Methods

4.2.1. Detection of miRNA Using the RCA–ERA Assay

The ligation reaction was performed in a 10 μL reaction mixture containing 1× ligation buffer (50 mM Tris–HCl (pH 7.5@25 °C), 2 mM MgCl2, 1 mM DTT, 400 μM ATP), 1 U/μL RNase inhibitor, 10 nM padlock probe, 2 U T4 RNA ligase 2, and different concentrations of target miRNA. Before the ligase and ligation buffer were added, the reaction mixture was heated at 95 °C for 5 min and cooled slowly to 37 °C over a 30 min period. After that, T4 RNA ligase 2 and ligation buffer were added to the mixture and incubated at 37 °C for 2 h.

The RCA reaction was carried out in a 20 μL reaction mixture containing 10 μL of ligation reaction products, 1× phi29 DNA polymerase reaction buffer [10 mM MgCl2, 50 mM Tris–HCl, 4 mM DTT, 10 mM (NH4)2SO4 (pH 7.5@25 °C)], 1 U/μL RNase inhibitor, 0.2 mg/mL BSA, 0.5 mM of each dNTPs, and 10 U phi29 DNA polymerase. The mixture was incubated at 37 °C for 4 h, 65 °C for 10 min.

50 μL of reaction solution containing 20 μL RCA products, 1× NEBuffer 3 (10 mM MgCl2, 50 mM Tris–HCl, 1 mM DTT, 100 mM NaCl (pH 7.9@25 °C)), 200 nM TaqMan probe, and 0.2 U/μL Endo IV was prepared and incubated at 37 °C for 1 h. Then RNase-free water was added into the reaction with a final reaction volume of 100 μL for fluorescence detection. The fluorescence spectral measurements were recorded using a quartz cuvette on an F-7000 fluorescence spectrophotometer (Hitachi, Japan) at room temperature. The excitation wavelength was set at 495 nm, and the spectra were recorded from 505 to 600 nm with a slit of 5 nm for both excitation and emission.

4.2.2. Gel Electrophoresis Analysis

The RCA reaction products were analyzed using 3% agarose gel electrophoresis which was stained by 0.5 μg/mL GoldView and 0.5 μg/mL ethidium bromide. The gel electrophoresis was carried out at a constant voltage of 101 V for 90 min in 0.5× TBE buffer at room temperature with a load of 10 μL of each sample into the lanes. The image of the gel was visualized using a Tanon 4200SF gel imaging system (Tanon Science &Technology Co., Ltd., China).

4.2.3. Cell culture and Total RNA Extraction

Human mammary epithelial cell line (MCF-10A), human cervical carcinoma cell line (HeLa), and human breast cancer cell line (MCF-7) were cultured in RPMI 1640 medium supplemented with 15% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. All of the cells were cultured at 37 °C in a humidified incubator containing 5% CO2. Total RNA was extracted from each cell line by employing the UNlQ-10 column Trizol total RNA purification kit (Sangon Biotech Co., Ltd., Shanghai, China). The extracts were stored at −80 °C for analysis by utilizing our proposed method.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (21906077), the Natural Science Foundation of Hunan Province (2020JJ5474), the Scientific Research Fund of Hunan Provincial Education Department (20B508), the Key R&D Program of Hunan Province (2018SK2029), the Research Initiation Fund of University of South China (190XQD008), the Fund of Hengyang Key Laboratory (2018KJ110), and the Natural Science Foundation of Shandong Province (ZR2019MH054).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c05141.

  • Sequences of miRNAs and DNA probes and condition optimization (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao0c05141_si_001.pdf (779.5KB, pdf)

