Evaluation of DNA Methyltransferase Activity and Inhibition via

Herein, we developed an isothermal, enzyme-free and autonomous strategy for analyzing ... evaluation of DNA MTase and fast screening its inhibitors ar...
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Evaluation of DNA Methyltransferase Activity and Inhibition via Isothermal Enzyme-Free Concatenated Hybridization Chain Reaction Qing Wang, Min Pan, Jie Wei, Xiaoqing Liu, and Fuan Wang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00168 • Publication Date (Web): 14 Jun 2017 Downloaded from http://pubs.acs.org on June 19, 2017

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Evaluation of DNA Methyltransferase Activity and Inhibition via Isothermal Enzyme-Free Concatenated Hybridization Chain Reaction Qing Wang, Min Pan, Jie Wei, Xiaoqing Liu, Fuan Wang* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, P. R. China *

To whom correspondence should be addressed. E-mail: [email protected] Keywords: Sensor, Enzyme-free, Amplification, Isothermal, Methylation ABSTRACT: Methyltransferase (MTase)-catalyzed DNA methylation plays a vital role in biological epigenetic process of key diseases and has been attracted increasing attentions, making the amplified detection of MTase activity of great significance in clinical disease diagnosis and treatment. Herein, we developed an isothermal, enzyme-free and autonomous strategy for analyzing MTase activity based on concatenated hybridization chain reaction (C-HCR)-mediated Förster resonance energy transfer (FRET). In a typical C-HCR procedure without MTase (Dam), Y-shaped initiator DNA activates upstream HCR-1 to assemble doublestranded DNA (dsDNA) copolymeric nanowire consisting of multiple tandem DNA trigger units that motivate downstream HCR-2 to successively bring a fluorophore donor/accepter (FAM/TAMRA) pair into close proximity, leading to the generation of an amplified FRET readout signal. The target Dam MTase and auxiliary DpnI endonuclease can sequentially and specifically recognize/methylate and cleave the Y-shaped initiator oligonucleotide, respectively, and thus prohibits the C-HCR process and FRET signal generation, resulting in the construction of a signal-on sensing platform for MTase assay. Our proposed isothermal enzymefree C-HCR amplification approach was further utilized for screening MTase inhibitors. Furthermore, the proposed C-HCR approach can be easily adapted for probing other different MTases and for screening the corresponding inhibitors just by changing the recognition sequence of Y-shaped initiator DNA through a “plug-and-play” format. It provides a versatile and robust tool for highly sensitive detection of various biotransformations and thus holds great promise in clinical assessment and diagnosis.

DNA methylation represents one of the most significant and critical epigenetic process and plays a crucial role in the regulation of gene transcription and expression in both eukaryotes and prokaryotes.1,2 The methylation reaction involves a sequence-specific methyltransferase (MTase)-catalyzed methyl transfer from S-adenosyl-L-methionine (SAM) to adenine or cytosine residues in the recognition sequence of DNA substrate.3,4 DNA methylation always executes a function of gene protection. However, aberrant DNA methylation is proven to associate with various human diseases,5,6 such as systemic lupus erythematosus,7 leukemia,8 hodgkin lymphoma9 and even fatal cancers.10,11 Consequently, DNA methylation has been recognized as a new generation of cancer biomarkers and the corresponding DNA MTases are regarded as a new family of pharmacological targets for antitumor therapy.5,12 In addition, the inhibition of DNA methylation is demonstrated to reactivate apoptotic pathways, making cancer cells sensitized to previously ineffective chemotherapies. Therefore, accurate evaluation of DNA MTase and fast screening its inhibitors are of great significance in clinical diagnosis and treatment.13 Conventional methods for detecting DNA MTase mainly include methylation-specific polymerase chain reaction (MSP),14 methylation-specific restriction enzyme polymerase chain reaction (MS-RE-PCR),15 and bisulfite sequencing pol-

