Highly sensitive assay of methyltransferase activity based on an

Oct 17, 2018 - Methyltransferase-involved DNA methylation is one of the most important epigenetic processes, making the ultrasensitive MTase assay hig...
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Highly sensitive assay of methyltransferase activity based on an autonomous concatenated DNA circuit Chunxiao Li, Huimin Wang, Jinhua Shang, Xiaoqing Liu, Bi-Feng Yuan, and Fuan Wang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00738 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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Highly sensitive assay of methyltransferase activity based on an autonomous concatenated DNA circuit Chunxiao Li,‡ Huimin Wang,‡ Jinhua Shang, Xiaoqing Liu, Bifeng Yuan, 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: Enzyme-free, Amplification, Isothermal, Methylation, Endonuclease ABSTRACT: Methyltransferase-involved DNA methylation is one of the most important epigenetic processes, making the ultrasensitive MTase assay highly desirable in clinical diagnosis as well as biomedical research. Traditional single-stage amplification means often achieve linear amplification that might not fulfil the increasing demands for detecting trace amount of target. It is desirable to construct multi-stage cascaded amplifiers that allow for enhanced signal amplifications. Herein, a powerful nonenzymatic MTase-sensing platform is successfully engineered based on a two-layered DNA circuit, in which the upstream catalytic hairpin assembly (CHA) circuit successively generates DNA product that could be used to activate the downstream hybridization chain reaction (HCR) circuit, resulting in the generation of a dramatically amplified fluorescence signal. In the absence of M.SssI MTase, HpaII endonuclease could specifically recognize the auxiliary hairpin substrate and then catalytically cleave the corresponding recognition site, releasing a DNA fragment that triggers the CHA-HCR-mediated FRET transduction. Yet the M.SssI-methylated hairpin substrate could not be cleaved by HpaII enzyme, and thus prohibits the CHA-HCR-mediated FRET generation, providing a substantial signal difference with that of MTase-absent system. Taking advantage of the high specificity of multiple-guaranteed recognitions of MTase/endonuclease and the synergistic amplification features of concatenated CHA-HCR circuit, this method enables an ultrasensitive detection of MTase and its inhibitors in serum and E. coli cells. Furthermore, the rationally assembled CHA-HCR also allows for probing other different biotransformations through a facile design of the corresponding substrates. It is anticipated that the infinite layer of multi-layered DNA circuit could furtherly improve the signal gain of the system for accurately detecting other important biomarkers, thus holds great promise for cancerous treatment and biomedical research.

DNA methylation participates in the crucial epigenetic processes and gets involved in many essential biological processes including cell proliferation, genomic imprinting and gene transcription in all of eukaryotic and prokaryotic cells.1 In a typical methyltransferases (MTases)-catalyzed methylation process, the methyl group was donated from S-adenosylmethionine (SAM) to the recognition adenine/cytosine sites of a given target.2 Aberrant methylation is demonstrated to closely relate to many human diseases, including systemic lupus erythematosus, hodgkin lymphoma, leukemia, and even lethal cancers. Abnormal MTase expression has thus been recognized as an emerging cancerous indicator and the associated methyltransferase has been considered as a new pharmacological object in antitumor treatments.3-5 Moreover, the targeting inhibition of methylation reaction can reactivate apoptotic pathways, and sensitize cancerous cells with chemotherapy resistance. Therefore, highly sensitive and accurate evaluation of MTase and its inhibitor is especially important in clinical diagnosis and epigenetic treatment. Conventional MTase assays mainly include bisulfite sequencing polymerase chain reaction,6 methylation-specific melting curve analysis,7 and methylation-specific single-strand conformation analysis.8 However, these indirect bisulfite-

based strategies suffer heavily from laborious and tedious sample preparation procedures. A possible incomplete bisulfite-mediated conversion may also lead to limited sensitivity which is crucial for detecting trace amount of MTase as acquired from invaluable clinical specimens. Besides, plenty of new approaches have been utilized for reliable MTase detection, including liquid chromatography,9 mass spectrometry,10 high performance capillary electrophoresis,11 microarray12 and radioactive [methyl-3H]-SAM labeling.13 However, some limitations, including discontinuous and time-consuming operations, expensive and sophisticated apparatus, limited resolutions, and even radioactive contaminations, largely prevent their extensive application in practical MTase assay.14 Besides the above traditional methods, several optical techniques, such as chemiluminescence,15 colorimetry,16-18 and fluorescence,19 have also been introduced for MTase assay. For example, DNA methylation-mediated gold nanoparticles (AuNPs) assembly,20 and functional AuNPs-involved click chemistry21 were respectively utilized for optical MTase assay. Similarly, a methylation-mediated spatial separation between fluorescent DNA substrate and graphene oxide scaffold was constructed for evaluating MTase activity.22 Nonetheless, these optical sensors suffer with insufficient sensitivity and miscellaneous 1

