Colorimetric PCR-Based microRNA Detection Method Based on Small

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Colorimetric PCR-based microRNA detection method based on small organic dye and single enzyme Juan Dong, Gangyi Chen, Wei Wang, Xin Huang, Huipan Peng, Qinlin Pu, Feng Du, Xin Cui, Yun Deng, and Zhuo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01111 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on May 31, 2018

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Analytical Chemistry

Colorimetric PCR-based microRNA detection method based on small organic dye and single enzyme Juan Dong, † Gangyi Chen, † Wei Wang, † Xin Huang, † Huipan Peng, † Qinlin Pu, † Feng Du, † Xin Cui, † Yun Deng‡* and Zhuo Tang†* †

Natural Products Research Center, Chengdu Institution of Biology, Chinese Academy of Science, Sichuan, Chengdu 610041, PR China ‡ State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resource, Chengdu University of TCM, Chengdu 611137, PR China *To whom correspondence should be addressed. Tel: +86 28 82890648; Fax: +86 28 82890648; Email: [email protected] Correspondence may also be addressed to Yun Deng. Tel: +86 28 61800234; Fax: +86 28 61800234; Email: [email protected]

ABSTRACT: microRNAs (miRNAs) have been a class of promising disease diagnostic biomarkers and therapeutic targets for their important biological functions. However, because of the high homology, interference from precursors (pri-miRNA, premiRNA) as well as limitations in the current assay technologies, it poses high demand and challenge for specific, efficient and economic miRNA assay method. Here, we propose a new miRNA detection method based on a label-free probe and a small organic dye with sequence dependence, realizing the sequence-specific and colorimetric detection of target miRNA. What is pleasantly surprised, only one enzyme is enough to propel the whole miRNA assay process, greatly simplifying the reaction component and detection process. Together with PCR amplification for the high enough sensitivity and three checks for specificity control, a detection limit of 5fM was obtained and even one mutation could be discriminated visually. Overall, the new method makes much progress in convenience and economy of PCR-based miRNA assay method so that miRNA assay is going to be more friendly and affordable.

microRNAs (miRNAs) are a class of small and endogenous RNAs about ~22nt in length which play an important role in the regulation of mRNA expression and finally influence a wide range of biological processes1. Recently, increasing studies have found that miRNAs have close relationship with tumour growth, progression, metastasis and drug resistance. And, a plenty of miRNAs have been demonstrated to be disease biomarkers for early diagnosis as well as timely and effective prognosis2,3. However, because miRNAs are highly homologous to each other, in addition to the interference from their precursors (pri-miRNA, pre-miRNA), it poses a huge challenge to miRNA assay methods for specific, effective and economic analysis of interest miRNA. RT-PCR, an exponential amplification method, has always been considered as a “gold standard” method for miRNA quantification because of its high sensitivity, specificity, and excellent ability in accurate quantification by fluorescence. The stem-loop RT-PCR4 is the most representative RT-PCR technology for miRNA detection. The predominant merit is its high specificity which comes from the ingenious and meticulous designs, including the stem-loop RT-primer, forward primer and TaqMan probe. TaqMan probe is a classic sequence-specific fluorescent probe chemically modified with fluorophore and quencher as well as a MGB-group5. It is the most significant part to verify the PCR amplification product and report the detection result. Take the exponential amplification merit of PCR together, the stem-loop RT-PCR is by far the well-known most sensitive, specific, and reliable miRNA

