Detection of 3′ → 5′ exonuclease activity using a metal-based luminescent switch-on probe

Methods 64 (2013) 218–223

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Detection of 30 ? 50 exonuclease activity using a metal-based luminescent switch-on probe Hong-Zhang He a, Weng-I Chan b, Tsun-Yin Mak a, Li-Juan Liu b, Modi Wang a, Daniel Shiu-Hin Chan a, Dik-Lung Ma a,⇑, Chung-Hang Leung b,⇑ a b

Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China

a r t i c l e

i n f o

Article history: Available online 21 August 2013 Keywords: G-quadruplex Luminescence Iridium Exonuclease Metal complex

a b s t r a c t A luminescent iridium(III) complex has been discovered to be selective for G-quadruplex DNA, and was employed in a label-free G-quadruplex-based detection assay for 30 ? 50 exonuclease activity in aqueous solution. A proof-of-concept of this assay has been demonstrated by using prokaryotic exonuclease III (ExoIII) as a model enzyme. In this assay, a G-quadruplex-forming hairpin oligonucleotide (hairpin-G4 DNA, 50 -GAG3TG4AG3TG4A2GCAGA2G2ATA2CT2C4AC3TC4AC3TC-30 ) initially exists in a duplex conformation, resulting in a low luminescence signal due to the weak interaction between the iridium(III) complex and duplex DNA. Upon digestion by ExoIII, the guanine-rich sequence is released and folds into a G-quadruplex, which greatly enhances the luminescence emission of the iridium(III) probe. This method was highly sensitive for 30 ? 50 exonuclease over other DNA-modifying enzymes. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction DNA-modifying enzymes play vital roles in fundamental biological processes such as replication, recombination, repair, and transcription, and are important for maintaining genomic stability and integrity [1–3]. In particular, enzymes containing 30 ? 50 exonuclease activities are involved in many important biological processes, such as DNA proofreading and repair [4–7]. 30 ? 50 exonuclease inhibitors have the potential to potentiate the action of DNA-alkylating antitumor drugs by inhibiting DNA repair [7–9]. At the same time, 30 ? 50 exonucleases have been utilized to provide signal amplification in oligonucleotide-based sensing platforms for the detection of various analytes [10–12]. Radioactive labeling in conjunction with gel electrophoresis is the most commonly-used technique for assaying 30 ? 50 exonuclease activity [13,14]. However, these protocols are generally discontinuous, time-consuming, require multiple steps, and necessitate the use of stringent safety procedures to control radiographic exposure. Consequently, it is desirable to develop efficient strategies to assay 30 ? 50

Abbreviations: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; ctDNA, calf-thymus DNA; ExoIII, exonuclease III; T7 Exo, T7 exonuclease; k Exo, lambda exonuclease; T4 PNK, T4 polynucleotide kinase; UDG, uracil-DNA glycosylase; BSA, bovine serum albumin. ⇑ Corresponding authors. E-mail addresses: [email protected] (D.-L. Ma), [email protected] (C.-H. Leung). 1046-2023/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymeth.2013.08.011

exonuclease activity, which could facilitate the screening of modulators of such enzymes as potential drugs and biochemical tools. DNA oligonucleotides have attracted tremendous interest for the construction of sensing platforms due to their low cost, ease of synthesis, high solubility, biocompatibility and stability in aqueous solution and biological media [15–37]. In particular, G-quadruplexes have attracted intense attention for the development of various analytical assays. The G-quadruplex is a non-canonical DNA secondary structure that consists of planar stacks of four guanines stabilized by Hoogsteen hydrogen bonding [38–40]. G-quadruplexes show a rich diversity in structural topologies that can be sensitive to several factors, such as base sequence, loop connectivity, or cations in solution [41]. The extensive structural polymorphism of G-quadruplexes has rendered them as versatile biological sensing elements for the construction of colorimetric, chemiluminescent, or fluorescent DNA-based sensing platform for metal ions, small molecules, and biomolecules [42–44]. In recent years, a number of luminescent DNA-based assays for 30 ? 50 exonuclease activity have been reported. For example, our group has developed a label-free, G-quadruplex-based switch-on fluorescence assay for 30 ? 50 exonuclease activity by using the organic dye crystal violet as G-quadruplex-binding probe [45]. Zhao and co-workers reported a fluorescence assay for monitoring 30 ? 50 exonuclease activity in living cells by employing a photoinduced electron transfer process between stacked guanine bases and a fluorescent probe [46]. Min and co-worker developed a fluorescence assay for the detection of 30 ? 50 exonuclease activity by

