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Fluorometric Detection of MicroRNA using Isothermal Gene Amplification and Graphene Oxide Chaesun Hong, Ahruem Baek, Sang Soo Hah, Woong Jung, and Dong-Eun Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00046 • Publication Date (Web): 23 Feb 2016 Downloaded from http://pubs.acs.org on February 25, 2016
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Analytical Chemistry
Fluorometric Detection of MicroRNA using Isothermal Gene Amplification and Graphene Oxide Chaesun Hong,a Ahruem Baek,a Sang Soo Hah,b Woong Jung,c Dong-Eun Kim*,a a b
Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea Department of Chemistry and c Department of Emergency Medicine, Kyung Hee University, Seoul 130-701, Korea
ABSTRACT: We have developed a facile fluorometric system for the detection of microRNA (miRNA), using rolling circle amplification (RCA), graphene oxide (GO), and fluorescently labeled peptide nucleic acid (F-PNA). The padlock probe DNA complementary to a target miRNA was selectively ligated to form circular DNA that was then used as the template for RCA. F-PNAs complementary to the target miRNA were annealed to multiple sites of the isothermally amplified single-stranded RCA product (RCAP) containing multiple target miRNA sequences. This F-PNA/RCAP duplex is less adsorbed onto the GO monolayer, thus attenuating the quenching of F-PNA fluorescence by GO. In the absence of target miRNA (and hence the absence of RCA and duplex formation), the free F-PNA is completely adsorbed onto the GO monolayer and fluorescence quenching ensues. Thus, GO-based fluorescence detection coupled with isothermal gene amplification would be a simple and convenient method for the quantitative detection of miRNA.
MicroRNAs (miRNAs) are single-stranded (ss) small noncoding ~25-nucleotide RNAs that play important roles as endogenous gene regulators by binding to the 3’ untranslated region of target mRNAs, causing target mRNA degradation and regulation of protein expression. Because they are involved in various biological processes, such as cell proliferation, differentiation, stress resistance, and cell death, miRNAs are closely associated with the pathogenesis of human diseases, including malignant tumors.1-4As miRNAs are present in serum, plasma, and whole blood, their detection is important for further understanding of their biological functions and for the diagnosis of cancer.5,6 The northern blot technique7 and microarrays8,9 as well as real-time PCR10, the standard miRNA detection methods currently used, have limitations, such as low sensitivity, low selectivity, and labor-intensive steps. The unique characteristics of miRNAs, including their short length, low abundance, and sequence homology among the miRNA family, also make them difficult to analyze. Recently, several amplification methods for miRNA, using chemical or enzymatic modifications, have been developed to improve on assay sensitivity and selectivity, including fluorescence labeling assay11-13 and nanoparticle-amplified detection.14,15 In particular, rolling circle amplification (RCA) is commonly used for the detection of DNA, RNA, and protein. RCA is an isothermal enzymatic process used to synthesize long ssDNA molecules with tandem repeats. Owing to its simplicity and high sensitivity and specificity, RCA is regarded as a promising DNA amplification tool in various applications such as biosensing, diagnosis, and genomics.16 Graphene oxide (GO) is a water-soluble derivative of graphene, consisting of carbon, oxygen, and hydrogen. GO shows high affinity to single-stranded nucleic acids through hydrogen bonds and pi-stacking interactions, whereas it has weak affini-
ty to double-stranded nucleic acids because the nucleobases are hidden inside the double helix.17,18 In addition, GO efficiently quenches nearby fluorescence via long-range energy transfer from the fluorophore to the pi-system of the GO molecule.19 Owing to these properties, We have recently used GO as fluorescence quenching material for discerning fluorescently labeled single-stranded oligonucleotide from doublestranded nucleic acids.20-22 Peptide nucleic acids (PNAs), which are artificially synthesized nucleic acids similar to DNA or RNA,23 was previously used as probe oligonucleotide in the GO-based assay.24 The lack of the phosphate group in PNA allows it to hybridize strongly with its complementary nucleic acid, by the elimination of charge repulsion during hybridization.25 Thus, in our study, single-stranded RCA product (RCAP) containing the amplified miRNA sequences was to be sensitively detected with fluorescently labeled PNAs (F-PNAs) and GO as the oligonucleotide probe and fluorescence quencher, respectively.
