Letter pubs.acs.org/ac
Isothermal Nucleic Acid Amplification Strategy by Cyclic Enzymatic Repairing for Highly Sensitive MicroRNA Detection Dian-Ming Zhou, Wen-Fang Du, Qiang Xi, Jia Ge, and Jian-Hui Jiang* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China S Supporting Information *
ABSTRACT: Technologies enabling highly sensitive and selective detection of microRNAs (miRNAs) are critical for miRNA discovery and clinical theranostics. Here we develop a novel isothermal nucleic acid amplification technology based on cyclic enzymatic repairing and strand-displacement polymerase extension for highly sensitive miRNA detection. The enzymatic repairing amplification (ERA) reaction is performed via replicating DNA template using lesion bases by DNA polymerase and cleaving the DNA replicate at the lesions by repairing enzymes, uracil-DNA glycosylase, and endonuclease IV, to prime a next-round replication. By utilizing the miRNA target as the primer, the ERA reaction is capable of producing a large number of reporter sequences from the DNA template, which can then be coupled to a cyclic signal output reaction mediated by endonuclease IV. The ERA reaction can be configured as a single-step, close-tube, and real-time format, which enables highly sensitive and selective detection of miRNA with excellent resistance to contaminants. The developed technology is demonstrated to give a detection limit of 0.1 fM and show superb specificity in discriminating single-base mismatch. The results reveal that the ERA reaction may provide a new paradigm for efficient nucleic acid amplification and may hold the potential for miRNA expression profiling and related theranostic applications.
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quadratic enzymatic amplification,12 exponential amplification reaction,13 and duplex-specific nuclease signal amplification.14,15 Most of these amplification reactions involve a key mechanism of DNA nicking, in which a phosphodiester bond is cleaved cyclically by a nickase such that 3′ end at the nick site can act as a primer for a DNA polymerase to initiate the product of numerous DNA replicates. However, it is reported that the combination of nickase and DNA polymerase may lead to nonspecific amplification even in the absence of DNA templates,16,17 possibly causing false positive signal in the detection. Hence, the development of alternative mechanisms that enable highly efficient polymerase-based amplification with low nonspecific background remains a great challenge in the area. Herein, we develop a novel isothermal nucleic acid amplification technology based on cyclic enzymatic repairing and strand-displacement polymerase extension for highly sensitive miRNA detection. The enzymatic repairing amplification (ERA) reaction is primed specifically by the target miRNA and performed cyclically to amplify the designed DNA template into a large number of DNA replicates. The cyclic amplification
icroRNAs (miRNAs), a group of short (approximately 19−25 nucleotides) and endogenous nonprotein-coding RNA molecules, play crucial regulatory roles in gene expression that are critical to various biological processes. Aberrant expression of miRNAs is closely implicated in various diseases, including cancers, diabetes, neurological disorders, cardiovascular, and autoimmune diseases.1 It is revealed that miRNAs has emerged as a new class of promising biomarkers for clinical diagnostics and targets for drug development and disease therapy.2 MiRNAs have several unique characteristics, such as short lengths, sequence homology among family members, lability to degradation, and low abundance in total RNA samples,3 which increases the difficulty in miRNA-related target discovery and clinical theranostics. For the reasons, it is very important to develop sensitive and selective technologies for miRNA detection. Detection of miRNA has been conventionally implemented using Northern blotting technology4 and DNA microarrays5 without a preliminary amplification step. The unfavorably low sensitivity of these techniques, however, precluded their applications in clinical theranostics. Recently, there is increasing interest in the development of nucleic acid amplification technologies to improve the sensitivity in miRNA analysis, such as reverse transcription quantitative PCR,6 rolling circle amplification,7−9 exponential strand-displacement amplification,10 modified invader amplification,11 hairpin-mediated © 2014 American Chemical Society