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using the graphene oxide and a singly-labeled oligonucleotide substrate [47]. These reports demonstrate that DNA oligonucleotides can be integrated as useful functional and structural elements for the construction of sensitive detection platforms for 30 ? 50 exonuclease activity. Meanwhile, luminescent transition metal complexes have found use in various chemical and biological sensors in view of their useful photophysical properties such as, (i) tunable excitation and emission maxima over the visible region; (ii) large Stokes shift for facile separation of excitation and emission wavelengths and elimination of self-quenching; and (iii) relatively long phosphorescent lifetimes that can be distinguished from a short-lived autofluorescence background through the use of time-resolved spectroscopy [48–52]. Luminescent transition metal complexes have been used to detect DNA [53], RNA [54], protein [55], small molecules [56], and metal ions [57]. For example, the group of Zhang utilized the ‘‘molecular light switch’’ complex [Ru(phen)2 (dppz)]2+ (where phen = phenanthroline and dppz = dipyrido[3,2a:2’,3’-c]phenazine) for mercury ion detection [58]. Our previous 30 ? 50 exonuclease activity assay was limited by the significant affinity of crystal violet for duplex DNA [59,60], which led to a high fluorescent background signal in the absence of enzyme. We envisaged that the conformational change of oligonucleotides induced by 30 ? 50 exonuclease could be more effectively monitored by luminescent transition metal complexes displaying a greater selectivity for the G-quadruplex motif over other DNA conformations. In this work, the luminescent cyclometallated iridium(III) complex [Ir(phq)2(BPhen)]+ (1, where phq = 2phenylquinoline and BPhen = bathophenanthroline, Fig. 1) was discovered to be selective for G-quadruplex DNA. We therefore employed complex 1 to develop a ‘‘mix-and-detect’’ assay for 30 ? 50 exonuclease activity that is simple, convenient, label-free, and that does not require expensive instrumentation or extensive sample preparation. 2. Materials and methods 2.1. Chemicals and materials Reagents were purchased from Sigma–Aldrich (St. Louis, MO) and used as received. Iridium chloride hydrate (IrCl3xH2O) was purchased from Precious Metals Online (Australia). All oligonucleotides were synthesized by Techdragon Inc. (Hong Kong, China) (Table 1). 2.2. General experimental Mass spectrometry was performed at the Mass Spectroscopy Unit at the Department of Chemistry, Hong Kong Baptist

Table 1 DNA sequences. Sequence Hairpin-G4 DNA PS2.M ss DNA Pu27 Pu22 Hairpin-G4 DNAm1

5-GAG3TG4AG3TG4A2GCAGA2G2ATA2CT2C4AC3TC4AC3TC-3 5-GTG3TAG3CG3T2G2-3 5-GA3T2CT2A2GTGCGATCGAG-3 5-TG4AG3TG4AG3TG4A2G2-3 5-GAG3TG4AG3TG4A2G-3 5-T2AGT3GTA4GCAGA2G2ATA2CT4ACA3CTA2-3