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preparation of nanoprobes. Recently, different isothermal enzymatic amplification strategies, such as strand displacementbased signal amplification,23 exonuclease-mediated analyte regeneration,24-26 rolling circle amplification,27 and polymerase-assisted amplification17 have thus been adapted for sensitive MTase assay. Especially, the efficient and rapid amplification operation of these isothermal amplifiers is achieved at a constant temperature without complicated thermocycling procedures which is prerequisite for conventional polymerase chain reaction. Unfortunately, these enzyme-involved schemes always require stringent operation conditions that might otherwise inhibit the activity of enzymes. Thus, it is highly desirable to develop new nonenzymatic sensing platforms for homogeneous MTase assay. CHA28 and HCR29 are characteristic isothermal nonenzymatic nucleic acid circuits with inherently modular and scalable features, which require only the facile design of reacting strands. As two efficient and homogeneous amplification strategies, CHA mediates the target-catalysed assembly of hairpin substrates into dsDNA structures, while HCR involves the target-initiated autonomous cross-hybridization of hairpin reactants to produce chained DNA nanowires. Each of the CHA or HCR circuit can transduce molecular recognitions into distinct readout signals upon their integration with varied transducing approaches. Recently, various concatenated amplifiers were constructed with substantially amplified biosensing performance.30-32 For example, the first amplification layer produced massive intermediators to furtherly motivate the second amplification layer for transducing the primary sensing event, e.g., a concatenated HCR or/and CHA circuit33-35 has been engineered with significant amplification efficiencies by its integration with RNA-cleaving DNAzyme36 or hemin/Gquadruplex DNAzyme.37 Take the inspirations from two-stage signal amplification, we reported on a general straightforward strategy for detecting MTase and for screening MTase inhibitors based on CHAHCR-mediated Förster resonance energy transfer (FRET). Taking advantage of these multiple-guaranteed specified recognitions of MTase/endonuclease and the synergistic amplification feature of concatenated CHA-HCR circuit, this method enables an ultrasensitive detection of MTase and its inhibitors from complicated biological systems, including serum and bacterial cells. The universal and rational design of our method is anticipated to provide a promising toolbox for studying other biotargets associated with key diseases in future tumor treatments and anticancer drug discovery researches. MATERIAL AND METHODS Reagents. All DNA were HPLC-purified and purchased from Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China) (Table S1). HpaII and DpnI endonucleases, M.SssI and Dam MTases, and SAM were supplied by New England BioLabs (Ipswich, MA). 5-Fluorouracil (5-FU) and 5-Azacytidine (5-Aza) were supplied by Sigma-Aldrich (Beijing, China). BCA protein assay kit and RIPA cell lysis buffer were obtained from Beyotime Institute of Biotechnology (Shanghai, China). All stock solutions need to be kept at 20 °C prior to these experiments. The used water is purified through an ultra-purification system (Millipore, Bedford, MA). M.SssI MTase Assay. Each DNA hairpin (4 µM) was respectively heated at 95 °C for 5 min and then cooled down to room