detection method for early disease diagnosis and prognosis. However, these advantages are at the cost of tedious twoenzymatic reaction, expensive fluorophore and MGB modification, as well as high dependence on advanced real-time fluorescent quantitative PCR machine, which limit its application in routine tests and screening. Therefore, a large family of label-free6,7, colorimetric8-12, electrochemical13-16 miRNA detection methods have been developed, which are almost isothermal amplification methods, in order to address the similar problems in RT-PCR. Even though these methods can be performed without precise control of temperature cycling, labelfree or reporting signal easily, the nature that the specificity control most relies on simple one-check hybridization between target miRNA and their recognition element makes them less reliable for the detection of miRNAs as disease biomarkers. DISC2(5) is one kind of cyanine derivative and was disclosed to be able to bind within the minor groove of dsDNA at sequences consisting of alternative adenine/thymidine (AT) pairs (such as ATATA/TATAT as well as its repeating units) to offer an obvious shifting from 647 to 590 at the maximum light absorption peak17, showing great colorimetric reporting potential. However, to our knowledge, no naked eyes observable colorimetric detection method for PCR product based on the sequence-specific interaction between the small organic dye DISC2(5) and dsDNA rich in alternative AT pairs (“ATdsDNA” for short) has been reported to date. Herein, we developed a new miRNA detection method based on the sequence-specific interaction between the small

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Figure 1. Schematic of the detection principle. Step 1: miRNA was recognized by fragment “c” of AT-probe and elongated through miRNA extension on AT-probe by RNA extension enzyme to form alternative AT-rich RNA-DNA chimera (CH); Step 2: taking CH as template, reverse primer (RP) elongated by TaqM1 DNA polymerase to obtain its cDNA (Ts); Step 3: PCR amplification of cDNA (Ts) with primer FP and RP, yielding duplex products (T/T’); step 4: adding the small organic dye DISC2(5) into PCR amplification products to report the detection result in colorimetric way through displaying different colors.

organic dye DISC2(5) and AT-dsDNA, on the one hand to provide a new choice for miRNA assay which could improve the convenience, visibility and economy of PCR-based miRNA detection methods, on the other hand to verify the ability and practicability of DISC2(5) for sequence-specific colorimetric report of PCR products rich in alternative AT pairs. Figure 1 illustrates the work principle of the new strategy. Step 1: A label-free stem-loop probe (AT-probe) composed of four fragments (a,b,c,d) was designed for the specific recognition and signal transformation of target miRNA. Fragment “a” (black) is an upturned head. Fragment “b” (prunosus) is a self-hybridized stem-loop region rich of alternative AT pairs. Fragment “c” (blue) is designed to be complement to the 3’-half of target miRNA. Fragment “d” (grey) is an additional unbound poly-A tail. In presence of miRNA, AT-probe could catch the target miRNA through hybridization of fragment “c” with 3’-part of target miRNA and form a RNA/DNA duplex, like the target recognition principle of most isothermal detection methods. In order to increase the specificity between target miRNA and AT-probe further, stem-loop structure was constructed in the way of self-hybridization of fragment “b”. The stem-loop structure was learned from the RT primer in RT-PCR which was demonstrated to be at least 100 more times higher in efficiency and specificity4. Unlike stem-loop RT-PCR to start the amplification using reverse transcriptase reaction, our strategy initiates the amplification through elongating miRNA on AT-probe with DNA polymerase to produce a RNA-DNA chimera sequence (CH). To start the amplification in this manner could exclude the interference of miRNA precursors (pri-miRNA, pre-miRNA) as well as those miRNAs with different or extra bases at 3’-end compared to the target miRNA effectively, because miRNA extension should