University, Hong Kong (China). Melting points were determined using a Gallenkamp melting apparatus and are uncorrected. Deuterated solvents for NMR purposes were obtained from Armar and used as received. 1 H and 13C NMR were recorded on a Bruker Avance 400 spectrometer operating at 400 MHz (1H) and 100 MHz (13C). 1H and 13 C chemical shifts were referenced internally to solvent shift (CD3CN: 1H d 1.94, 13C d 118.7; d6-DMSO: 1H d 2.50, 13C d 39.5). Chemical shifts (d) are quoted in ppm, the downfield direction being defined as positive. Uncertainties in chemical shifts are typically ±0.01 ppm for 1H and ±0.05 for 13C. Coupling constants are typically ±0.1 Hz for 1H–1H and ±0.5 Hz for 1H–13C couplings. The following abbreviations are used for convenience in reporting the multiplicity of NMR resonances: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. All NMR data was acquired and processed using standard Bruker software (Topspin). 2.3. Synthesis [Ir(phq)2(BPhen)]PF6 was synthesized according to the reported literature method [61] and was characterized by 1H NMR, 13C NMR and high resolution mass spectrometry (HRMS). [Ir(phq)2(BPhen)]PF6 (1). A suspension of [Ir2(ppyr)4Cl2] (0.2 mmol) and bathophenanthroline (0.44 mmol) in a mixture of DCM:methanol (1:1, 20 ml) was refluxed overnight under a nitrogen atmosphere. The resulting solution was allowed to cool to room temperature and filtered to remove unreacted dimer. To the filtrate was added an aqueous solution of ammonium hexafluorophosphate in excess, and the filtrate was reduced in volume by rotary evaporation until precipitation of the crude product occurred. The precipitate was then filtered and washed with several portions of water (2  50 ml) followed by diethyl ether (2  50 ml). The product was recrystallized by acetonitrile:diethyl ether vapor diffusion to yield the titled compound as an orange solid. Yield: 67%. 1H NMR (400 MHz, CD3CN): 8.61 (d, J = 8.0 Hz, 2H), 8.37 (q, J = 8.0 Hz, 4H), 8.22 (d, J = 8.0 Hz, 2H), 7.86 (s, 2H), 7.78 (d, J = 8.0 Hz, 2H), 7.75 (d, J = 8.0 Hz, 2H), 7.58–7.55 (m, 6H), 7.47–7.44 (m, 4H), 7.33–7.22 (m, 6H), 6.94–6.86 (m, 4H), 6.67 (d, J = 8.0 Hz, 2H); 13C NMR (400 MHz, CD3CN): 171.6, 152.6, 151.9, 149.8, 149.1, 148.6, 147.7, 141.5, 137.0, 136.0, 132.1, 131.9, 131.1, 130.5, 130.4, 129.9, 129.2, 128.9, 128.4, 128.1, 127.0, 125.6, 124.4, 119.5; MALDI-TOF-HRMS: Calcd for C54H36IrN4 [MPF6]+: 933.2568. Found: 933.2606. Anal. Calcd for C54H40F6IrN4O2P: C, 58.22; H, 3.62, N, 5.03. Found: C, 58.47; H, 3.50; N, 5.09. 2.4. Emission response of 1 toward different forms of DNA

Fig. 1. Chemical structure of the cyclometallated luminescent iridium(III) complex 1.

The G-quadruplex-forming sequences (PS2.M, Pu22, and Pu27) were annealed in Tris–HCl buffer (20 mM Tris, 100 mM KCl, pH 7.0) and were stored at 20 °C before use. Complex 1 (1 lM) was added to 5 lM of ssDNA, ctDNA or G-quadruplex DNA (PS2.M, Pu22, and Pu27) in Tris–HCl buffer (20 mM Tris, pH 7.0). Emission spectra were recorded in 510–700 nm range using an excitation wavelength of 360 nm.

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2.5. Emission titration for the detection of 30 ? 50 exonuclease activity in buffered solution

3.2. Principle of luminescent G-quadruplex-based probe for 30 ? 50 exonuclease activity detection

The random-coil oligonucleotide (hairpin-G4 DNA, 50 lM) was incubated in Tris buffer (20 mM, 100 mM NaCl, pH 7.0). The solutions were heated to 95 °C for 10 min, cooled to room temperature at 0.1 °C/s, and further incubated at room temperature for 1 h to ensure formation of the duplex substrate. The annealed product was stored at 20 °C before use. For assaying 30 ? 50 exonuclease activity, 50 lL of 1  NEBuffer 1 (10 mM Bis-Tris-Propane–HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.0) and the indicated concentrations of ExoIII were added to the solution containing the duplex substrate. The mixture was heated to 37 °C for 30 min to allow the oligonucleotide digestion reaction to take place. The mixture was allowed to cool and was diluted using Tris buffer (20 mM Tris, 50 mM KCl, pH 9.0) to a final volume of 500 lL. Finally, 0.5 lM of complex 1 was added to the mixture. Luminescence emission spectra were recorded on a PTI QM-4 spectrofluorometer at 25 °C. The luminescence emission intensity at 510–700 nm was monitored after excitation of the sample at 360 nm.