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temperature (25 °C) for more than 2 h in 10 mM HEPES buffer (pH 7.2, containing 1.0 M NaCl and 50 mM MgCl2). To investigate the M.SssI MTase activity, HM (10 nΜ) was incubated with SAM (0.16 mΜ) and different concentrations of M.SssI in 1 × NEBuffer for 2 h at 37 °C, and then was reacted with HpaII (11 U/mL) in 1 × CutSmart buffer for 2 h at the same temperature. The M.SssI/HpaII-treated solution was then successively introduced with hairpins H1-H6 (300 nM each) to execute homogeneous CHA-HCR reaction under a constant room temperature (25 °C), of which the fluorescence was acquired by a Cary Eclipse spectrometer (Varian Inc.). M.SssI MTase Inhibitors Assay. For screening of M.SssI MTase inhibitors, HM (10 nΜ) was incubated with SAM (0.16 mΜ), M.SssI MTase (7 U/mL) and different inhibitors in 1 × NEBuffer at 37 °C for 2 h. The following endonuclease treatment and CHA-HCR transduction operations were the same as described before. The following equation was used to evaluate the relative activity (RA) of M.SssI MTase: RA = (F2 – F0)/(F1 – F0) where F0, F1 and F2 corresponded to the fluorescence readout of CHA-HCR amplifier activated by HpaII alone, M.SssI/HpaII, and M.SssI/HpaII/MTase inhibitors, respectively. M.SssI MTase Assay in Serum. The procedure of MTase assay was the same as those described above except that the methylation procedure was carried out in 10% human serum. The recovery ratio (R) was estimated by using the following equation: R = C2/C1 × 100% where C1 and C2 referred to the determined M.SssI concentrations in the absence and presence of human serum, respectively. Detection of MTase from Bacterial Cells. The preparation of bacterial cells and the corresponding cell extracts are based on a reported procedure with minor modifications.25 The Dampositive (GW5100) and Dam-negative (JM110) E. coli cells was cultivated in culture-medium (5 g/L yeast extract, 10 g/L trypton, 10 g/L NaCl) for 12 h at 37 °C, and was then diluted by 20 times for an additional 2.5 h of cultivation. Subsequently, each of the E. coli cells was collected by centrifugation (6000 rpm, 5 min). After rinsing with PBS (pH 7.4) for three times, these harvest cells were lysed by RIPA lysis buffer. The total extracted protein was quantified with the assistance of the BCA protein assay kit while the contained MTase is measured according to the M.SssI MTase assay procedure as mentioned before. RESULTS AND DISCUSSION Design of CHA-HCR-Based Signal Amplification Strategy. Herein, a CHA-HCR cascade was established for signal amplification. As schematically depicted in Figure 1A, the CHAHCR system consists of the first layer of catalytic hairpin assembly circuit (CHA) and the second layer of hybridization chain reaction (HCR). Based on a powerful toehold-mediated strand displacement principle, the entropy-driven CHA28 and HCR29 circuits dictate self-assembly of hairpin reactants, at a constant temperature, into sophisticated isolated and connected double-stranded DNA (dsDNA) nanostructures, respective2

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ACS Sensors ly. The upstream isothermal CHA catalyzes the continuous hybridizations between two hairpin substrates to yield multiple DNA duplexes (detailed working principle, see Figure S1), while the downstream isothermal HCR motivates the successive and sequential cross-opening of its hairpin reactants to yield dsDNA concatamers (detailed working principle, see Figure S2). In order to integrate CHA and HCR circuits into a compact CHA-initiated HCR system, the trigger strand of HCR needs to be engineered and grafted onto one of the CHA hairpin substrates. For purpose of monitoring the CHA-HCR transduction, FAM (acting as fluorescence donor) is attached to the 5'-end of hairpin H5 while TAMRA (acting as fluorescence acceptor) is attached to the 3'-end of hairpin H3. Based on a universal toehold-mediated strand-displacement, the entropy-driven upstream CHA trigger T-mediated multiple cycles of H1·H2 hybridizations lead to the reinforced generation of H1·H2 duplex, where the caged ssDNA fragment c*-d* of H2 was exposed (nominating as intermediate trigger I) for motivating the subsequent entropy-driven HCR amplifier. In downstream HCR circuit, the newly generated trigger I mediates the cyclic and sequential hybridizations among these hairpin H3-H6 reactants, generating a dsDNA concatemeric nanochain that was decorated with numerous FRET-transducing FAM/TAMRA fluorophore pairs, and producing a tremendously amplified FRET signal at a constant room temperature (detailed characterization, see Figure S3). During this process, each H1·H2 pair hybridization event produces a HCR trigger to assemble one copolymeric dsDNA nanowire while each H3·H4·H5·H6 reaction results in one readout signal. Accordingly, the overall hybridization reaction of CHA-HCR system could be successively accelerated from CHA to HCR and could be featured with dramatic amplification performances. The encoded HCR trigger strand needs to stay in a caged state in the initial hairpin configuration so that the undesired signal leakage from CHA layer to HCR layer could be strictly eliminated. In the meantime, this caged HCR trigger could only be activated to assemble the dsDNA concatemeric nanowires and to generate an amplified readout signal only through CHA approach, thus enabling an efficient CHA-HCR operation. The synergistic enhancement effect of CHA and HCR circuits facilitates their ultrasensitive biosensing and biomedical applications. The components of CHA-HCR amplifier could coexist and stay in metastable state without external trigger, due to their stable hairpin configurations, and are theoretically38 and experimentally optimized to avoid any undesirable hybridization. Nearly no florescence variation was revealed in CHAHCR system without trigger T (curve a of Figure 1B), indicating that these hairpins are extremely robust without spontaneous cross-hybridization reactions. Yet a remarkable fluorescence change was shown in the T-triggered CHA-HCR system, and it reached to a saturation value after ~50 min (curve b of Figure 1B), which was chosen as the optimized reaction time to acquire the fluorescence spectra (Figure 1C) for all of the subsequent experiments. Here the fluorescence of FAM was adapted as the readout signal in the present isothermal entropy-driven CHA-HCR amplifier, resulting in a “turn off” detection of the DNA analyte. Additionally, the H6-expelled CHA-HCR amplifier was in fact a traditional CHA circuit, and its performance was also studied with a moderate fluorescence readout (Figure S4). It is interpretable since the entropy-driven CHA-HCR amplifier holds a higher amplification efficacy (1/N2) as compared with

