Metallointercalators for Real-time Monitoring of DNA Amplification

chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assays are commercially available, most of them rely on organic parent molecule...
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Letter Cite This: Anal. Chem. 2019, 91, 8777−8782

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Structurally Defined Ru(II) Metallointercalators for Real-Time Monitoring of DNA Amplification Reactions Qinfeng Xu,*,† Jing Dong,† Xiya Ma,† Yanni Zhao,† Chen-chen Li,‡ and Chun-yang Zhang*,‡ †

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School of Food and Biological Engineering, National R&D Center for Goat Dairy Products Processing Technology, Shaanxi University of Science and Technology, Xi’an 710021, P. R. China ‡ College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, P. R. China S Supporting Information *

ABSTRACT: The low cost and convenience of fluorescent DNA binding dyes make them widely used for real-time DNA analysis. Even though several dyes for real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assays are commercially available, most of them rely on organic parent molecules with an indefinite chemical structure, making it difficult to predict their behavior in nucleic acid amplification and to get the best performance. Herein, we demonstrate for the first time the use of the structurally defined dipyridophenazine complexes of ruthenium(II) for real-time monitoring of PCR and LAMP. These inorganic metallointercalators exhibit less inhibition on DNA amplification. They can quantify a range of initial template concentrations and provide the product melting curve to differentiate the unspecific amplification and even perform multiplex assays. These ruthenium(II) complexes have the advantages of the defined structure, nontoxicity, good stability, and facile synthesis, and they may act as excellent alternative DNA binding dyes with wide applications in the field of nucleic acid research. eal-time polymerase chain reaction (PCR) was first developed in 1993 by Higuchi et al.1 and has become a powerful tool with wide application in the fields of basic research, clinical diagnosis, food safety, and environment monitoring.2,3 Due to the exponential amplification power of PCR and the fluorescent monitoring of amplification progress in a closed-tube, this technology is able to amplify, identify, and quantify the target DNA sequence in a single step with excellent sensitivity, repeatability, rapidity, low contamination risk, and ease of operation. The fluorescent double-stranded DNA (dsDNA) binding dyes and fluorophore-labeled probes (e.g., TaqMan probe and molecular beacon) are usually used as the reporters for real-time PCR.2 In comparison with the fluorophore-labeled probes, the dyes display less specificity since their binding to dsDNA is nonspecific,4,5 but they can offer greater simplicity, universality, and reduced cost and even allow for multiplex PCR6 and high-resolution melting genotyping.7 Although a variety of fluorescent dsDNA dyes (e.g., SYBR Green, EvaGreen, LC Green, TOTO-3, POPO-3 and SYTO family of dyes) have become commercially available, only a limited number of dyes (e.g., SYBR Green I (SGI),5 EvaGreen,8 LC Green7 and SYTO dyes9−11) are suitable for real-time PCR, because the dyes should meet following three requirement: (1) have a preferential binding for the amplified dsDNA to generate a fluorescence signal, (2) do not inhibit the

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© 2019 American Chemical Society

PCR reaction, and (3) be chemically stable under overall thermal PCR cycles.8,10 All these characteristics are dominated by the chemical structure of dyes, but the exact structures of organic dyes are undisclosed and kept a trade secret.11 Only the empirical data available make it difficult to predict the behavior of organic dyes in nucleic acid amplification and to explore their better performance.10,11 Therefore, the development of new structure-defined dyes for real-time PCR is highly desirable. As an alternative to organic dsDNA binding dye, metal ion complexes may provide an attractive set of DNA binding and optical properties.12,13 Ruthenium(II)−dipyrido [3,2-a:2′,3′-c] phenazine (dppz) complexes (Ru−dppz complexes) are known as the molecular light switches for DNA, and they possess distinct advantages of strong fluorescence enhancement upon DNA binding (>104), water-soluble, and chemically stable.12,14 The Ru−dppz complexes have been used for molecular recognition15−18 and bioanalysis,19−26 in vivo imaging,27−31 and anticancer drug development,31−33 but they have not been explored in real-time PCR, except for the real-time electrochemical PCR/LAMP using osmium−dppz complexes.34−37 We postulate that the enhanced fluorescence Received: May 13, 2019 Accepted: July 3, 2019 Published: July 3, 2019 8777