base on perfect complement between fragment “c” and 3’-half of miRNA. To meet this objective, the DNA polymerase needs not only the extension ability on DNA template with RNA primer, but also the 5’-3’ strand displacement activity to open the stem-loop structure, simultaneously without 3’-5’ exonuclease activity ideally. Step 2: We intended to reverse transcribe the sequence information of the elongated miRNA (CH) to its cDNA. In this process, CH/AT-probe duplex will be denatured by heating, and then a reverse primer (RP, same with fragment “a” of AT-probe in sequence) could bind to chimera sequence CH to carry on a successive reaction that the first is extension of primer (RP) taking DNA part of CH as template and the following is reverse transcription of RNA part of CH to synthesize the cDNA. To fulfill this job, a mutated Taq DNA polymerase (TaqM1) is applied because it owns both RNA reverse transcriptase activity and the 5’-3’ DNA polymerase activity18,19. Step 3: Once the sequence information of RNA-DNA chimera (CH) was copied to its cDNA (Ts), the final PCR template (T) could be synthesized by a 5’-end lengthened forward primer FP, taking Ts as template. Here, the 3’-part (blue) of FP is the same sequence of the left 5’-half of target miRNA. Only if the reverse transcribed sequence at 3’-end of Ts is originated from the left 5’part sequence of target miRNA, primer FP can bind to Ts correctly and extent to form the final PCR template (T), meaning that the forward primer FP is another specific check of target miRNA besides AT-probe (AT-probe checks the 3’-half of miRNA and FP checks the 5’-half). Then, in presence of template T, PCR amplification could be carried out by TaqM1, producing a large amount of PCR amplicons (T/T’ duplex). Step 4: As the sequence rich in alternative AT pairs has been


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Analytical Chemistry terval provided the best extension efficiency. Surprisingly, in the following trials, we found that TaqM1 is also capable of extension of RNA on DNA template to form the RNA-DNA chimera sequence, at relative lower temperature (30 oC) than the reported optimal extension temperature (60-70 oC) for DNA-primered DNA synthesis 19, by accident. Thus, the extension ability of Bsm, TaqM1 and Taq DNA polymerase for miRNA extension along AT-probe (Prs12) was compared at 30 oC (Figure 2A). According to the yield of the extension product (68nt, Figure 2B), TaqM1 revealed much weaker extension ability compared with Bsm DNA polymerase (Lane 2 vs Lane 3, Figure 2B), while Taq DNA polymerase was absolutely out of service (Lane 1, Figure 2B), to produce the RNA-DNA chimera sequence CH (Figure 2A). Nevertheless, once combining the extension step with following reverse transcription and PCR amplification in which TaqM1 would be still used, the two-enzyme system (Bsm and TaqM1) and the single enzyme system (TaqM1) produced almost the same amount of PCR amplicons (lane 4 vs lane 2, Figure 2C), indicating that miRNA amplification by the new strategy with only one enzyme (TaqM1) is totally feasible. Using the optimized single enzyme system, 100pM of miR21 was extended at 30 oC for 30min, and then amplified with PCR for 25cycles. Ultimately, after adding of 12mM DISC2(5) into the PCR

Figure 2. Verification of the strategy. (A) Schematic of the extension of 5’ isotope-labeled miR21 on AT-probe. (B) PAGE analysis of 5’ isotope-labeled miRNA extension by three different DNA polymerases. M: 25nt miR21 marker and 68nt miR21 extension product marker; Lane 1: Taq DNA polymerase; Lane 2: TaqM1; Lane 3: Bsm DNA polymerase; Lane 4: reaction without DNA polymerase. (C) miRNA amplification with two-enzyme system (Lane 3 and 4) and single enzyme system (Lane 1 and 2). M: DNA ladder; Lane 1,3: reactions without miR21(-ve PCR); Lane 2,4: reactions containing 100pM miR21(+ve PCR). (D) Colorimetric result (left) and light absorption spectrum (right) of the amplification reaction with single-enzyme, corresponding to lane 1 and 2 in Fig 2C. “+ve PCR”: tests with miRNA; “-ve”: tests without miRNA.

seeded in fragment “b” of AT-probe beforehand, all PCR amplicons originated from miRNA targets own the fragment rich in alternative AT pairs (red) too. Therefore, once DISC2(5) dye is added, samples with or without target miRNA could appear to be different in color and be colorimetric discriminated by naked eyes. Here, the sequence-specific interaction between DISC2(5) and PCR product containing AT-dsDNA could be considered as the third check to discriminate the right amplicons from non-specific PCR product. Moreover, a polyA tail has been added to 3’-end of AT-probe to prevent undesired extension during the whole process to further guarantee the specificity of the strategy. To prove the feasibility of the proposed strategy, miR21 was taken as the model target. Firstly, the extension of miR21 on AT-probe was investigated. We found that isothermal DNA polymerase (Bsm), with 5’-3’ strand displacement activity but no 3’-5’ exonuclease activity20, can successfully catalyze the extension reaction on AT-probe using miR21 as primer. Then, the screening of the optimal probe for extension was carried out. Five AT-probes for miR21 with different length of fragment “c” (10-14nt) and interv2al between “c” and “b” (0-6nt) has been tested. AT-Probe (Prs12) with 12nt “c” and 3nt in-