ymerase chain reaction (BSP).16 However, these indirect sensing approaches need tedious bisulfite pretreatment and laborious PCR, and lack the sensitivity for analyzing trace amount of MTase derived from precious tumor samples. Recently, a straightforward MS-qFRET17,18 (methylation-specific quantum dot fluorescence resonance energy transfer) or MethylBEAMing19 (beads, emulsion, amplification, and magnetics) technology was proposed to facilitate the direct detection of low-abundance MTase. Meanwhile, several techniques have been developed for MTase assay, including gel electrophoresis,20 radioactive labeling,21 high-performance liquid chromatography (HPLC),22 and electrochemical method.23 However, these methods are discontinuous, time-consuming, or operational inconvenient as they usually require radio-labeled substrates. Hence, various nanomaterial-amplified biosensing methods have been developed for detecting DNA MTase. For example, MTase-responsive DNA-modified gold nanoparticles (AuNPs) were prepared for optical MTase assay based on DNA methylation-mediated AuNPs assembly.24 Similarly, graphene oxide (GO)/DNA nanocomposite was constructed for evaluating MTase activity based on the methylationinvolved detachment of the fluorophore-labeled DNA substrate from GO surface and the subsequent recovery of the fluorescence of DNA.25 Nonetheless, these MTase-targeting 1

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nanobiosensors are suffered with insufficient sensitivity and tedious preparation of nanomaterials. To overcome the limitations of these methods described above, different enzymemediated isothermal signal amplification strategies, such as rolling circle amplification (RCA),26 strand displacement amplification (SDA),27 polymerase-assisted signal amplification28 and exonuclease-assisted signal amplification29,30 have thus been constructed for amplified MTase assay. These enzymemediated amplifications provide a rapid and efficient tool at a constant temperature without thermocycling. Unfortunately, these enzyme-involved schemes have the limitations of operating under conditions that might otherwise inhibit protein enzymes. Thus, there is still an urgent demand to develop isothermal, sensitive, selective and enzyme/nanomaterial-free sensing platforms for MTase assay. Hybridization chain reaction (HCR) is a characteristic isothermal, enzyme/nanomaterial-free signal amplification strategy. Conventional HCR always involves the nucleic acidtriggered autonomous cross-opening of two DNA hairpins into long dsDNA copolymers, and it has been successfully utilized for detecting metal ions,31 proteins32,33 and nucleic acids34-36 analytes. Here, we demonstrated a novel concatenated hybridization chain reaction (C-HCR)-mediated Förster resonance energy transfer (FRET) for measuring DNA MTase and for screening its inhibitors. A Y-shaped DNA initiator substrate was specifically designed to be selectively methylated/cleaved by DNA MTase and restriction endonuclease, and was intentionally introduced to trigger the autonomous C-HCR system. In the absence of MTase and endonuclease, initiator-motivated HCR-1 produced numerous tandem triggers for HCR-2 that successively bring the fluorophore accepter (TAMRA) and donor (FAM) into closed proximity to generate FRET signal, thus amplifying the fluorescence readout signal. DNA MTase and auxiliary endonuclease can successively methylate and cleave the Y-shaped initiator oligonucleotide, thus prohibiting the C-HCR process and FRET signal generation, leading to the construction of a signal-on sensing platform for MTase. It is a general straightforward MTase assay since only the initiator substrate needs to be replaced with an appropriate sequence that can be recognized by other sequence-specific biotransformations through a “plug-and-play” fashion. By taking advantage of the signal amplification features of synergistic HCR, we anticipate that C-HCR can provide an important tool for MTase assay and thus should hold great promise for accurate MTase sensing and reliable MTase inhibitors screening. MATERIAL AND METHODS Reagents. Dam and M.SssI MTases, DpnI and HpaII restriction endonucleases, and S-adenosyl-L-methionine (SAM) were purchased from New England Biolabs (Ipswich, MA, USA). Gentamycin and 5-fluorouracil were purchased from Sigma-Aldrich (Beijing, China). The water used throughout the study was purified by using a Milli-Q apparatus (Millipore, Bedford, MA). All oligonucleotides used in this study were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) (Table S1, Supporting Information). Other chemicals with analytical grade were purchased from Sigma-Aldrich (Beijing, China). These nucleic acids and enzymes stock solutions were stored at -20 °C before use. Fluorescence Assay. All the assays were prepared in reaction buffer (10 mM HEPES, 1 M NaCl, and 50 mM MgCl2, pH 7.2). Each DNA hairpin (4 µM) was heated to 95 °C for 5 min and then allowed to cool to room temperature (25 °C) for