References

  1. Thomou T.; Mori M. A.; Dreyfuss J. M.; Konishi M.; Sakaguchi M.; Wolfrum C.; Rao T. N.; Winnay J. N.; Garcia-Martin R.; Grinspoon S. K.; Gorden P.; Kahn C. R. Adipose-derived circulating miRNAs regulate gene expression in other tissues. Nature 2017, 542, 450–455. 10.1038/nature21365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Shi L.; Lei J.; Zhang B.; Li B.; Yang C. J.; Jin Y. Ultrasensitive and facile detection of microRNA via a portable pressure meter. ACS Appl. Mater. Interfaces 2018, 10, 12526–12533. 10.1021/acsami.8b02551. [DOI] [PubMed] [Google Scholar]
  3. He L.; Hannon G. J. MicroRNAs: small RNAs with a big role in gene regulation. Nat. Rev. Genet. 2004, 5, 522–531. 10.1038/nrg1379. [DOI] [PubMed] [Google Scholar]
  4. Luby B. M.; Zheng G. Specific and direct amplified detection of microRNA with microRNA: Argonaute-2 cleavage (miRACle) beacons. Angew. Chem., Int. Ed. 2017, 56, 13704–13708. 10.1002/anie.201707366. [DOI] [PubMed] [Google Scholar]
  5. Rossi J. J. New hope for a microRNA therapy for liver cancer. Cell 2009, 137, 990–992. 10.1016/j.cell.2009.05.038. [DOI] [PubMed] [Google Scholar]
  6. Volinia S.; Calin G. A.; Liu C.-G.; Ambs S.; Cimmino A.; Petrocca F.; Visone R.; Iorio M.; Roldo C.; Ferracin M.; Prueitt R. L.; Yanaihara N.; Lanza G.; Scarpa A.; Vecchione A.; Negrini M.; Harris C. C.; Croce C. M. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2257–2261. 10.1073/pnas.0510565103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Tricoli J. V.; Jacobson J. W. MicroRNA: potential for cancer detection, diagnosis, and prognosis. Cancer Res. 2007, 67, 4553–4555. 10.1158/0008-5472.can-07-0563. [DOI] [PubMed] [Google Scholar]
  8. Duan R.; Zuo X.; Wang S.; Quan X.; Chen D.; Chen Z.; Jiang L.; Fan C.; Xia F. Lab in a tube: ultrasensitive detection of microRNAs at the single-cell level and in breast cancer patients using quadratic isothermal amplification. J. Am. Chem. Soc. 2013, 135, 4604–4607. 10.1021/ja311313b. [DOI] [PubMed] [Google Scholar]
  9. Dong H.; Lei J.; Ding L.; Wen Y.; Ju H.; Zhang X. MicroRNA: function, detection, and bioanalysis. Chem. Rev. 2013, 113, 6207–6233. 10.1021/cr300362f. [DOI] [PubMed] [Google Scholar]
  10. Zhang C.-H.; Tang Y.; Sheng Y.-Y.; Wang H.; Wu Z.; Jiang J.-H. Ultrasensitive detection of microRNAs using catalytic hairpin assembly coupled with enzymatic repairing amplification. Chem. Commun. 2016, 52, 13584–13587. 10.1039/c6cc07116c. [DOI] [PubMed] [Google Scholar]
  11. Chen A.; Gui G.-F.; Zhuo Y.; Chai Y.-Q.; Xiang Y.; Yuan R. Signal-off electrochemiluminescence biosensor based on Phi29 DNA polymerase mediated strand displacement amplification for microRNA detection. Anal. Chem. 2015, 87, 6328–6334. 10.1021/acs.analchem.5b01168. [DOI] [PubMed] [Google Scholar]
  12. Zhou M.; Teng X.; Li Y.; Deng R.; Li J. Cascade transcription amplification of RNA aptamer for ultrasensitive microRNA detection. Anal. Chem. 2019, 91, 5295–5302. 10.1021/acs.analchem.9b00124. [DOI] [PubMed] [Google Scholar]
  13. Várallyay É.; Burgyán J.; Havelda Z. MicroRNA detection by northern blotting using locked nucleic acid probes. Nat. Protoc. 2008, 3, 190–196. 10.1038/nprot.2007.528. [DOI] [PubMed] [Google Scholar]
  14. Schwarzkopf M.; Pierce NA Multiplexed miRNA northern blots via hybridization chain reaction. Nucleic Acids Res. 2016, 44, e129 10.1093/nar/gkw503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lee J. M.; Jung Y. Two-temperature hybridization for microarray detection of label-free MicroRNAs with attomole detection and superior specificity. Angew. Chem., Int. Ed. 2011, 50, 12487–12490. 10.1002/anie.201105605. [DOI] [PubMed] [Google Scholar]