Figure 3. Sensitivity and practicability. (A) A series of concentrations of miR21 (0, 5fM, 50fM, 500fM, 5pM, 50pM) were amplified and analyzed by agarose gel electrophoresis. (B) Corresponding light absorption spectrum. (C) Light absorption values at 590nm were taken to plot against the concentration of miR21. (D) Discrimination of miRNA for its precursor (pre-miR21). Upper: sequence and structure information of pre-miR21, in which the red-marked is the sequence of miR21. Lower: colorimetric detection result of miR21, pre-miR21 and “-ve” (negative control with no miRNA). (E) Colorimetric analysis of miR21 in total RNA extract and cell lysate from HCC cell line HuH-7. “+ve” are tests with cell samples; “-ve” are tests without cell samples.



products amplified with single enzyme, the reaction with miR21 (+ve PCR) appeared to be blue and that without miR21 was light purple (Figure 2D-left). Correspondingly, the light absorption at 590nm showed an obvious increase when miR21 was detected (Figure 2D-right). Therefore, our new strategy is capable of colorimetric detection of miRNA with single enzyme. With the single enzyme promoted colorimetric detection strategy, a series of concentrations of miR21 (0, 5fM, 50fM, 500fM, 5pM, 50pM) were amplified three times in parallel to study the sensitivity and reproducibility. Amplification products were analyzed with agarose electrophoresis, and the band intensity reveals a graded enhancement as miR21 concentration increased (Figure 3A). The corresponding light absorption spectrum was recorded as well (Figure 3B). The values at 590nm were taken to plot against the concentration of miR21, showing good linear relationship and reproducibility (Figure 3C). The detection limit of 5fM miR21 ensured a satisfactory sensitivity for clinical samples21. To investigate whether the new strategy is effective to differentiate miRNA from its precursor, pre-miR21 containing the same sequence with miR21 (Figure 3D-upper) was chosen to be amplified under the same condition. As shown in Figure 3D-lower, the tube containing pre-miR21 afforded the same colorimetric result with negative control tube (-ve) containing no any targets. Basing on this positive premise, the performance of the new strategy for real sample analysis was investigated. HCC (Hepatocellular Carcinoma) cell line HuH7 that has been reported to be miR21 over-expressed22,23 was used as cell material. The cell samples were treated in two ways: one is to be extracted to get total RNA and another is to be lysed directly for the detection. miR21 in about 500ng total RNA extract and cell lysate from about 7x104 cancer cells were analyzed. As shown in Figure 3E, miR21 in both of the two mixtures could be easily tested with obvious blue color, indicating promising potential for practical application.