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at least 2 h before use. For sensitive detection of DNA by means of C-HCR scheme, DNA initiator was introduced into the H1 + H2 + H3 + H4 + H5 + H6 mixture (100 nM each) to trigger the self-assembly process at 25 °C. Unless specifically indicated, all of the control experiments were performed without changing the concentration of DNA. The fluorescence measurements were performed by using a Cary Eclipse spectrometer (Varian Inc). The emission spectra were acquired by exciting the samples at 490 nm. Time-dependent fluorescence changes were recorded at a fixed wavelength of 520 nm. C-HCR-based Dam or M.SssI MTase Assay. The Y-type initiator substrate was prepared by annealing a mixture consisting of 10 µM I1 and 10 µM I2 in annealing buffer (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, and 1.0 mM EDTA, PH 7.5) at 95 °C for 5 min and then cooling down slowly to room temperature. Then a MTase reaction mixture consisting of 400 nM initiator substrate, 160 µM SAM, 5 U DpnI or HpaII auxiliary endonuclease, and Dam or M.SssI MTase was incubated in 1×CutSmart buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 0.1 mg/ml BSA, pH 7.9) at 37 °C for 1 h. To achieve the best performance, the incubation time and temperature of Dam MTase were optimized. 10 µL of each MTase-incubated mixture was added to 190 µL of H1 + H2 + H3 + H4 + H5 + H6 mixture in reaction buffer, and the resulting mixture was kept at room temperature (25 °C) to execute the C-HCR process and to acquire the respective fluorescence spectra. C-HCR-based Dam MTase inhibitors evaluation. For Dam MTase inhibition evaluation, the procedure was similar to the aforementioned Dam MTase assay, except an additional inhibitor was introduced into the first MTase incubation step. The relative activity (RA) of Dam MTase was defined as (RA) = [F(inhibitor) – F0]/ [F(no inhibitor) – F0] in which the F(inhibitor) and F(no inhibitor) referred to the fluorescence emission intensity of FAM with and without inhibitor, respectively, and F0 referred to the fluorescence emission intensity of C-HCR system with initiator DNA. Gel Electrophoresis Verification of Dam MTasemediated DNA Methylation. The gel electrophoresis sample consisting of 0.4 µM initiator DNA, 1 U of Dam MTase, 5 U of DpnI and 160 µM SAM was incubated in 1×CutSmart buffer at 37 °C for 1 h. 10 µL of the sample was then mixed with loading buffer and loaded into the notches of the freshly prepared 12% native polyacrylamide gel. Electrophoresis was performed at a constant voltage of 100 V in 1×TBE buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 8.3) for 3 h. The gel was then stained with GelRed and imaged by FluorChem FC3 (ProteinSimple, USA) under 365 nm irradiation. RESULTS AND DISCUSSION Construction of Concatenated Hybridization Chain Reaction. Herein, we proposed a concatenated hybridization chain reaction (C-HCR)-mediated Förster resonance energy transfer (FRET) to evaluate DNA MTase activity and inhibition. As illustrated in Figure 1A, this C-HCR strategy is composed of two concatenated hybridization chain reactions, upstream HCR-1 (consisting of hairpins H1 and H2, see Table S1) and downstream HCR-2 (consisting of hairpins H3, H4, H5 and H6, see Table S1). H2 is elongated with two isolated DNA segments d and e at its 5'- and 3'-ends, respectively. Only through HCR-1 approach then the adjacent DNA segments d 2

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Figure 1. (A) Scheme for the isothermal concatenated hybridization chain reaction (C-HCR). (B) Time-dependent fluorescence changes of the C-HCR system shown in Figure 1(A) without (a) and with (b) 20 nM initiator I. (C) Fluorescence spectra generated by the C-HCR system outlined in Figure 1(A) in the absence of the initiator (a), and upon analyzing 20 nM initiator (b). The system consisting of H1 + H2 + H3 + H4 + H5 + H6 mixture (100 nM each) was carried out in reaction buffer for a fixed time interval of 2 h.