Received: May 19, 2014 Accepted: June 20, 2014 Published: June 20, 2014 6763
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Scheme 1. Illustration of ERA Strategy for miRNA Detection
displaced primers from P2 are then annealed on P1 of another DNA template, triggering a cycle of the polymerizationrepairing amplification. This cyclic ERA reaction cannot only generate a large number of copies of the primer for highly efficient amplification but also produce a great quantity of reporter sequences that are responsible for efficient signal output. To deliver an activated fluorescence signal, a fluorescence-quenched signal probe24−26 is designed to have a tetrahydrofuran abasic site mimic (TAP site) flanked in close proximity by nucleotides modified with a fluorophore (FAM) and a quencher (Dabcyl). This signal probe is reported to be a stable substrate for Endo IV cleavage.27 When annealed on the reporter sequence, the signal probe can be specifically and efficiently cleaved by Endo IV. The resulting two fragments, one carrying the fluorophore and the other bearing the quencher, are both too short to be stably annealed on the reporter sequence. The dissociation of these two fragments from the reporter sequence, therefore, activates the fluorescence signal while enables signal amplification via the cyclic annealing and cleavage of the signal probe on the reporter sequence. On the basis of this principle, the developed miRNA detection technology can be implemented by a single-step mixing of the miRNA sample and the enzyme system followed by a real-time monitoring of the fluorescence signal with no need for additional steps for reagent inputs and reactions. This single-step, closed-tube and real-time configuration can offer excellent resistance to possible contaminants. Moreover, because of the combination of cyclic ERA amplification and Endo IV mediated signal amplification, the developed technology indeed provides a new paradigm for highly efficient nucleic acid amplification enabling very sensitive miRNA detection. To demonstrate the feasibility of this technology, we choose miR-21 (Table S1 in the Supporting Information) as the model system. MiR-21 has been reported to be overexpressed in varying human cancers, including glioblastoma, cholangiocarcinoma, multiple myeloma cells, and breast cancer.28 A major advantage of the developed technology is the efficient inhibition for nonspecific amplification from templateor primer-independent DNA synthesis (Figure S1 in the Supporting Information). It was observed that, in the presence of nickase Nt.BstNBI and DNA polymerase (Bst exo− or Vent exo−) as well as four nucleotides dATP, dGTP, dCTP, and dTTP, a great amount of DNA was synthesized at 60 °C even in cases when no DNA was added as the template or primer.
reaction comprises two steps: the replication of the DNA template using lesion bases catalyzed by a DNA polymerase and the scissoring at the lesions to create a new priming site by two DNA repairing enzymes, uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV). Because nonspecific polymerization of nucleotides may incorporate too many lesions to form long DNA templates that can be efficiently amplified, the developed ERA may have the potential to combat nonspecific background. Moreover, by using site-specific cleavage of a fluorescence-quenched probe by Endo IV, we can directly incorporate a real-time fluorescence-activation strategy for specific and efficient detection of the amplified DNA replicates. This provides an additional advantage of further signal amplification over current nickase-based amplification reactions, which generally rely on double-strand fluorescence staining13,18 or molecular beacon design19,20 for signal output. The analytical principle of the developed ERA technology for miRNA detection is illustrated in Scheme 1. A DNA template is designed to comprise four regions and two lesion incorporating sites: a target annealing region (T) that is complementary to a given fragment at 3′ end of target miRNA, a primer-annealing region (P1) that is used to hybridize with the primers for cyclic amplification, a reporter coding region (R) that is used to generate a reporter sequence complementary to the fluorescence-quenched signal probe, and a primer-producing region (P2) that has an identical sequence as P1 and is used for primer production in cyclic amplification. In the assay, the miRNA is annealed on the DNA template in the T region, which can act as a primer