the traditional CHA system (1/N). The synergistic CHA and HCR reaction leads to the successive hybridization processes and a concomitantly effective FRET generation at a constant room temperature. Polyacrylamide gel electrophoresis (PAGE) and atomic force microscope (AFM) were also applied to verify the underlying molecular mechanism of the isothermal CHA-HCR amplifier and their individual CHA and HCR reactions. Clearly, only their respective initiators or triggers could activate the corresponding CHA, HCR, and CHA-HCR systems (Figure S5). In addition, AFM was used to examine the morphological properties of the generated DNA nanostructures. Only tiny, spherical and monodisperse spots were observed for non-triggered CHA-HCR mixture. As expected, numerous linear dsDNA structures were emerged for triggered CHA-HCR system, demonstrating the robust working principle of our proposed strategy (Figure S6). Thus the homogeneous CHA-HCR system was successfully implemented with remarkable signal gain feature, implying that the present isothermal strategy could then be adapted for sensitively evaluating MTase activity as well as MTase inhibition.

Figure 1. (A) Schematic representation of the isothermal nonenzymatic cascaded CHA-HCR system. (B) The variations of FAM fluorescence (at λ = 520 nm) with time and (C) the corresponding ultimate fluorescence spectra of the CHA-HCR amplifier (a) without and (b) with 10 nM trigger T. The H1-H6 (300 nM each)-consisting CHA-HCR system was carried out at room temperature for 50 min unless specified. Adaption of CHA-HCR Circuit for Amplified MTase Assay. Given the high amplification capacity and the general applicability of our CHA-HCR circuit, the present concatenated system was envisaged to be especially suitable for sensitively detecting MTase and for efficiently evaluating its inhibitors. Here M.SssI (CpG MTase) and HpaII were respectively selected as the model methyltransferase and auxiliary endonuclease. Figure 2A schematically illustrates the principle of the updated CHA-HCR system for detecting M.SssI MTase. A foreigner hairpin HM was encoded, in loop and partial stem regions, with a locked trigger sequence to be released for motivating CHA-HCR amplifier, and in partial stem region with a symmetric 5′-CCGG-3′ domain that corresponds to the sub3