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Scheme 1. Schematic illustration of the miRNA detection process using the graphene oxide (GO)-based fluorometric assay combined with RCA-based miRNA amplification.
The miRNA intended for detection is isothermally amplified using RCA (Scheme 1). Herein, a rolling circle template (RCT) acting as the padlock probe DNA is designed to be complementary to both ends of the target miRNA. In the presence of target miRNA, the RCT would hybridize with it to be used as a template, and circularization is achieved by the addition of T4 DNA ligase. RCA is initiated by phi29 DNA polymerase, using the circular DNA as the template, and miRNA now becomes the primer for DNA synthesis. As RCA progresses, a long ssDNA sequence is synthesized, bearing multiple copies of the target miRNA sequence. However, in the absence of target miRNA, circular DNA formation and synthesis of the long ssDNA by phi29 DNA polymerase do not take place. The RCAP thus synthesized in the presence of target miRNA is then subjected to detection via the fluorescence-quenching platform, using GO and F-PNA (Scheme 1). The F-PNA, designed to be complementary to the target miRNA, can then be annealed to multiple sites of the RCAP to form an FPNA/RCAP duplex molecule. With F-PNA/RCAP hybrid formation in the presence of miRNA, the annealed duplex form of the F-PNA probe cannot adsorb onto the GO monolayer, and thus the F-PNA fluorescence is not quenched. When the target miRNA is not present and its corresponding RCAP is thus not formed, the free single-stranded F-PNA probes would adsorb onto the GO monolayer surface, and significant fluorescence quenching by GO would ensue. Thus, miRNAs can be quantitatively detected simply by measuring the fluorescence signal after adding GO to the reaction mixture. Before the assay was employed, atomic force microscopy (AFM) was used to verify the uniform monolayer structure of the GO used in this study (Supporting Figure S1). We selected miRNA21 as the target miRNA for optimization of the experimental conditions, because miRNA21 is overexpressed in many lung-cancer patients.26 miRNA16 was used as a reference miRNA for data normalization.6 We first examined for formation of the circular DNA as the template for RCA. Circular DNA was formed only in the presence of the target miRNA (miRNA21) via ligation of the linear DNA (RCT21) complementary to the target miRNA (Figure 1a). Because both ends of RCT21 were complementary to miRNA21, the end segments of RCT21 were connected by the ligation reaction, forming the circular DNA. In contrast, when RCT21 was mixed with miRNA16, only a single RCT21 band was observed (lane 3). We checked further for circular DNA formation using digestion with Exonuclease I, which digests ssDNA in a 3′-to-5′ direction but does not digest doublestranded or circular DNA. RCT21 was degraded upon addition of Exonuclease I to the ligation reaction mixture (lane 4); the band corresponding to circular DNA remained intact (lane 2). This indicates that circularization of RCT specifically occurred in the presence of target miRNA. We next examined whether the target miRNA sequence was specifically amplified through ligation and subsequent RCA. Ligation and RCA were carried out in the presence of target miRNA21 or non-target miRNA16. A large, high-molecularweight DNA molecule was observed only in the presence of miRNA21, suggesting that the RCAP generated by RCA was a long ssDNA containing possibly multiple copies of the target
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miRNA21 sequence (Figure 1b, lane 3). In contrast, this large DNA product was not observed in the absence of miRNA21 or in the presence of non-target miRNA16 (lanes 1 & 2, respectively). These results suggested that the ligation and RCA reaction only occurred in the presence of the padlock template and corresponding miRNA with complementary sequences. To validate our GO-based fluorometric detection system, we monitored the fluorescence change in FITC-labeled PNA21 (F-PNA21; complementary to miRNA21) by adding various RCAPs and GO to the F-PNA21 mixture (Figure 1c). When GO was added to a solution containing F-PNA21 only, fluorescence was markedly quenched. In contrast, addition of GO to the F-PNA21/RCAP21 duplex did not result in fluorescence quenching. Interestingly, when the F-PNA was hybridized to corresponding ssDNA, PNA fluorescence was more enhanced than that of the ssPNA (data not shown). As a negative control, RCAP16 with multiple copies of the miRNA16 sequence was also incubated with F-PNA21 (which was not complementary to RCAP16). The fluorescence of the F-PNA21 and RCAP16 mixture was significantly quenched in the presence of GO, as compared with the F-PNA21 and RCAP21 mixture. In addition, F-PNA21 fluorescence in the RCA reaction mixture without miRNA21 was also quenched to a similar extent as RCAP16, setting a background fluorescence.