and e can be brought together and converted as intermediate trigger for HCR-2. H3 is functionalized at its 3'-end with a fluorescence acceptor (TAMRA) while H5 is modified at its 5'-end with a fluorescence donor (FAM). Only through HCR-2 approach then the two fluorophores (FAM and TAMRA) can be brought into close proximity that enables the Förster resonance energy transfer (FRET) process and generates an amplified FRET readout signal. Initiator DNA-motivated HCR-1 propagates a cross-opening reaction between H1 and H2 to consequently assemble dsDNA copolymeric nanowire and to concomitantly bring the separated segments d and e into close proximity, resulting in the formation of tandem DNA triggers to initiate HCR-2. Each of the HCR-1-generated DNA trigger contributes to the subsequent alternating hybridization of H3, H4, H5 and H6, yielding a long DNA concatamer carrying a large number of adjacent FAM and TAMRA, producing a remarkable FRET signal. Note that HCR-2 can be simplified and designed to include two hairpins as HCR-1, while a sophisticated and expensive internal modification of the hairpins is usually needed to readout the HCR system. The isothermal C-HCR, consisting of six hairpin components, stays metastable in the absence of the initiator, due to the firmly formation of the hairpin structures. The synergistic effect of HCR-1 and HCR-2 thus generates amplified fluorescence signal for analyzing various biologically important biomarkers. In a primary design, all six hairpins were theoretically (by Mfold software) 37 and experimentally optimized to avoid any

unexpected hybridizations. It is necessary to protect the toehold or/and binding regions of output sequences of upstream HCR-1 in order to prevent their possible interactions with downstream HCR-2 before exposure. The proof-of-concept demonstration of the optimized C-HCR strategy was first examined. As shown in Figure 1B, no fluorescence change was observed in the absence of the initiator I (curve a), indicating these hairpins are metastable without obvious signal leakage (spontaneous hybridization chain reactions). However, a dramatically decreased fluorescence was observed for initiator I and it leveled off after ca. 2 h (curve b). Accordingly, the resulting fluorescence spectra were recorded after 2 h (Figure 1C). This tremendous decreased fluorescence of initiator Itriggered C-HCR is attributed to synergistic effect of HCR-1 and HCR-2, indicating the C-HCR-mediated successive crossopening of the hairpin mixtures and a concomitant effective FRET generation. The performance of the H6-excluded CHCR system (as conventional HCR control) was also examined for analyzing the same initiator (details see Figure S1). As compared with C-HCR system, a moderate signal response was observed for conventional HCR system (Figure S2), which was reasonable since the amplification efficacy of the C-HCR (1/N2) was overwhelmingly enhanced over that of the traditional HCR (1/N). AFM and gel electrophoresis experiments were further carried out to verify the two-layered CHCR circuit and their individual upstream HCR-1 and downstream HCR-2 systems (see Figures S3 and S4). This, on the 3

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Figure 2. (A) Schematic representation of the methylation/cleavage of the initiator substrate and the subsequent C-HCR readout. (B) Fluorescence spectra generated by the C-HCR-based MTase sensing platform outlined in Figure 2(A) in the presence of 20 nM initiator I (a), 20 nM initiator I, 5 U/mL Dam MTase and 25 U/mL DpnI (b), 20 nM initiator I1 (c) and 20 nM initiator I2 (d). (C) Native gel electrophoresis characterization of C-HCR-based MTase assay as shown in Figure 2(A): (a) hairpins H1-H6, initiator I, Dam MTase and DpnI; (b) hairpins H1-H6 and initiator I; (c) hairpins H1-H6. Unless specified, the system consisting of H1 + H2 + H3 + H4 + H5 + H6 mixture (100 nM each), initiator I (20 nM), Dam (5 U/mL) and DpnI (25 U/mL) were carried out in reaction buffer for a fixed time interval of 2 h.