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strate sequence of M.SssI and HpaII enzymes. Thus the hairpin HM could execute as a general precursor of the CHA-HCR initiator by acting as a universal substrate of M.SssI (MTase) and HpaII (endonuclease). Without M.SssI, the endonuclease HpaII can specifically cleave the nicking domain of intact HM substrate, resulting in the release of trigger sequence to activate the CHA-HCR amplifier by an entropy-driven toeholdmediated strand displacement principle as described before. While the M.SssI MTase could specifically methylate hairpin substrate HM, resulting in the methylated HM that could not be cleaved by HpaII endonuclease. The CHA-HCR trigger still remains in a caged configuration in methylated HM substrate where its primary toehold region is still blocked in the stem region, thus prohibiting the subsequent CHA-HCR-mediated FRET transduction. Obviously, the cleavage of substrate HM plays a crucial role for amplified MTase assay. This HMinvolved biotransformation is confirmed by a preliminary gel electrophoresis measurement (Figure S7). The mixture of HM, M.SssI, and HpaII showed only one band of the methylated hairpin HM (lane 3) which is close to the intact hairpin HM (lane 1), demonstrating that the M.SssI-methylated HM could not be cleaved by the auxiliary endonuclease HpaII. As a comparison, two new bands with faster migration rates were observed for the mixture of HM and HpaII (lane 2), indicating that the auxiliary endonuclease HpaII could mediate the efficiently biocatalytic cleavage of hairpin HM for generating CHA-HCR trigger. Thus the MTase-mediated distinct configurations of MTase substrate (precursor of CHA-HCR trigger) enable a reliable and sensitive M.SssI MTase assay. The CHA-HCR strategy was then examined for fluorescent MTase assay. Considering the primary MTase-mediated blockage of CHA-HCR initiator and the subsequent initiatormotivated fluorescence “turn-off” feature of CHA-HCR amplifier, then the present CHA-HCR-mediated MTase assay represents a fluorescence “turn-on” procedure. As indicated in Figure 2B, no fluorescence change was observed for M.SssImethylated HM, no matter whether HpaII exists (curve c) or not (curve b), upon its exposure to the CHA-HCR mixture. And the fluorescence signals are nearly the same as that of the blank control (curve a). It is reasonable since the restriction endonuclease HpaII cannot cleave methylated HM that is unable to produce the CHA-HCR amplifier and to generate FRET signal. While a significant fluorescence change is observed for HpaII-treated intact HM as a result of the successive hybridization reactions of CHA-HCR circuit (curve d). The methylation of HM was thus proved to prohibit the CHA-HCR-stimulated FRET transduction, implying that M.SssI MTase indeed blocks the HpaII-catalyzed digestion of hairpin substrate and the following homogeneous CHA-HCR system. These fluorescence results validate the successful implementation of our CHA-HCR approach for amplified detection of MTase. The whole working principle of our CHA-HCR-amplified M.SssI assay was furtherly evaluated by native PAGE experiment, Figure 2C. The homogeneous M.SssI biosensing system was investigated by incubating CHA-HCR reactants with the intact or treated HM substrate, respectively. No new band was revealed in CHA-HCR mixture that was introduced with M.SssI- or M.SssI/HpaII-treated HM substrate (lanes 9 and 11, respectively), which is similar to the intact HM-triggered or non-triggered CHA-HCR mixture (lanes 8 and 7, respectively). This implies that the methylated HM substrate could not be

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digested by HpaII and thus failed to motivate the homogeneous CHA-HCR system. In contrast, higher-molecular-weight DNA products were emerged while the monomers were efficiently consumed for the CHA-HCR mixture that was exposed to HpaII-treated HM substrate (lane 10). This phenomenon is the same as that of a predesigned trigger-motivated CHA-HCR system (lane 12), validating the CHA-HCR-assembled dsDNA copolymers without MTase treatment of the corresponding hairpin substrate. This can be explained by the HpaII-mediated cleavage of unmethylated HM and the concomitant generation of cleaved HM fragment product, which then acts as a universal trigger to stimulate the subsequent CHA-HCR process. This gel electrophoresis result shows a good accordance to the previous fluorescence measurement (Figure 2B), thus verifying the viability of our autonomous CHA-HCR system for accurate MTase detection.

Figure 2. (A) Scheme for CHA-HCR-amplified M.SssI detection. (B) Fluorescence spectra of the CHA-HCR amplifier upon its incubation with (a) HM; (b) M.SssI-treated HM, (c) M.SssI/HpaIItreated HM, and (d) HpaII-treated HM. (C) PAGE investigation of the CHA-HCR-motivated M.SssI detection: hairpins H1-H6 (lane 1), hairpins H1-H6 and HM (lane 2), hairpins H1-H6, HM, and M.SssI (lane 3), hairpins H1-H6, HM, and HpaII (lane 4), hairpins H1-H6, HM, M.SssI, and HpaII (lane 5), hairpins H1-H6 and T (lane 6). The CHA-HCR operation was carried out at a constant room temperature (25 °C) for 50 min.