Figure 1. Validation of the graphene oxide (GO)-based fluorometric assay combined with rolling circle amplification (RCA)-based miRNA amplification. (a) Analysis of rolling circle template (RCT) circularization. RCT21 was mixed with miRNA21 (target miRNA) or miRNA16 (non-target miRNA). o After incubation at 25 C for 1 h, linear DNA in the mixture was degraded by adding Exonuclease I. The reaction product was analyzed by denaturing 15% PAGE. (b) Agarose gel electrophoresis images for the RCA product (RCAP). RCA was conducted in the presence or absence of miRNA21 (target miRNA) with RCT21. In addition, RCA was performed in the presence of miRNA16 (non-target miRNA). The RCAP was analyzed by 1% agarose gel electrophoresis and visualized with ethidium bromide staining. M: molecular weight marker DNAs. (c) Fluorescence emission spectra (λex= 495 nm) of 2
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FITC-labeled PNA21 in the presence or absence of RCAP (16 or 21) or RCT after incubation with GO.
To augment the fluorescence signal difference between the background fluorescence coming from the F-PNA probes and the unquenched fluorescence of the F-PNA probes hybridized to the target miRNA sequence, the optimal concentration of GO was determined to be 3–10 µg/mL (Supporting Figure S2). Next, to evaluate the sensitivity of the GO-based miRNA detection method, fluorescence intensity was measured at increasing miRNA21 concentrations (0–10 nM). Figure 2 shows the relationship between fluorescence intensity and the logarithm of miRNA21 concentrations. The fluorescence intensity of RCAP21 increased gradually with increasing miRNA concentration, showing a good linear correlation from 1 fM to 50 pM (Figure 2b). The limit of detection (LOD) was estimated from the equation LOD = 3.3 × (SD/S) with a 1% confidence level,23 where SD is the standard deviation of the response and S is the slope of the standard curve in the linear region. The LOD was calculated to be 0.4 pM, which indicates that our method can detect miRNA in amounts as low as 0.4 pM. The amount of miRNA21 was also quantified in a mixture of cellular RNA. Total cellular RNAs were prepared from cultivated lung cancer cells (A549 cells), and a 1-µg sample was then spiked with synthetic miRNA21 at increasing concentrations (0–100 nM), following which the fluorescence intensity of F-PNA complementary to RCAP21 was measured. The F-PNA fluorescence quantitatively increased with increasing logarithm of miRNA21 concentrations in the cellular RNA mixture, showing a good linear correlation from 5 fM to 50 pM (Supporting Figure S3). The LOD of the GO-based miRNA detection for the total RNA sample was 0.7 pM. This result was compared with quantitative real-time PCR (qRT-PCR) analysis, using the same sample containing total cellular RNA with various concentrations of miRNA21 (Supporting Figure S4). The GO-based miRNA detection system was superior to qRT-PCR for quantitative detection of target RNA at low concentrations (less than 100 pM). This result indicates that miRNA can be quantitatively detected by the GO-based miRNA detection through isothermal amplification of miRNA sequences by RCA with high sensitivity unattainable with contemporary qRT-PCR.
Figure 2. Sensitivity of the graphene oxide (GO)-based fluorometric miRNA detection. (a) Fluorescence emission spectra (λex = 495 nm) obtained from the RCAP21/F-PNA21 hybrid in the presence of GO, upon addition of miRNA21 at different concentrations (0–10 nM). The maximal emission fluorescence at 518 nm was measured. (b) The linear relationship between the fluorescence intensity (λem = 518 nm) and the logarithm of target miRNA concentrations in the range from 1 fM to 0.5 pM. Error bars reflect three separate measurements.