other hand, demonstrates the robustness of our C-HCR system. These results clearly demonstrate the successful implementation of C-HCR that leads to the significant amplified signal changes. Thus we adapt the C-HCR strategy for evaluating Dam MTase activity and inhibition. Adaption and optimization of C-HCR for Dam MTase assay. The C-HCR strategy can be utilized as a general sensing platform for MTase assay. Here DNA adenine methylation (Dam) MTase and DpnI were chosen as the model MTase and auxiliary restriction endonuclease, respectively. To develop a general MTase sensing platform, the initiator I was designed as a partially hybridized dsDNA consisting of two ssDNAs I1 and I2 (I = I1/I2), as illustrated in Figure 2A. I1 includes sequences m and a* while I2 includes a complementary sequence m* and a protruding sequence b*. Thus the resulting I1/I2 initiator DNA yields a dsDNA domain m/m* and two protruding ssDNA domains a* and b*. The firmly assembled 16-bp long dsDNA domain m/m* connects domains a* and b* to assemble an integrated I for triggering C-HCR (path a, Figure 2A) and generating FRET signal (curve a, Figure 2B), yielding scarcely no fluorescence emission. The dsDNA domain m/m* was encoded with MTase substrate sequence (5'-G-A-T-C-3') to be recognized by Dam MTase and transformed into a methylated sequence (5'-G-Am-T-C-3'). The intermediated methylated DNA can then be successively recognized and cleaved by a specific auxiliary DpnI endonuclease, resulting in splitted ssDNA fragments (path b, Figure 2A). Thus the coupled Dam MTase and DpnI endonuclease biotransformations lead to the degradation of domain m/m* of I1/I2 initiator and the production of the disconnected DNA fragments a* and b* that unable to trigger C-HCR for generating FRET signal (curve b, Figure

2B), thus a MTase-motivated turn on of fluorescence spectra could be anticipated. In addition, each strand of the initiator DNA alone, I1 and I2, cannot initiate the C-HCR process (curves c and d). This on the other hand demonstrates that it is the m/m* duplex-connected substrate not the isolated fragments a* and b* that facilitate the C-HCR procedure, validating the robustness of C-HCR for MTase assay. Polyacrylamide gel electrophoresis (PAGE) was further applied to evaluate the feasibility of the proposed C-HCR strategy for Dam assay. The C-HCR was investigated by incubating the hairpins mixture with the original and Dam/DpnI-treated initiator I, respectively. As shown in Figure 2C, many bright new bands with much lower electrophoretic mobility were immerged while the monomer hairpins bands turned to be weakened and even vanished for the original initiator-motivated C-HCR, demonstrating the successful execution of the C-HCR to assemble long dsDNA copolymer-like products (lane b). In contrast, scarcely a small amount of long dsDNA copolymer-like products was formed for the Dam/DpnI-treated-initiatormotivated C-HCR (lane a), and it is nearly the same as that without initiator DNA (lane c).These results were consistent with that of fluorescence spectra (Figure 2B), validating the feasibility of the present C-HCR strategy for MTase assay. Control experiments showed that weak fluorescence intensities were observed when the adapted C-HCR system was incubated with Dam or DpnI alone, or with both of Dam and DpnI but a mutant initiator (Im = I1m/I2m) without methylation site (Figure S5A). The methylation of initiator was proved to dominate the prohibition of C-HCR and FRET signal generation, implying that a successive MTase-mediated 4

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Figure 3. Optimization of the initiator substrate based on the fluorescence intensity ratio (FDam/F0) of the C-HCR-based MTase sensing platform. FDam refers to the fluorescence of FAM for the methylated/cleaved initiator substrate and F0 refers to the fluorescence of FAM for the unmethylated initiator substrate. The system consisting of H1 + H2 + H3 + H4 + H5 + H6 mixture (100 nM each), initiator I (20 nM), Dam (5 U/mL) and DpnI (25 U/mL) were carried out in reaction buffer for a fixed time interval of 2 h. Error bars were derived from n = 5 experiments.