Optimization of Methylation for MTase Assay. For higher performance of M.SssI assay, the effects of methylation time and HpaII concentration were carefully investigated to acquire the most appropriate reaction conditions. After the HM substrate was treated with M.SssI and HpaII enzymes at 37 °C (an optimized reaction temperature, Figure S8(B)) for different methylation durations, the as-generated product was used to trigger the autonomous CHA-HCR circuit, Figure S8(A). As a result, the fluorescence intensified with an extended methylation duration, and then leveled off until 2 h. The successive M.SssI-methylated HM substrate turned out to dominate the prohibition of HpaII-mediated HM cleavage and the subsequent CHA-HCR-motivated FRET generation, making the intensified fluorescence with prolonged methylation duration. 4

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ACS Sensors This result indicated that a 2 h incubation is long enough to ensure a complete methylation of HM substrates and is chosen as the most appropriate reaction duration. Similarly, HpaII was also investigated for optimized M.SssI sensing purpose. Clearly, the optimized cleavage time of 2 h and the optimized HpaII concentration of 11 U/mL were revealed in Figures S8(C) and S8(D), respectively.

much better than a majority of the existing M.SssI assay systems (Table S2). This tremendously improved sensing performance is originated from the collaborative signal gain between HCR and CHA circuits.

Figure 4. (A) Inhibitory effect of 5-FU and 5-Aza on M.SssI MTase as revealed by fluorescence measurements. (B) Relative M.SssI (7 U/mL) activity against varied 5-Aza concentrations. Error bars were acquired from 5 parallel experiments. The RA is presented by the fluorescence ratio of the present sensing platform in the presence and absence of the corresponding inhibitors.

Figure 3. (A) Fluorescence spectra generated by the CHAHCR-amplifier upon their incubation with varied concentrations of M.SssI MTase. (B) Full range and (C) the corresponding linear calibration curves of (a) the CHA-HCR- and (b) CHA-mediated MTase detection as plotting fluorescence change (F–F0) versus M.SssI concentration. (D) Selectivity of our homogenous CHA-HCR-based M.SssI detection: (a) control; (b) Dam (7 U/mL); (c) Dam (35 U/mL); (d) M.SssI (7 U/mL). Error bars were acquired from 5 parallel experiments. The H1-H6-containing CHA-HCR transduction system (300 nM each) and the H1-H5-containing CHA control (300 nM each) were implemented in reaction buffer at 25 °C for 50 min. Amplified CpG M.SssI Assay. The optimized reaction condition ensures a sensitive M.SssI assay through the homogeneous CHA-HCR amplifier. Also, the H6-expelled CHA-HCR amplifier was applied for quantitative M.SssI detection (Figure S9). As illustrated in Figure 3A, the fluorescence intensified progressively and it leveled off upon increasing the concentration of M.SssI MTase up to 7.0 U/mL for the CHAHCR-amplified M.SssI assay. As shown in Figure 3B, the M.SssI concentration-dependent fluorescence variations were summarized for the compact CHA-HCR and conventional CHA strategies. The fluorescence of CHA-HCR amplifier intensified gradually as the increasing concentrations of M.SssI MTase with a more prominent upward trend than conventional CHA control system. A good linear relationship was obtained for M.SssI ranging from 0 to 0.004 U/mL with a derived equation expressed as Y = 93200X - 0.6 (R2 = 0.999). Based on the engineered hairpin substrate and the homogeneous CHA-HCR amplifier, the detection limit of M.SssI MTase was corresponded to 1.2 × 10-4 U/mL according to a traditional 3σ calculation method (Figure 3C), which is tremendously lower than that of conventional CHA system (0.18 U/mL) and is also