  16. Babak T.; Zhang W.; Morris Q.; Blencowe B. J.; Hughes T. R. Probing microRNAs with microarrays: tissue specificity and functional inference. RNA 2004, 10, 1813–1819. 10.1261/rna.7119904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Yin B.-C.; Liu Y.-Q.; Ye B.-C. One-step, multiplexed fluorescence detection of microRNAs based on duplex-specific nuclease signal amplification. J. Am. Chem. Soc. 2012, 134, 5064–5067. 10.1021/ja300721s. [DOI] [PubMed] [Google Scholar]
  18. Chen C.; Ridzon D. A.; Broomer A. J.; Zhou Z.; Lee D. H.; Nguyen J. T.; Barbisin M.; Xu N. L.; Mahuvakar V. R.; Andersen M. R.; Lao K. Q.; Livak K. J.; Guegler K. J. Real-time quantification of microRNAs by stem–loop RT–PCR. Nucleic Acids Res. 2005, 33, e179 10.1093/nar/gni178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tian Q.; Wang Y.; Deng R.; Lin L.; Liu Y.; Li J. Carbon nanotube enhanced label-free detection of microRNAs based on hairpin probe triggered solid-phase rolling-circle amplification. Nanoscale 2015, 7, 987–993. 10.1039/c4nr05243a. [DOI] [PubMed] [Google Scholar]
  20. Li D.; Zhang T.; Yang F.; Yuan R.; Xiang Y. An efficient and exponential rolling circle amplification molecular network leads to ultrasensitive and label-free detection of microRNA. Anal. Chem. 2019, 92, 2074–2079. 10.1021/acs.analchem.9b04585. [DOI] [PubMed] [Google Scholar]
  21. Jonstrup S. P.; Koch J.; Kjems J. A microRNA detection system based on padlock probes and rolling circle amplification. RNA 2006, 12, 1747–1752. 10.1261/rna.110706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Cheng Y.; Zhang X.; Li Z.; Jiao X.; Wang Y.; Zhang Y. Highly sensitive determination of microRNA using target-primed and branched rolling-circle amplification. Angew. Chem., Int. Ed. 2009, 48, 3268–3272. 10.1002/anie.200805665. [DOI] [PubMed] [Google Scholar]
  23. Cui L.; Zhu Z.; Lin N.; Zhang H.; Guan Z.; Yang C. J. A T7 exonuclease-assisted cyclic enzymatic amplification method coupled with rolling circle amplification: a dual-amplification strategy for sensitive and selective microRNA detection. Chem. Commun. 2014, 50, 1576–1578. 10.1039/c3cc48707e. [DOI] [PubMed] [Google Scholar]
  24. Xu H.; Zhang Y.; Zhang S.; Sun M.; Li W.; Jiang Y.; Wu Z.-S. Ultrasensitive assay based on a combined cascade amplification by nicking-mediated rolling circle amplification and symmetric strand-displacement amplification. Anal. Chim. Acta 2019, 1047, 172–178. 10.1016/j.aca.2018.10.004. [DOI] [PubMed] [Google Scholar]
  25. Li Q.; Zeng F.; Lyu N.; Liang J. Highly sensitive and specific electrochemical biosensor for microRNA-21 detection by coupling catalytic hairpin assembly with rolling circle amplification. Analyst 2018, 143, 2304–2309. 10.1039/c8an00437d. [DOI] [PubMed] [Google Scholar]
  26. Xue T.; Bongu S. R.; Huang H.; Liang W.; Wang Y.; Zhang F.; Liu Z.; Zhang Y.; Zhang H.; Cui X. Ultrasensitive detection of microRNA using a bismuthene-enabled fluorescence quenching biosensor. Chem. Commun. 2020, 56, 7041–7044. 10.1039/d0cc01004a. [DOI] [PubMed] [Google Scholar]
  27. Oishi M. Comparative study of DNA circuit system-based proportional and exponential amplification strategies for enzyme-free and rapid detection of miRNA at room temperature. ACS Omega 2018, 3, 3321–3329. 10.1021/acsomega.7b01866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ma C.; Liu H.; Wu K.; Chen M.; Zheng L.; Wang J. An exonuclease I-based quencher-free fluorescent method using DNA hairpin probes for rapid detection of microRNA. Sensors 2017, 17, 760. 10.3390/s17040760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lu J.; Getz G.; Miska E. A.; Alvarez-Saavedra E.; Lamb J.; Peck D.; Sweet-Cordero A.; Ebert B. L.; Mak R. H.; Ferrando A. A.; Downing J. R.; Jacks T.; Horvitz H. R.; Golub T. R. MicroRNA expression profiles classify human cancers. Nature 2005, 435, 834–838. 10.1038/nature03702. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

ao0c05141_si_001.pdf (779.5KB, pdf)

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