methylation and DpnI-catalyzed cleavage of initiator might plays an important role in this process. Methylation-induced cleavage of initiator substrate was further verified by gel electrophoresis experiments. Only Dam-treated initiator substrate was efficiently cleaved by the corresponding restriction endonuclease DpnI (Figure S5B). The cleavage of initiator DNA could be prohibited without Dam MTase or/and DpnI enzymes. These results clearly demonstrated that the cleaved substrate initiator is indeed originated from the coupled biotransformation of Dam and DpnI enzymes, and the encoded methylation sequence can be only specifically recognized and transformed by its corresponding MTase and restriction endonuclease enzymes. The robustness and feasibility of the CHCR approach was thus validated for detecting Dam MTase activity. To achieve a MTase sensing platform of higher performance, the initiator structure needs to be further optimized. The ratio of the fluorescence intensity of the C-HCR system for methylated and unmethylated initiator (FDam/F0) was utilized to evaluate the performance of the present MTase sensing platform. First, the single-stranded domains a* and b* were optimized. The whole initiator sequence and the connecting domain m/m* were kept constant while the split and grafting position varied. That means the initiator sequence was encoded into ssDNA domains a* and b* (24 nt) with different distribution of nucleotides, such as 12+12 (I = I1/I2), 10+14 (Ia = I1a/I2a), 8+16 (Ib = I1b/I2b) and 6+18 (Ic = I1c/I2c). As shown in Figure 3, the single-stranded domains a* and b* with a distribution of 12+12 (I) leads to the best performance of the MTase sensing platform (FDam/F0 = 17.2, ten times higher than the others. Details see Figures S6A and S6B), which is consistent with the gel electrophoresis results (Figure S6C). This is presumably due to the secondary structures of the initiator DNA and a better synergistic effect of the equational distribution of the initiator sequence. After the ssDNA domains were

Figure 4. (A) Fluorescence profile of the C-HCR-mediated MTase assay with different concentrations of Dam MTase. (B) Resulting calibration curve of the C-HCR-mediated MTase assay. Inset: expanded linear calibration curve. The system consisting of H1 + H2 + H3 + H4 + H5 + H6 mixture (100 nM each), initiator I (20 nM), DpnI (25 U/mL), and different Dam concentrations were carried out in reaction buffer for a fixed time interval of 2 h. Error bars were derived from n = 5 experiments.

fixed with an optimized distribution of 12+12, then the connecting dsDNA domain m/m*was further optimized by changing the distance between the methylation/cleavage site and ssDNA tethers. That is a feasible way to precisely modulate the stabilities of the methylated/cleaved initiator substrates. The methylation sequence of one initiator (I) was placed in the middle of the dsDNA domain m/m* while that of the other (Id = I1d/I2d) was encoded closer to the ssDNA tethers a* and b*. It is expected that the one with closer methylation encoding might generate a much distinct signal for MTase assay due to a much less stability of the degraded connecting dsDNA m/m* domain. However, the result showed that there was no significant difference between these two distinct initiator structures (Figure 3 and Figure S6D), which was attributed to the welldesigned limited stabilities of the dsDNA domain m/m* (16-bp long). Thus the optimized initiator I was chosen as the most 5

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Figure 5. Selectivity of the present Dam assay for different MTases. The system consisting of H1 + H2 + H3 + H4 + H5 + H6 mixture (100 nM each), initiator I (20 nM) and DpnI (25 U/mL) were carried out in reaction buffer for a fixed time interval of 2 h. Error bars were derived from n = 5 experiments.