To assess specificity, our detection method was tested using miRNA21, miRNA16, and two other miRNAs (miRNA31 and miRNA155). Padlock template DNAs (RCT) and corresponding PNA probes with different fluorophores were designed for the amplification and detection of the different miRNAs. When each RCT was incubated with its corresponding target miRNA, large high-molecular-weight DNA molecules (RCAP) were readily synthesized through RCA, as determined by agarose gel electrophoresis (Supporting Figure S5). To further test the specificity of the RCT to its corresponding target miRNA, each RCT or a mixture of RCTs (RCT21, RCT31, and RCT155) was used in the ligation and RCA reaction with a mixture of miRNAs (miRNA21, miRNA31, and miRNA155). Strong fluorescence signals were selectively observed in the RCA reaction containing the appropriate RCT designed for annealing to each target miRNA and its corresponding fluorescent PNA probe (Figure 3a). Next, to test the specificity of each fluorescent PNA probe, a mixture of PNAs labeled with different fluorophores (FITCPNA21, ATTO550-PNA31, and Cy5-PNA155) was incubated with one or a mixture of RCAPs. Strong fluorescence signals were selectively observed only for the target RCAP hybridized to its corresponding fluorescent PNA (Figure 3b). These results demonstrated that multiple probes with different fluorophores could be used to allow multiplexed detection of different targets in a single reaction vessel using appropriate fluorescence detection channels.
Figure 3. Specificity of the graphene oxide (GO)-based fluorometric miRNA detection system, tested in a multiwell plate. The upper panel shows the reaction scheme used to test the miRNA detection specificity. The relative fluorescence intensity was obtained by setting the fluorescence of PNA probe in the presence of GO with and without the corresponding RCT or RCAP pair as “100” and “0”, respectively. (a) A mixture of miRNAs (miRNA21, miRNA31, and miRNA155; 10 nM each) was incubated with diverse rolling circle target (RCT) DNAs, and RCA was subsequently carried out. An F-PNA probe (labeled with different fluorophores: FITC-PNA21, ATTO550PNA31, and Cy5-PNA155) corresponding to each RCT was added to the reaction mixture, and GO was added for fluo3
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rescence quenching in a 96-well plate. The fluorescence signal was quantified using a multiplate reader. (b) A mixture of the three F-PNA probes was incubated with their respective RCAP (path (b) in the reaction scheme). (c) Multiplexed detection of miRNA in a multiwell plate. Fluorescence images of multiwell plates containing the respective RCT DNA and F-PNA probe for each miRNA to be detected. The fluorescence image was visualized using a fluorescence imaging system (IVIS-Lumina II).
We applied our miRNA detection strategy to a multiplex detection mode, in which optical fluorescence images were monitored and quantified in 96-well plates. Each padlock DNA corresponding to a different target miRNA was placed in each well for miRNA annealing, ligation and subsequent RCA reaction. After the RCA, as described in the experimental section (Supporting Information), the RCA product was mixed with FITC-labeled PNA (F-PNA) and the annealing between RCAP and F-PNA was allowed to occur. An aliquot of each reaction mixture was transferred to a 96-well plate containing GO (5 µg/mL). The RCT and F-PNA set for detection of each miRNA were arranged in separate rows. Either single miRNA or mixtures of miRNA samples were tested for detection in each column, and the fluorescence intensities were visualized using a fluorescence imaging system (Figure 3c). Strong fluorescence signals were selectively observed in wells containing the target miRNAs amplified and hybridized with the corresponding F-PNAs. However, the fluorescence in multiwell plates containing non-target miRNA was quenched as no duplexes were formed.