DOI: 10.1021/acs.analchem.9b02244 Anal. Chem. 2019, 91, 8777−8782

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Analytical Chemistry

widely used organic dyes,1,5 except for the use of real-time PCR instrument with the excitation channel of 400−500 nm and emission channel of 550−750 nm for Ru−dppz dyes (Figure S1). Three ruthenium(II) complexes (Scheme 1, bottom) including [Ru(bpy)2dppz]2+ and its derivatives ([Ru(phen)2dppz]2+ and [Ru(phen)2dppz(CH3)2]2+ (bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline)) were synthesized. All them display the well-known “light switch” effect, but the diverse ancillary ligands (bpy or phen) and the intercalating ligands (dppz or dppz(CH3)2) endow them with different “light switch” efficiencies42,44,46 and DNA binding affinities,43,47,48 which may affect their performance in real-time PCR reaction. We investigated the luminescence stability of these Ru− dppz complexes under thermal PCR cycling and excitation conditions. These Ru−dppz dyes can provide stable fluorescence signal under high temperature and continuous excitation, and their stability is much higher than that of organic dye SGI (Figure S2). The inhibition effect of these Ru−dppz complexes upon PCR process was investigated by gel electrophoresis analysis of the end-PCR products. The PCR amplification of a 231-bp DNA fragment of the foodborne pathogen (Staphylococcus aureus, S. aurues (S. aur)) was performed in the presence of various concentrations of [Ru(bpy)2dppz]2+. As shown in Figure 1a, the obvious inhibition is observed when the concentration of [Ru(bpy)2dppz]2+ is more than 6.0 μM, which is much higher than the SGI dye (>1.6 μM, Figure S3), suggesting that the Ru−dppz dye exhibits less inhibitory effect on PCR amplification. This inhibition may be ascribed to the strong binding of [Ru(bpy)2dppz]2+ with dsDNA, which can hinder the replication of DNA by polymerase.8,34 Nevertheless, a higher dye concentration can induce a higher fluorescence

intensity, the DNA binding affinity, and the binding selectivity of Ru−dppz complexes in real-time PCR can be modulated by the modifications of ancillary ligands38−40 and intercalating ligands.41−44 The principle of metallointercalator-based real-time PCR and their chemical structures are shown in Scheme 1. These Scheme 1. Principle of Real-Time Fluorescence Monitoring of PCR Amplification Using Three Structurally Defined Ruthenium(II) Metallointercalators

inorganic DNA binding dyes have negligible fluorescence in aqueous solution due to the quenching effect from the hydrogen bonding between water and the dppz intercalating ligand, but they do display intense emission in the presence of the amplified dsDNAs which can protect the dppz ligand from water.12,23,45 With the addition of these dyes to the PCR reaction mixture, the kinetic progress of DNA amplification can be monitored for the detection of initial DNA concentration. This principle is analogous to that of the

Figure 1. Feasibility vindication of [Ru(bpy)2dppz]2+ in real-time PCR. (a) Gel image of PCR amplification products in response to various concentrations of [Ru(bpy)2dppz]2+. (b−d) Real-time monitoring of PCR amplification in the absence (S. aur−) and presence (S. aur+) of DNA template, (b) fluorescence emissions recorded during the PCR amplification, (c) corresponding amplification curves by plotting the fluorescence intensity at 625 nm as a function of cycle number, and (d) negative first derivative melting curves and gel image (inset) of PCR products. 8778

DOI: 10.1021/acs.analchem.9b02244 Anal. Chem. 2019, 91, 8777−8782

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Figure 2. Effect of ligand structures of Ru(II) metallointercalator upon the performance of real-time PCR. The amplification curves (a) and their corresponding cycle threshold (Ct) values (b), the melting curves (c), and their corresponding fwhm (full width at half-maximum) (d) in response to various concentrations of Ru-1 ([Ru(bpy)2dppz]2+), Ru-2 ([Ru(phen)2dppz]2+), Ru-3 ([Ru(phen)2dppz(CH3)2]2+), and SYBR Green I (SGI).