for Bst DNA polymerase to initiate DNA extension in the presence of four nucleotides dATP, dGTP, dCTP, and dUTP. (Note: dUTP is used instead of dTTP to incorporate lesions). The replication of the DNA template incorporates in the DNA extension product two uracil (dU) nucleotides. These two lesion sites are excised by UDG via catalyzing the hydrolysis of the N-glycosylic bond joining the uracil base to the deoxyribose,21,22 creating two abasic sites (AP site) that are then cleaved by Endo IV at the phosphodiester bond upstream to the abasic site.23 The repairing reaction, which generates a hydroxyl group at the 3′ terminus, will reprime the polymerase extension from the lesion site, thereby generating another copy of the DNA template and displacing the first copy away from the template. The repetition of the polymerase extension and the DNA repairing, therefore, results in a linear amplification of the DNA template into numerous copies of the primerproducing region P2 and the reporter coding region. The 6764
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a steep increase after ∼131 min. The background responses are ascribed to the primer-independent replication of the DNA template, which is frequently involved in polymerase reaction.30 Because the background responses required much longer time to reach the inflections than that for the positive sample, we could define the point of inflection (POI), the time corresponding to the maximum slope in the fluorescence curve, for the quantification. Accordingly, we obtained a POI value of 49 min for 1 nM target and a POI value of 131 min for the backgrounds. Such a big difference of the POI values revealed that the ERA technique enabled effective differentiation of target miR-21 from nonhomologous targets. In addition to the real-time fluorescence monitoring of the ERA reaction, the steady-state fluorescence spectra were also collected after 90 min. The data confirmed that the ERA amplification delivered a high signal-to-background ratio for target miRNA and a nonhomologous target (Figure S2 in the Supporting Information). Gel electrophoresis analysis also verified the effective amplification of the reporter sequences and the primers in the ERA reaction (Figure S3 in the Supporting Information). The ERA reaction enabled quantitative detection of target miR-21 using the real-time fluorescence curve, as shown in Figure 2. It was observed that the real-time fluorescence curves all displayed the sigmoidal shape, and the corresponding inflection regions appeared at shorter reaction time with increasing concentrations of target miRNA in a wide
Such nonspecific DNA amplification is attributed to the DNA polymerase mediated ab initio synthesis29 and elongation of DNA duplexes in which the short-sequence recognition site of nickase will be randomly incorporated and used as seeds for further elongation.16,17 The repeated cycles of nickase mediated digestion and polymerase based elongation thus allow exponentially amplification of nonspecific DNA under isothermal conditions. On the other hand, when DNA polymerase reacted with four nucleotides in the absence of nickase, no substantial DNA synthesis was obtained. This result implied that the cooperative reactions of nickase and DNA polymerase were essential to the nonspecific DNA synthesis. In contrast, for the ERA reaction in which DNA polymerase, UDG, and Endo IV were incubated with four nucleotides dATP, dGTP, dCTP, and dUTP, nonspecific DNA was not detectable, suggesting the ability of the ERA technology to effectively avoid nonspecific amplification. Presumably, the ERA reaction can incorporate in the ab initio synthesis of DNA many dU nucleotides that can be excised as lesion sites by UDG. This prevents the formation of long double-stranded DNA that can be used as templates for further amplification and hence avoids effective amplification in the absence of DNA template. Figure 1 depicts the typical real-time fluorescence curves of the ERA technology in the detection of miR-21. It was
Figure 1. Real-time fluorescence curves of ERA reactions. (a) 1 nM miR-21, 500 pM template, 100 nM signal probe, 0.05 U/μL Bst DNA polymerase, 0.08 U/μL UDG, 0.16 U/μL Endo IV, and 500 nM dNTPs (dATP, dGTP, dCTP, dUTP); (b) control with no DNA template; (c) control with no UDG; (d) control with no Endo IV; (e) control with no DNA polymerase; (f) control with dUTP replaced by dTTP; (g) control with no miRNA target; (h) control with miR-21 replaced by miR-141.