Figure 4. Selectivity of the graphene oxide (GO)-based fluorometric miRNA detection system, assessed using mutant miRNAs containing single-base mismatches. (a) Sequences of miRNA21 (wild-type miRNA21 and mutant-type miRNA21) and their corresponding RCT21 DNA (blue-colored sequence). Bases differing from the miRNA21 sequence are marked in red. (b) Agarose gel electrophoresis image for the rolling circle amplification product (RCAP), analyzed on 1 % agarose gel and visualized with ethidium bromide staining. RCAPs were synthesized by using each miRNA (10 nM). The RCAP band was observed for the wild-type miRNA21 only. (c) Analysis of the multiwell plate fluorescence signal for respective reactions using RCT21 DNA and F-PNA21 probe in the presence of GO. The graph inset shows a fluorescence image
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of the 96-well plate, where the fluorescence was quantified using a multilabel plate reader (λex = 485 nm and λem = 535 nm).
We have further attempted to distinguish miRNA sequences with high homology (1 or 2-nt difference) to verify the specificity of our GO-based fluorometric method. Single-base mismatches were introduced into the target miRNA, at the binding site of the 5′ or 3′ end of the padlock DNA template (miRNA21_G and miRNA21_A, respectively, in Figure 4a). Ligation and RCA were performed using RCT21 with either target miRNA21 or miRNA21 containing the single-base mismatch. miRNA16 (non-target miRNA) was also used as a negative control. As shown in Figure 4b, the high-molecular-weight product was observed in the presence of wild-type miRNA21 but not of non-target miRNA16 or miRNA21s containing a single-base mismatch. RCAPs were generated in the presence of each miRNA type and fluorescence was measured with FPNA21 complementary only to miRNA21 in a 96-well plate. Strong fluorescence was observed only for wild-type miRNA21; fluorescence for the miRNA21s containing a singlebase mismatch was similar to that of miRNA16. This result indicates that the target miRNA was readily distinguished from other miRNAs with a single-base difference. Thus, our GO-based fluorometric detection method can be applied for the quantitative detection of high-homology miRNA sequences. In conclusion, we have developed a high-sensitivity, highselectivity method to detect miRNA by coupling a GO-based fluorometric assay with RCA-based miRNA amplification. By observing differences in fluorescence intensity between target and non-target miRNAs, we have verified that the ligation of padlock probe DNA and the subsequent RCA reaction are target-specific. In addition, miRNA can be quantitatively detected at amounts as low as 0.4 pM. This assay has several advantages over previously reported methods that have detection limitations and low sensitivity, and are complicated and time-consuming. First, via isothermal amplification (i.e., RCA), a small amount of miRNA can be amplified to a long stretch of ssDNA harboring multiple copies of the target miRNA sequence. In the absence of target miRNA, RCA will not occur because the circular DNA for amplification is not formed, thereby ensuring the specificity of the system. Second, the GO-based fluorescence-quenching platform enables us to easily detect the presence of miRNA by simply evaluating the presence or absence of fluorescence in a 96-well plate, without requiring other cumbersome processes. Third, the high specificity and selectivity of our assay enables the efficient detection of multiple miRNAs in a mixture, using a simple 96-well format that can be completed within an hour. This GO-based fluorescence detection system combined with RCA-based miRNA amplification would be a simple and convenient method for detecting cancer-related miRNAs for the diagnosis of diseases such as lung cancer.
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ASSOCIATED CONTENT Supporting Information Experimental methods, materials information, and supplementary data figures (PDF). This material is available free of charge on the ACS Publications website.
AUTHOR INFORMATION
(23) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O.; Science. 1991, 254, 1497-1500. (24) Park, J. S.; Baek, A.; Park, I.-S.; Jun, B.-H.; Kim, D.-E. Chem. Commun. 2013, 49, 9203-9205. (25) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature. 1993, 365, 566-568. (26) Si, M.-L.; Zhu, S.; Wu, H.; Lu, Z.; Wu, F.; Mo, Y.-Y. Oncogene. 2007, 26, 2799-2803.
Corresponding Author *E-mail:
[email protected]; Tel.: +82-2-2049-6062
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This research is supported by a National Research Foundation grant funded by the Korean Government (NRF2014R1A2A1A-11051361).
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