Our label-free G-quadruplex-based luminescent switch-on assay for 30 ? 50 exonuclease activity is illustrated in Scheme 1. The prokaryotic 30 ? 50 ExoIII was chosen as a model enzyme to demonstrate the proof-of-concept of our assay. ExoIII catalyzes the removal of mononucleotides from the 30 -terminus of doublestranded DNA [65]. However, ExoIII is unable to digest singlestranded DNA. We therefore designed a G-quadruplex-forming sequence (hairpin-G4 DNA, 5-GAG3TG4AG3TG4A2GCAGA2G2ATA2CT2C4AC3TC4AC3TC-3) consisting of a G-quadruplex-forming sequence at the 50 -terminus and its complementary cytosine-rich sequence at the 30 -terminus connected by a short linker region. Initially, hairpin-G4 DNA is self-hybridized to form a duplex structure. In the absence of ExoIII, the iridium(III) complex 1 interacts weakly with the DNA duplex, resulting in a low luminescence signal due presumably to non-radiative decay of the excited state of 1 by complex–solvent interactions. The addition of ExoIII results in digestion of the cytosine-rich strand from the 30 -terminus. However, ExoIII is arrested at the linker region as it is unable to accept single-stranded DNA as substrate. This results in the release of the guanine-rich sequence, which is able to fold into a G-quadruplex structure under the conditions employed. The nascent G-quadruplex is recognized by the iridium(III) complex 1 with an enhanced luminescence response, due to the selective interaction between complex 1 and G-quadruplex DNA.

3. Results and discussion 3.1. Characterization of complex 1 We initially examined the ability of complex 1 to interact with different forms of DNA by emission titration. Complex 1 exhibited weak luminescence in aqueous buffered solution (Tris 20 mM, 100 mM KCl, pH 7.0), presumably due to non-radiative decay of the excited state by complex–solvent interactions (Fig. 2). Minute changes in the luminescence signal of 1 was observed in the presence of calf-thymus DNA (ctDNA) and single-stranded DNA (ssDNA), indicating that the complex interacted weakly with duplex or random coil DNA structures. Intriguingly, a dramatic enhancement in the luminescence of 1 was observed in the presence of various G-quadruplexes, including PS2.M [62], Pu22 [63] and Pu27 [64] sequences. This result indicates that complex 1 is able to discriminate G-quadruplex DNA from duplex or random coil structures. To our knowledge, complex 1 has not yet been reported to be a G-quadruplex-selective luminescent probe. We envisage that the selective affinity of 1 for the G-quadruplex motif arises from the presence of the large aromatic ligands of the iridium(III) complex, which can form end-stacking interactions with the terminal guanine quartets.

Fig. 2. Luminescence response of complex 1 (1 lM) in 20 mM Tris buffer (pH 7.0) in the presence of 5 lM ssDNA, 5 lM ctDNA, 5 lM Pu27, 5 lM Pu22, and 5 lM PS2.M. Pu27, Pu22, and PS2.M were pre-annealed in Tris buffer (20 mM, 100 mM KCl, pH 7.0).

Scheme 1. Schematic representation of the G-quadruplex-based luminescence turn-on detection strategy for 30 ? 50 exonuclease activity using the G-quadruplexselective iridium(III) complex 1. In the absence of ExoIII, the G-quadruplex is not formed, and the emission of the iridium(III) complex 1 is low. However, in the presence of ExoIII, ExoIII will specifically digest the cytosine-rich sequence from the 30 -terminus in the duplex stem region, but is arrested at the linker region due to its inability to accept single-stranded DNA as substrate. The nascent G-quadruplex formed after the release of the guanine-rich sequence is subsequently recognized by the G-quadruplex selective iridium(III) complex 1 with an enhanced luminescence response.