verify that the metallointercalator [Ru(bpy)2dppz]2+ can be used for real-time PCR assay. We further investigated the effect of three Ru(II) metallointercalators on PCR amplification inhibition and melting temperature. All Ru(II) metallointercalators exhibit concentration-dependent fluorescence emission and eventually level off with a delayed amplification (Figure 2a). The delayed cycle threshold (Ct) values demonstrate the inhibitory effect of highconcentration Ru(II) metallointercalators on the amplification (Figure 2b). The threshold concentration that can generate a high fluorescence signal without inhibiting PCR is 6.0 μM for [Ru(bpy)2dppz]2+, consistent with the value obtained by electrophoresis analysis (Figure 1a). The threshold concentration is 4.0 μM for [Ru(phen)2dppz]2+ and 2.5 μM for [Ru(phen)2dppz(CH3)2]2+, consistent with the results obtained by gel analysis (Figure S4). Among them, [Ru(bpy)2dppz]2+ gives the narrowest melting peak and the minimal nonspecific amplification (Figure 2c,d). Notably, all

signal. Thus, it is required to balance the dye concentrationdependent inhibitory effect and the detectable signal response. [Ru(bpy)2dppz]2+ (3.0 μM) was added to the PCR reaction mixture, and the accumulation of amplified DNAs was realtime monitored. The fluorescence emission of [Ru(bpy)2dppz]2+ enhances with the increasing cycles of PCR amplification in the presence of DNA template, but little fluorescence enhancement is observed even at the PCR cycle >30 in the absence of DNA template (Figure 1b). A characteristic exponential amplification curve is obtained when plotting the fluorescence intensity at the maximum emission wavelength against the PCR cycle number (Figure 1c). In addition, the melting temperature (Tm) of the specific 231-bp amplicon is defined at 82.3 °C, and the melting peak from the nonspecific amplification of primer dimers is observed at 70−75 °C (Figure 1d), suggesting the differentiation of specific amplicon from the nonspecific products can be easily achieved through melting curve assay. These results clearly 8779

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Figure 3. Quantitative (a,b) and multiplexed (c,d) real-time PCR assays using [Ru(bpy)2dppz]2+ as the DNA-binding dye. (a) Amplification curves in response to various initial copies of S. aur DNA template in three replicates and (b) the linear plot of Ct values against the logarithm of DNA copies. (c,d) Amplification (c) and melting (d) curves of the duplex PCR assay in response to the singleplex S. aureus, C. sak, and duplex S. aur + C. sak, respectively. NTC (no template control) shows the absence of any targets.

Figure 4. Application of [Ru(bpy)2dppz]2+ for real-time and multiplexed LAMP: (a) schematic principle of real-time LAMP, (b) real-time amplification curves in response to a serial of initial copies of S. aur DNA, and (c,d) amplification (c) and melting (d) curves of duplex LAMP assay in response to singleplex S. aur, C. sak, and duplex S. aur + C. sak, respectively.

dimer,8,10 and the Ru(II) metallointercalators can be exploited for real-time PCR application due to their defined structure and tunable affinity to both ssDNA and dsDNA by ancillary modification.40 To further evaluate the feasibility of Ru(II) complexes to quantify target DNA, a series of parallel amplification curves in response to the various initial DNA copies are observed (Figure 3a), similar to those obtained by conventional SGI dye (Figure S5b). These amplifications exhibit good reproducibility

three Ru(II) metallointercalators exhibit less inhibition and narrower melting peak than the SGI organic dye (Figure 2b−d, Figure S3). This ligand structures-dependent PCR performance can be explained by their different affinities to ssDNA and dsDNA with an order of [Ru(bpy) 2 dppz] 2+ < [Ru(phen)2dppz]2+ < [Ru(bpy)2dppz(CH3)2]2+.43,47,48 The high affinity of Ru(II) metallointercalators to dsDNA and ssDNA may lead to their strong inference and inhibition on DNA replication as well as the ease of formation of primer− 8780

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DNA-binding reporters for various nucleic acid assay applications.