appropriate substrate for the subsequent Dam MTase assay. Effect of methylation/cleavage time and DpnI concentration for MTase assay. The methylation time was studied by incubating a fixed concentration of Dam MTase and auxiliary DpnI with the initiator for varied reaction times. Then the enzyme-treated product was introduced into the six hairpins mixture to trigger C-HCR process, Figure S7. It showed that the fluorescence intensity of FAM increased gradually with prolonged enzymatic reaction time under 37 °C (an optimized methylation temperature, details see Figure S8), and finally reached a plateau with an incubation time of 60 min. The successive Dam-methylated and DpnI-cleaved initiator substrate turned out to dominate the prohibition of C-HCR reaction and FRET signal generation, making the intensified fluorescence of FAM with increasing incubation time. The result suggested that the initiator substrate was increasingly methylated and cleaved with prolonged enzymatic biotransformation time until the incubation time reached 60 min when all the initiator substrate was completely degraded. Thus a fixed incubation time of 60 min was then chosen as the optimized reaction time for the subsequent experiments. DpnI endonuclease as used to verify the methylation performance of Dam MTase through cleaving the methylated sequence 5'-G-Am-T-C-3'. It plays an indispensable role for probing MTase activity and the effect of DpnI concentration needs to be investigated. Figure S9, showed that the fluorescence signal intensified with increasing concentrations of DpnI and leveled off after ca. 25 U/mL. Thus, 25 U/mL of DpnI endonuclease was chosen as the optimized cleavage condition for the following experiments. Amplified assay of Dam MTase activity. The optimized C-HCR strategy was utilized for sensing Dam MTase of different concentrations under the optimized reaction conditions. Figure 4A showed that the fluorescence of FAM intensified upon increasing Dam concentration up to 5 U/mL. The correlationship between fluorescence intensity change and the concentration of Dam was summarized and depicted in Figure 4B. A linear relationship was obtained at a Dam concentration

Figure 6. (A) Effect of 5-fluorouracil (50 µM) and gentamycin (50 µM) on the activity of Dam MTase. The inhibitor screening system without inhibitors was used as control. (B) Relative activity of Dam MTase against concentration of gentamycin. The system consisting of H1 + H2 + H3 + H4 + H5 + H6 mixture (100 nM each), initiator I (20 nM), Dam (5 U/mL), DpnI (25 U/mL), 5fluorouracil (50 µM) or different concentrations of gentamycin were carried out in reaction buffer for a fixed time interval of 2 h. Error bars were derived from n=5 experiments.

range of 0−1.0 U/mL with a good correlation coefficient R2 = 0.993. Based on a specific initiator substrate design and the CHCR-aided signal amplification, the detection limit for Dam MTase was calculated to be 0.015 U/mL (based on 3σ calculation method), which is comparable to and even lower than most of the existing Dam MTase assay approaches (Table S2). We further investigated the selectivity of the C-HCR strategy for Dam MTase assay. M.SssI MTase, which has a specific recognition sequence of 5'-CCGG-3', was chosen as an interference MTase to evaluate the specificity of this method. Under the same conditions, Dam can induce a significant fluorescence signal while no distinct fluorescence signal was observed upon introducing even four-fold more amount of M.SssI MTase (Figure 5). It is clear that the proposed MTase sensing platform exhibits a good selectivity for probing Dam MTase activity, due to the highly specific recognition between Dam MTase and the corresponding initiator substrate. Evaluation of Dam MTase Activity Inhibition. Since DNA methylation is closely related to human diseases, the screening of DNA MTase inhibitors is of great significance in cancer therapies. The MTase-mediated methyl-transfer from 6