Selective CpG M.SssI Detection. The present MTase assay was not merely sensitive, but also selective. Dam MTase, which corresponds to a different recognition sequence, was selected as interfering methyltransferase for investigating the selectivity of the MTase sensing system. As illustrated in Figure 3D, a significant fluorescence change was observed upon addition of M.SssI MTase while a negligible fluorescence response was revealed upon introducing even 5-fold amount of Dam MTase. Clearly, our proposed CHA-HCR strategy could easily discriminate M.SssI MTase from other MTase interferers, which is attributed to the high specificity of multipleguaranteed recognitions and transformations of MTase/endonuclease on its substrate. Evaluation of MTase Inhibitors. Aberrant methylation has been considered as an indicator for large varieties of human diseases, the screening of MTase inhibitors thus holds great promise for therapeutic applications.39,40 Herein, we examined two well-known FDA-approved anticancer agents, 5-Aza (5azacitidine) and 5-FU (5-fluorouracil), as model inhibitors on M.SssI. These MTase inhibitors could effectively prohibit the methylation of hairpin substrate, thus facilitating the subsequent HpaII-mediated cleavage of unmethylated substrate to activate the ultimate CHA-HCR circuit. These inhibitors can facilitate the CHA-HCR execution to export a valid fluorescence readout. Indeed, the fluorescence intensity decreased for inhibitor-involved MTase sensing system as compared with that of inhibitor-free system, Figure 4A. Interestingly, 5-Aza revealed a more obviously inhibitory effect on MTase as compared with 5-FU and its dose-dependent inhibition effect was furtherly investigated (Figure 4B). The activity of M.SssI declined with increasing dosage of 5-Aza. The IC50 value,30 the corresponding concentration of inhibitors for inhibiting M.SssI activity by 50%, was achieved and corresponded to 15 µM. These two small-molecule inhibitors are demonstrated to catabolically incorporated into DNA and eventually exert their inhibitory effects by covalent cross-linking with the corresponding methyltransferases. Also, these nucleobase analogs drugs may also bind to the active site of methyltransferase and inhibit its activity, considering that a high concentration of 5

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inhibitors is required to induce an appreciable inhibition on methyltransferase activity.41 Considering that both of HpaII and M.SssI enzymes are involved in the assay system, control experiment is carried out to clarify that these inhibitors indeed interact with the corresponding MTase not restriction endonuclease (Figure S10). It is shown that 5-Aza and 5-FU have no influence on HpaII activity even their concentration reaches to 90 µM, implying that these inhibitors indeed only inhibit M.SssI activity. Certainly, our CHA-HCR strategy can be employed to screen new inhibitors of MTases and to explore more different antitumor agents.

Figure 5. (A) Schematic illustration of the CHA-HCRamplified detection of Dam MTase which facilitates the execution of CHA-HCR amplifier. (B) Fluorescence spectra corresponding to the optional Dam-targeted detection platform by using the mixtures of (a) CHA-HCR hairpins and HD, (b) CHA-HCR hairpins, HD and Dam MTase, (c) CHA-HCR hairpins, HD and DpnI, (d) CHA-HCR hairpins, HD, Dam MTase and DpnI. Universality of Our MTase-Sensing Strategy. The CHAHCR-mediated MTase assay is a universal biosensing system and is able to probe more sequence-specific biotransformations through redesigning only the hairpin substrate HM. For example, the present M.SssI/HpaII (MTase/endonuclease) pair can be substituted with another Dam/DpnI pair. Then the facile CHA-HCR strategy could be furtherly developed for evaluating Dam MTase by replacing the recognition site from 5'-CCGG-3' to 5'-GATC-3' in hairpin substrate that was specifically recognized by Dam and the supplementary DpnI (Figure 5A). Thus the CHA-HCR amplifier could be featured with universal sensing performance to evaluate other MTases and endonucleases. Different with the M.SssI/HpaII system, here DpnI endonuclease cannot cleave the intact hairpin substrate HD, and hence blocking the CHA-HCR-involved FRET transduction (curve c, Figure 5B). This signal response is the same as that of unmethylated and methylated HD-included CHA-HCR mixture (curves a and b, respectively, Figure 5B). The recognition site of substrate HD could be successively methylated and digested by Dam and HpaII, respectively, resulting in fragmented ssDNA TD that triggers the CHA-HCR circuit and FRET generation (curve d, Figure 5B). Clearly, the universal applicability of our CHA-HCR system enables us to analyze other MTases through reengineering the MTase substrate. These versatile functions of CHA-HCR amplifier are anticipated to play important roles in a large variety of important clinical applications. Feasibility of MTase Assay for Practical Samples. The feasibility of our proposed CHA-HCR-based MTase assay was then investigated under biological conditions, e.g., serum samples. The recovery efficiency of M.SssI was evaluated by spiking different amount of M.SssI (0.001–0.004 U/mL) into normal human serum. A recovery efficiency of 95.5–107% was obtained with a relative standard deviation (RSD) of