(Figure 3a) and good specificity (Figure S5a). Moreover, the Ct value is linearly dependent on the logarithm of initial DNA copies with a correlation coefficient of 0.9980 (Figure 3b). The detection limit is calculated to be 10 copies for [Ru(bpy)2dppz]2+ and is comparable with that obtained by using SGI dye (10 copies, Figure S5b). The [Ru(bpy)2dppz]2+ complexes are suitable for real-time multiplexed PCR due to their narrower melting peaks8 (the fwhm is 2.07 °C for [Ru(bpy)2dppz]2+ and 4.45 °C for SGI dye) (Figure 2d). As a proof-of-concept, we used S. aur and Cronobacter sakazakii (C. sakazakii, C. sak), which have resolvable Tm values, as two DNA targets. As shown in Figure 3c, it is not possible to distinguish amplicons between S. aur and C. sak from the amplification curves. Nevertheless, a single distinguishable melting peak appears in the melting curves of target S. aur or C. sak alone, and both peaks are observed for the mixture of S. aur and C. sak targets (Figure 3d). There is neither false-positivity nor cross-reactivity observed (Figure 3d), and the result is consistent with the gel electrophoresis analysis (Figure S6). These results clearly demonstrate that the [Ru(bpy)2dppz]2+ can be used to simultaneously measure multiple target sequences in a single reaction. The Ru(II) metallointercalator can be further applied for the monitoring of isothermal LAMP (Figure 4a). LAMP has the potential to provide a simple screening assay for the point-ofcare,49,50 which requires the real-time and closed-tube detection of templates and the elimination of crosscontamination.51,52 Because [Ru(bpy)2dppz]2+ with a concentration of more than 2.0 μM exhibits an obvious inhibition effect on LAMP amplification and both [Ru(phen)2dppz]2+ and [Ru(phen)2dppz(CH3)2]2+ can efficiently inhibit LAMP amplification at any concentration (Figure S7), the real-time LAMP was carried out in the presence of 1.0 μM [Ru(bpy)2dppz]2+. Distinct signal enhancement in the amplification curve is observed in the presence of S. aur DNA template, but no obvious enhancement is observed in the absence of S. aur DNA template (Figure 4b). For the detection of pathogen S. aur, a low detection limit (10 copies) and a large linear range from 10 copies to 107 copies are obtained, superior to SGI dye (Figure S8). Moreover, the duplexed LAMP assay can be easily achieved by the melting curve analysis of LAMP amplicons with distinct Tm values (Figure 4c,d), which is further verified by gel electrophoresis analysis (Figure S9). These results clearly demonstrate that [Ru(bpy)2dppz]2+ can be used for real-time multiplexed LAMP. In summary, we have demonstrated that Ru(II) metallointercalators can function as efficient fluorescent reporters for real-time monitoring of PCR and LAMP amplification. These Ru(II) metallointercalators are chemically stable and exhibit no inhibition on the amplification process at detectable concentration. The initial template of 10 copies can be successfully quantified, and the detection sensitivity may be down to single-copy DNA with the structure optimization of the Ru(II) complexes. The defined structure of Ru(II) metallointercalators together with the ancillary/intercalating ligand-dependent properties provides plenty of room for designing more efficient metallointercalators for real-time monitoring of the DNA amplification reaction. For example, the modification of ancillary ligands with carboxylic acids will improve the binding selectivity between dsDNA and ssDNA,40 and the methyl substituents of dppz ligand will increase the quantum yield.44 We believe that this finding will benefit the DNA research community who are searching for new ideal

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b02244.

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Details of materials and instruments, sequence information, experimental protocols, and additional plots (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Chun-yang Zhang: 0000-0002-8010-1981 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (Grant Nos. 21735003, 21527811, 21405169, and 31800328), the Hundred Talent Program of the Shaanxi Province (Grant SXBR9230), the Science and Technology Program of Shaanxi Province (Grants 2018KJXX-043, 2018JQ3017, and 2019NY-126), start-up funds from Shaanxi University of Science and Technology (Grant 2016QNBJ-01), and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China.

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