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SAM to DNA is effectively blocked by DNA MTase inhibitors, and the subsequent DpnI-motivated DNA-cleavage is prohibited. Thus DNA MTase inhibitors can promote the CHCR transduction and mediate the effective quenching of FAM. Two Dam MTase inhibitors, 5-fluorouracil (an anticancer drug) and gentamycin (broad-spectrum antibiotic), were chosen and examined. The fluorescence intensity of the inhibitors-involved C-HCR system decreased in the presence of MTase inhibitors (Figure 6A). It was demonstrated that gentamycin inhibits Dam MTase more effectively than 5fluorouracil, and its dose-dependent inhibition was further investigated (Figure 6B). The activity of Dam declined with increasing concentration of gentamycin. The IC50 value, the concentration of inhibitor required to reduce enzyme activity by 50%, was obtained to be 28.6 µM from the plot of relative activity (RA) of Dam MTase versus concentration of gentamycin. Control experiment was carried out to clarify that the inhibition effect indeed originates from the interactions between inhibitors and the corresponding MTase not restriction endonuclease. It showed that 5-fluorouracil and gentamycin have no influence on DpnI activity even reach a high concentration of 100 µM and 500 µM, respectively (Figure S10). Therefore, gentamycin and 5-fluorouracil indeed inhibit only Dam MTase activity in the present sensing platform. The proposed method has potential application for screening other MTase inhibitors and provides a useful tool for discovering new antibiotic drugs. General M.SssI MTase assay based on C-HCR scheme. The present C-HCR-mediated MTase assay can be further developed as a general sensing platform for probing other sequence-specific biotransformations through redesigning the Y-shaped DNA substrates. Then the proposed C-HCR approach was able to evaluate the activity of M.SssI MTase just by changing the recognition site from 5'-G-A-T-C-3' to 5'-CC-G-G-3' and by replacing the auxiliary restriction endonuclease from DpnI to HpaII, making the C-HCR a general sensing strategy for measuring other MTase or restriction enzymes (details see Figure S11 and the accompanying discussions). In contrast with the Dam MTase sensing strategy, HpaII endonuclease can’t cleave the intermediated methylated DNA initiator (path b, Figure S11A), which can still then trigger C-HCR for generating FRET signal (curve b, Figure S11B). While in the absence of M.SssI MTase, the unmethylated recognition sequence of DNA initiator could be successively recognized and cleaved by the auxiliary HpaII restriction endonuclease (path a, Figure S11A), resulting in cleaved ssDNA fragments, thus prohibiting the C-HCR process and FRET signal generation (curve a, Figure S11B). These results clearly demonstrate the versatile functions of C-HCR amplifier for potential applications of important clinical bioassays. CONCLUSIONS In summary, we have developed a novel nonenzymatic signal amplified strategy for DNA MTase assay and MTase inhibitors screening based on concatenated hybridization chain reaction (C-HCR)-triggered Förster resonance energy transfer (FRET). Y-shaped DNA initiator-motivated HCR-1 produced numerous tandem trigger for HCR-2 that successively generates and amplifies the FRET signal. The Y-shaped initiator was designed to be specifically recognized and transformed by MTase (Dam) to produce intermediated methylation initiator substrate that was subsequently cleaved by a restriction endonuclease (DpnI), leading to the prohibition of C-HCR process

and FRET generation. The synergistic effect of HCR enables the isothermal enzyme-free amplified detection of DNA MTase with low background and substantial signal enhancement capacity. This signal-on sensing strategy was utilized for selectively and sensitively measuring Dam MTase with a low detection limit of 0.015 U/mL in a range of 0-1.0 U/mL. Furthermore, this sensing strategy was successfully adapted for screening the inhibitors of methyltransferase, which is significant for drug discovery and clinic diagnostics. The Y-shaped DNA substrate can be redesigned and converted to probing other sequence-specific biotransformations, e.g., M.SssI MTase just by changing the recognition site, making the C-HCR a general sensing strategy for measuring other MTase or restriction enzymes in a “plug-and-play” fashion. Take advantage of the robust C-HCR system, our study avoids laborious preparation/modification of nanomaterials, additional enzymes, and high-precision thermal cycling. Besides the analytical implications of the system, the unprecedented C-HCR holds great promise in bioengineering and biomedicine fields.

ASSOCIATED CONTENT Supporting Information Gel electrophoresis and AFM characterizations of conventional HCR and C-HCR, control experiments for MTase assay, initiator structure optimization, methylation/cleavage condition optimization, DpnI concentration optimization, effect of MTase inhibitors on DpnI, M.SssI MTase assay, DNA sequences and a summary of the present MTase signal amplification methods. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *

To whom correspondence should be addressed. E-mail: [email protected].

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

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by National Basic Research Program of China (973 Program, 2015CB932601), Hubei Provincial Natural Science Foundation of China (2015CFB503), Jiangsu Provincial Natural Science Foundation of China (BK20161248, BK20160381), National Natural Science Foundation of China (21503151, 81602610), Wuhan Youth Science and Technology Plan (2016070204010131) and 1000 Young Talent (to F.W. and X.L.).

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