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Fig. 3. (a) Luminescence spectra of the 1/hairpin-G4 DNA system in response to various concentrations of ExoIII: 0, 1, 2.5, 5, 10, 25, 50, 100, and 200 U/ml. Experimental conditions: hairpin-G4 DNA (0.25 lM) and 0.5 lM complex 1 in 20 mM Tris buffer (50 mM KCl, pH 9.0). (b) The relationship between luminescence intensity and ExoIII concentration. Inset: linear plot of the change in luminescence intensity at k = 560 nm vs. ExoIII concentration.

Fig. 4. Selectivity of the G-quadruplex-based 30 ? 50 exonuclease activity assay over other DNA-modifying enzymes and BSA. The concentration of ExoIII was 100 U/ml, the concentrations of the DNA modifying enzymes were 100 U/ml, and the concentration of BSA was 1.5 nM. Luminescence response of complex 1 (0.5 lM) with hairpin-G4 DNA (0.25 lM). Hairpin-G4 DNA (50 lM) was treated with ExoIII, T7 Exo, k Exo, T4 PNK, UDG or BSA at 37 °C for 30 min.

Table 2 Comparison of sensitivity for 30 ? 50 exonuclease activity assays recently reported in the literature. Method

Sensitivity

Signal output

Refs.

G-quadruplex-based fluorescence Photoinduced electron transfer process Graphene oxide

5 U/ml 0.04 U/ml

Fluorescent Fluorescent

[45] [46]

Not reported 1 U/ml

Fluorescent

[47]

Present study

Luminescent

3.3. Luminescent detection of 30 ? 50 exonuclease activity in aqueous solution We sought to employ 1 as a luminescent G-quadruplex-based probe for the detection of 30 ? 50 exonuclease activity in aqueous solution. We first investigated the luminescence response of the iridium(III) complex 1 (0.5 lM) and hairpin-G4 DNA (0.25 lM) to ExoIII. Upon the incubation with ExoIII, the luminescence intensity of 1 at kmax = 560 nm was significantly enhanced, with a ca. 15-fold enhancement in emission intensity at [ExoIII] = 200 U/ml (Fig. 3a)

and excitation intensity changes (Fig. S1). We hypothesize that the luminescence enhancement of the system is due to ExoIII digestion of hairpin-G4 DNA, which liberates the G-quadruplex motif that is recognized by complex 1. The duplex-to-quadruplex transition of a hairpin oligonucleotide upon digestion by ExoIII was demonstrated by CD spectroscopy in our previous work [45]. To maximize the performance of the 30 ? 50 exonuclease detection platform, we investigated the effect of various parameters on the luminescence response of the system to ExoIII, including the concentration of the hairpin-G4 DNA, the concentration of complex 1, and pH of the solution. It was observed that the luminescence response of the system was highest at 0.25 lM of hairpin-G4 DNA (Fig. S2). Similarly, 0.5 lM of complex 1 offered the highest luminescence fold-change response compared to 0.25, 1 or 1.5 lM of complex 1 (Fig. S3). On the other hand, it was observed that the luminescence response of the system was highest at a pH value of 9.0 (Fig. S4). Under the optimized conditions (0.5 lM complex 1, 0.25 lM hairpin-G4 DNA, 20 mM Tris buffer, 50 mM KCl, pH 9.0), the system exhibited a linear range of detection for ExoIII from 0 to 25 U/ml (Fig. 3b). The detection limit of this assay for ExoIII was estimated to be 1 U/ml at a signal-to-noise ratio (S/N) of 3 (Fig. S5), which is higher than that of the previous method using crystal violet as a fluorescent probe [45]. We propose that the greater sensitivity of the present method for ExoIII can be attributed to the superior selectivity of the iridium(III) complex 1 for the G-quadruplex motif compared to crystal violet. Crystal violet also shows significant binding to duplex DNA, resulting in a higher fluorescent background signal. We envisage that the lower background of the present system, afforded by the extremely weak interaction between 1 and hairpin-G4 DNA, allows for a greater fold-change response when 30 ? 50 exonuclease is added. To verify that the observed luminescence enhancement was due to the formation of the G-quadruplex caused by the digestion of hairpin-G4 DNA by ExoIII, a number of control experiments were conducted. We incubated complex 1 with ExoIII in the absence of hairpin-G4 DNA substrate. However, up to 200 U/ml of ExoIII induced only minute changes in the luminescence of 1, suggesting that the complex did not interact with ExoIII directly (Fig. S6). We also designed a hairpin DNA sequence (hairpin-G4 DNAm1) that is unable to form G-quadruplex structures in the presence of ExoIII. As expected, only a slight enhancement in the luminescence of 1 was observed at 100 U/ml (Fig. S7). This result suggested the luminescence enhancement of the system originated from the specific interaction of 1 with the G-quadruplex motif.