It is well known that miRNAs are highly homologous, which is quite hard to be discriminated from each other. Therefore, specificity is the most important index in miRNA detection method. To investigate the specificity of the new strategy, we took miR21 as the right target and several other similar miRNAs (Figure 4A) as interference factors. Among these miRNAs, R-miR21, S-miR21 and T-miR21 are mutated species that contain more than 3 mutations compare to miR21, providing the similar colorimetric result with that of negative control reaction (tube 5-7, Figure 4B-upper). R-miR21 that contains only one mutation at the 3’-half of sequence (U to A, Figure 4A), theoretically speaking, would be discerned mainly by AT-probe at the first checkpoint. S-miR21 that has one mutation at the 5’-half sequence (C to A, Figure 4A) could be recognized and extended on AT-probe but discriminated by FP primer at the second checkpoint. Though the detection tube containing R-miR21 (tube 4) or S-miR21 (tube 3) displays quite faint blue, it is quite easy to distinguish them from the positive result (tube 2 containing miR21) with the naked eyes (Figure 4B-upper). The light absorption of A590 in column chart (Figure 4B-lower) shows a consistent result. Those results verify that our designation for specificity control (ATprobe, forward primer, sequence-specific interaction between DISC2(5) and alternative AT-rich dsDNA as well as to start the reaction from miRNA extension) works effectively. In conclusion, the novel miRNA detection method described here appears to have several promising features for research and diagnostic applications as follows: (i) To our knowledge, the colorimetric detection of miRNA was realized for the first time by using the sequence-specific colorimetric detection method based on DISC2(5) and AT-dsDNA, resulting the visualization and cost-reduction; (ii) A pleasant surprise of the method is that only one single enzyme (TaqM1) is enough to fulfill the whole miRNA assay, simplifying the reaction component and detection process to a large extent; (iii) Three checks designed in the strategy not only eliminated interference from miRNA precursor, but also achieved visual discrimination of even one mutation, guaranteeing the high specificity of this method; (iv) A detection limit of 5fM was obtained, which could satisfy the requirement for clinical detection; (v) As the detection result was reported as colorimetric signal, the method now is suited for visual analysis, providing qualitative (a “yes or no” answer) detection. Even then, the quantitative detection could be realized by using a common microplate reader. This colorimetric detection method can also be widely applied in all PCR-based strategies. In general, the new method makes much progress in convenience and economy of PCR-based miRNA assay method, so that it should be suitable for high-throughput screening assays and point-of-care diagnostics, as long as normal or portable PCR machine is equipped.

ASSOCIATED CONTENT Supporting Information Details of material and instrument, sequence information, experimental protocol, condition improvement. Figure 4. Specificity investigation. (A) Sequence information of miR21, R-miR21, S-miR21, T-miR21, miR210, miR221. Red characters are mutations different from miR21. (B) Upper: photograph of the colorimetric detection of different miRNAs. Lower: bar graph plotted by light absorption value at 590nm, with each measurement done in triplicate. “NC” means negative control containing no miRNA target.

The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author


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Analytical Chemistry * Email: [email protected]; Phone: +86 28 82890648; Fax: +86 28 82890648.

Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT We gratefully acknowledge the financial supports from the National Natural Science Foundation of China [21402189, 21572222, 81373961], China Postdoctoral Science Foundation [2014M560733], West Light Foundation of The Chinese Academy of Sciences [Y4C3021100], Department of Science and Technology of Sichuan Province [2014RZ0022] and Chengdu Municipal Bureau of Science and Technology [2016-HM01-00371-SF, 2015-HM02-00099-SF].

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Analytical Chemistry

Figure 2. Verification of the strategy. (A) Schematic of the exten-sion of 5’ isotope-labeled miR21 on ATprobe. (B) PAGE analysis of 5’ isotope-labeled miRNA extension by three different DNA polymerases. M: 25nt miR21 marker and 68nt miR21 extension product marker; Lane 1: Taq DNA polymerase; Lane 2: TaqM1; Lane 3: Bsm DNA polymerase; Lane 4: reaction without DNA polymerase. (C) miRNA amplification with two-enzyme system (Lane 3 and 4) and single enzyme system (Lane 1 and 2). M: DNA ladder; Lane 1,3: reactions without miR21(-ve PCR); Lane 2,4: reactions containing 100pM miR21(+ve PCR). (D) Colorimetric result (left) and light absorption spectrum (right) of the amplification reaction with singleenzyme, corresponding to lane 1 and 2 in Fig 2C. “+ve PCR”: tests with miRNA; “-ve”: tests without miRNA. 101x120mm (300 x 300 DPI)


Colorimetric PCR-Based microRNA Detection Method Based on Small

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