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0.57–1.29%, consisting with those of QDs-mediated FRET assay (98.6–102.8% of recovery efficiency and 1.04–2.62% of RSD), Table S3. These results indicate a moderate precision of the CHA-HCR circuit for quantifying MTase from human serum. The present CHA-HCR-amplified MTase sensing strategy was furtherly used to study the activity of endogenous Dam from GW5100 (Dam-positive) and JM110 (Damnegative) E. coli cells, respectively. After adding a certain amount of E. coli cells lysate to the reaction mixture, the activity of endogenous Dam MTase and the total amount of proteins were measured by using our CHA-HCR method and the BCA protein assay, respectively. As compared with a high Dam expression in GW5100 cells, a much lower Dam expression was observed in JM110 cells (Dam-negative control), which is nearly the same as that of lysis buffer only (Figure 6A). Obviously, the fluorescence readout of GW5100 cells is indeed originated from the endogenous methyltransferases, and is not from other interfering proteins. As can be seen from Figure 6B, the fluorescence variation intensified with elevated content of total extracted proteins in Dam-positive E. coli cells. There revealed a linear correlationship between the fluorescence change and the total protein contents in the range of 7.54-150.8 ng. The limit of detection was determined to be 4.36 ng for CHA-HCR-mediated Dam MTase assay as calculated by the traditional 3σ/λ method, suggesting a high sensitivity of this method. The robust MTase assay of practical complex samples is originated from the high specificity of multiple-guaranteed recognitions/biotransformations of MTase/endonuclease enzymes and the synergistically amplified transduction of cascaded CHA-HCR system. These results obviously illustrate that the CHA-HCR system is suitable for analytes quantification in practical complex biological samples, thus holding excellent promise for early diagnosis and environmental monitoring researches.

Figure 6. (A) Evaluation of the activity of Dam MTase extracted from GW5100 and JM110 E. coli cells with 150.8 ng total proteins. (B) The linear calibration trace of the CHAHCR-amplified Dam sensing platform as acquired from the correlationship between fluorescence variations and total protein contents. CONCLUSIONS In summary, we have successfully constructed a nonenzymatic cascaded CHA-HCR method for amplified MTase assay and inhibitor screening by rational integration of CHA and HCR circuits. An auxiliary hairpin HM was engineered and utilized as HpaII substrate and thus could release a fragmented product to trigger the CHA-HCR-mediated FRET signal generation. Yet the M.SssI-methylated substrate could not be cleaved by HpaII enzyme, thus prohibiting the subsequent CHA-HCR process. As an isothermal homogeneous sensing strategy, this CHA-HCR amplifier enables the selective and ultrasensitive 6

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ACS Sensors detection of M.SssI with a low detection limit corresponding to 1.2 × 10-4 U/mL and a linear detection range from 0 to 0.004 U/mL. The CHA-HCR circuit was able to screen MTase inhibitors, which were of great importance for future clinical diagnosis and new drug design. The CHA-HCR-based MTase assay strategy is a universal detecting system for probing more different enzyme-mediated biotransformations, and for analyzing other restriction enzymes, e.g., Dam, by altering merely the specific sequence of the auxiliary hairpin substrate. The high specificity of the multiple-guaranteed recognitions of MTase/endonuclease, together with the synergistic amplification feature of concatenated CHA-HCR strategy, contributes to the ultrasensitive detection of MTase and its inhibitors in complex biological environments, including serum and E. coli cells. Compared with traditional MTase-targeting systems, our method requires no intricate nanomaterials preparations, sophisticated operations, and fragile enzymes involvements. Our CHA-HCR circuit is thus anticipated to contribute significantly to early diseases diagnosis and drug discovery.

ASSOCIATED CONTENT Supporting Information Sequences of oligonucleotides, comparison among M.SssI MTase detection methods, comparison of CHA-HCR system with conventional CHA circuit, gel electrophoresis analysis, AFM characterization, validation and optimization of the CHA-HCR-mediated M.SssI detection, conventional CHA-mediated MTase assay and the effect of MTase inhibitors on HpaII. 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 ‡ C. Li and H. Wang contributed equally. 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, 2015CB932600), National Natural Science Foundation of China (21503151, 21874103 and 81602610), Jiangsu Provincial Natural Science Foundation of China (BK20161248, BK20160381), Fundamental Research Funds for the Central Universities (No. 2042018kf0210), Wuhan Youth Science and Technology Plan (No. 2016070204010131) and 1000 Young Talent (to F. W. and X. L.).

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