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3.4. Selectivity of G-quadruplex-based 30 ? 50 exonuclease activity assay The selectivity of our approach for 30 ? 50 exonuclease activity assay was evaluated by investigating the response of the system to other DNA modifying enzymes such as lambda exonuclease (k Exo), T7 exonuclease (T7 Exo), T4 polynucleotide kinase (T4 PNK), and uracil-DNA glycosylase (UDG). The results showed that only ExoIII could significantly enhance the luminescence emission of the complex 1/hairpin-G4 DNA system (Fig. 4). By comparison, no significant change in emission intensity was observed upon the addition of the other DNA-modifying enzymes. These results indicate that the system displays significant selectivity for ExoIII over DNA modifying enzymes, originating from the specific digestion reaction of ExoIII on the hairpin substrate from cytosine-rich stand at the 30 -terminus. We also investigated the robustness of this strategy toward other chemical species likely to be present in biological samples, such as bovine serum albumin (BSA). The assay showed a slight response to 1.5 nM of BSA, with a relative luminescence response about 20% that of ExoIII at 100 U/ml. 4. Conclusion In conclusion, we have discovered that the iridium(III) complex 1 could function as a G-quadruplex-selective luminescent probe. We utilized complex 1 to develop a label-free G-quadruplex-based assay for 30 ? 50 exonuclease activity. Our strategy for 30 ? 50 exonuclease activity assay is based on the high selectivity of complex 1 for G-quadruplex DNA over duplex and random coil DNA. In the absence of 30 ? 50 exonuclease, the oligonucleotide substrate adopts a duplex confirmation that interacts only weakly with complex 1. However, the luminescence of the system is dramatically enhanced upon the addition of 30 ? 50 exonuclease due to the structural transition of the duplex structure into a G-quadruplex topology induced by the specific digestion of the substrate at the 30 -terminus by 30 ? 50 exonuclease. This assay is highly selective for 30 ? 50 exonuclease over other DNA-modifying enzymes. This label-free G-quadruplex-based luminescence switch-on platform is cost-effective compared to conventional radiographic or other luminescent assays, which typically require relatively costly isotopic or fluorescent labeling. For easier access, a comparison showing the sensitivity of detection of ExoIII of recently reported analytical techniques have been summarized in Table 2. A notable advantage of complex 1 is its long-lived 3MLCT phosphorescent emission in the visible region (lifetime = 2.39 ls) [61] compared to organic Gquadruplex probes such as zinc protoporphyrin IX (ZnPPIX) (lifetime = 3 ns) [66]. This allows the emission of complex 1 to be readily distinguished from background fluorescence arising from endogenous fluorophores in the sample matrix by use of time-resolved luminescence spectroscopy. We envision that our newly developed method could facilitate the development of label-free G-quadruplex-based assays targeting other DNA-modifying enzymes for use in biochemical and biomedical research. Acknowledgments This work is supported by Hong Kong Baptist University (FRG2/ 11-12/009 and FRG2/12-13/021), Centre for Cancer and Inflammation Research, School of Chinese Medicine (CCIR-SCM, HKBU), the Health and Medical Research Fund (HMRF/11101212), the Research Grants Council (HKBU/201811, HKBU/204612, and HKBU/ 201913), the French National Research Agency/Research Grants Council Joint Research Scheme (A-HKBU201/12), the Science and Technology Development Fund, Macao SAR (001/2012/A), and the University of Macau (MYRG091(Y2-L2)-ICMS12-LCH and MYRG121(Y2-L2)-ICMS12-LCH).

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