observed that, in the presence of miR-21 (1 nM), the fluorescence intensity increases rapidly after ∼49 min and reaches a plateau after ∼90 min, displaying a typical sigmoidal response (curve a). In contrast, there was no substantial fluorescence activation in the control experiments where one of the reagents in the ERA reaction was not added. This result verified the essential roles of the DNA template, the DNA repairing steps and DNA polymerase in the ERA reaction. In another control where dUTP was replaced by dTTP in the reaction mixture, there was also no appreciable fluorescence increase throughout the reaction, implying the incorporation of dUTP lesions in DNA replication was necessary for the ERA reaction. In the experiments with blank sample (miR-21 target not added in the reaction) or nonhomologous target (miR141), we obtained sigmoidal fluorescence intensity curves with
Figure 2. (A) Real-time fluorescence curves of ERA strategy for miR21 detection. (B) Correlation of POI values to logarithmic miR-21 concentrations. Error bars are standard deviations of four repetitive experiments. 6765
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lines, which was in good agreement with the data previously reported.33 In conclusion, we developed a novel isothermal nucleic acid amplification technology, enzymatic repairing amplification (ERA), based on cyclic enzymatic repairing and stranddisplacement polymerase extension for highly sensitive miRNA detection. Compared with commonly used nickase based nucleic acid amplification technologies, the ERA reaction provided comparable amplification efficiency with improved resistance to nonspecific amplification independent of primers and templates. When used for miRNA detection, the ERA strategy could be implemented in a single-step, closed-tube, and real-time detection format, enabling convenient operation and resistance to contaminants. The developed strategy was shown to give a detection limit as low as 0.1 fM for miRNA detection across a wide dynamic range up to 1 nM. Moreover, it was demonstrated that this technique showed excellent specificity with the capability of discriminating single-base mismatch and had the potential to be used for miRNA detection in real complex samples. In virtue of these advantages, the proposed ERA reaction indeed provided a new paradigm for efficient nucleic acid amplification and might hold the potential for miRNA expression profiling and related theranostic applications.
concentration range from 1 fM to 1 nM. A plot of the POI values versus the logarithmic concentrations of miR-21 showed linear dependency in the miR-21 concentration range from from 100 fM to 1 nM. The detection limit was calculated to be 0.1 fM according to the POI value 3 times the standard deviation over the blank response. Such a low detection limit implied a superior sensitivity of the ERA strategy in miRNA detection over existing methods.8,14,31,32 The improved sensitivity might be attributed to the low background of the ERA reaction as well as its high amplification efficiency and the additional signal amplification mediated by Endo IV-based cleavage. The relative standard deviations of POI values were 4.9%, 3.2%, and 1.14%, respectively, for four repetitive assays of miR-21 of 100 pM, 1 pM, and 10 fM, implying excellent reproducibility of the ERA strategy. The ERA reaction not only provided improved detection sensitivity but also afforded ideal specificity for target miRNA detection, as shown in Figure 3. It was found that non-
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ASSOCIATED CONTENT
* Supporting Information S
Experimental details and additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 86-731-88821916. Fax: 86-731-88821916. E-mail:
[email protected].
Figure 3. Specificity evaluation of ERA strategy for miRNA detection. Each miRNA has a concentration of 1 pM. ΔPOI is defined as the difference of the POI values between the positive sample and the blank.
Notes
The authors declare no competing financial interest.
homologous miRNA such as let-7a and miR-141 gave the same POI value as the blank. Moreover, we observed that miRNA with the single-base mismatch at the 3′ terminus also did not cause any interference, while single-base mismatch at other sites caused slightly decreased POI values (7. This high specificity with mismatch discrimination ability was derived from the miRNA-primed extension step in the ERA reaction, which was dominated by the perfect match at the 3′ end and highly dependent upon the hybridization stability between the target and the template. To demonstrate the capability of the ERA technology for miRNA detection in complex samples, miR-21 in total RNA extracts from five human cancer cell lines was analyzed using the developed strategy (Figure S4 in the Supporting Information). These results showed that the ERA strategy gave quantification data consistent with those obtained by RTqPCR (The maximum relative deviation was 11%), implying the potential of the developed ERA technology for miRNA quantification in real complex samples. In addition, it was revealed that the miR-21 showed different expression levels in these cancer cell lines with the human breast cancer cell line MCF-7 having the highest expression of miR-21 in all the cell
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ACKNOWLEDGMENTS
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REFERENCES
This work was supported by NSFC (Grants 21025521, 21221003, 21205034, 21035001, 21190041, 91317312) and the National Key Basic Research Program (Grant 2011CB911000).
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