Ultrasensitive and Accurate Assay of Human Methyltransferase Activity

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Ultrasensitive and Accurate Assay of Human Methyltransferase Activity at the Single-Cell Level Based on A Single Integrated Magnetic Microprobe Haiyan Zhao, Lei Wang, Weiqi Li, Shumei Zhai, and Wei Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09631 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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ACS Applied Materials & Interfaces

Ultrasensitive and Accurate Assay of Human Methyltransferase Activity at the Single-Cell Level Based on A Single Integrated Magnetic Microprobe Haiyan Zhao,† Lei Wang,‡ Weiqi Li,† Shumei Zhai,† Wei Jiang*,†

†

Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of

Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China.

‡

School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China.

KEYWORDS: Human methyltransferase activity, integrated magnetic microprobe, in situ rolling circle amplification, single microbead fluorescence imaging, single-cell level

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ABSTRACT Human DNA methyltransferase (MTase) activity expression patterns and inhibition response are linked to related cancer initiation, progression and therapeutic responses. Sensitive and accurate human MTase activity assay in cancer cells, especially at the single-cell level, is essential for biological study, clinical diagnosis and therapy. Here, we developed an ultrasensitive and accurate DNA (cytosine-5)-methyltransferase 1 (Dnmt1) activity assay at the single-cell level based on a single integrated magnetic microprobe of functionalized double-stranded DNA (dsDNA) anchored to a single magnetic microbead surface. Functionalized dsDNA is designed with a hemimethylated DNA site for Dnmt1 recognition and a single-stranded tail to trigger in situ rolling circle amplification (RCA). Under the action of Dnmt1, hemimethylated dsDNA could be recognized and catalyzed to fully methylated dsDNA, which would protect them from the cleavage of BssHII. But the dsDNA without full methylation would be cut by BssHII, making single-stranded tail separated from the single integrated microprobe. Subsequently, full methylation-protected in situ RCA could be performed and multiple signal probes were hybridized to the single integrated microprobe for amplified signal accumulation. Finally, Dnmt1 activity could be evaluated by reading the fluorescence of the single integrated microprobe. Meanwhile, to minimize matrix interferences, magnetic separation was performed in the process. In this strategy, the single integrated magnetic microprobe was provided with integrated capacities of target recognition, signal amplification, signal accumulation and matrix isolation. Therefore, an ultralow detection limit of 0.007 U/mL Dnmt1 was obtained and accurate Dnmt1 activity assays in multiple cell lysates at the single-cell level were achieved. Furthermore, the inhibition effect of RG108 was evaluated conveniently. These results indicate that the single integrated magnetic microprobe-based strategy is an excellent candidate for sensitive monitoring of Dnmt1 activity and screening of anticancer drugs.


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INTRODUCTION

Human DNA methyltransferases, which can transfer a methyl group from the donor Sadenosylmethionine (SAM) to target cytosine residues in DNA sequences, are an important family of enzymes for establishing and maintaining DNA methylation patterns in cell.1-4 Among them, the most abundant human DNA methyltransferase and mainly responsible for genomic DNA methylation patterns in somatic cell is human DNA (cytosine-5)-methyltransferase 1 (Dnmt1). Studies have revealed that abnormal expression of Dnmt1 activity can lead to aberrant DNA methylation. Hypermethylation of CpG islands in promoter regions may silence the tumor suppressor genes and hypomethylation involving in repetitive DNA sequences may cause chromosome abnormalities, both would cause cancerous transformations.5-7 In particular, overexpression of Dnmt1 activity have been found in many cancer cells including breast, cervical, colorectal, lung and stomach cancer,8-11 and inhibition of Dnmt1 activity in cancer cells can also promote the tumor suppressor gene promoters demethylation and reactivate their expression.12 Therefore, Dnmt1 activity has been considered as a predictive biomarker and therapeutic target. Sensitive and accurate Dnmt1 activity detection in cancer cells, especially at the single-cell level, is essential for clinical diagnosis and inhibitors drug screening related to clinical treatment. The traditional method for Dnmt1 activity assay is radioactive labeling strategy based on [methyl-3H]-SAM.13 Other detection methods avoiding radioactive reagent, including fluorescent


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assays,14 colorimetric assays,15,16 chemiluminescent assays,17 have been also successfully developed to determinate Dnmt1 activity. However, these methods are mainly performed in clean buffer for purified Dnmt1 activity detection, and the Dnmt1 activity assay in cells has rarely been demonstrated.14,16,17 The possible reason may be that these methods cannot work well in complex cellular environment due to the optical interferences induced by coexisting light scattering and autofluorescence from cellular components. Recently, electrochemical detection methods18-20 with separation operation have been used for Dnmt1 activity evaluation from cancer cells, but the detection at the single-cell level is a significant obstacle owing to sensitivity limitations. One reason may be that Dnmt1 with ultralow activity cannot interact with probes sufficiently with high reproducibility in these large bulk electrochemical assays. The other reason is that these electrochemical assays for Dnmt1 activity lack a robust amplification strategy. Thus, the development of detection method with both capacities of high sensitivity and high tolerance capability towards complex biological matrices is essential for Dnmt1 activity analysis from single cancer cell. Magnetic microprobe, with functionalized nucleic acids anchored to magnetic microbead (MB) surface, has been recently developed for biosensing and biodiagnostics.21-23 Studies have revealed that magnetic microprobe is particularly interesting for complex clinical samples analysis by the nature of magnetic separation capability, specific molecule recognition capability and amplification availability of DNA.24-26 However, these magnetic microprobe-based assays for complex samples usually involve thousands of small-sized microprobes in one detection


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system. And when the concentration of target molecule is ultralow, only small number of signals are generated and diluted on a few microprobes that may be hidden in the bulk measurements, leading to a large bias and limited sensitivity. Recently, single microbead-based fluorescence imaging (SBFI) strategy has been proposed and proven powerful in ultrasensitive detection of protein kinase (PK) activity.27 In the SBFI strategy, a single large microbead functionalized with rare earth ions is utilized for PK-induced phosphopeptides enrichment, bringing in highly concentrated fluorescence signals and enhanced detection sensitivity. In our regard, the applicability and sensitivity of the SBFI strategy can be further promoted by integrating magnetic microprobe. On one hand, functionalized DNA in the magnetic microprobe is benefit to more wide range of biomolecules recognition. On the other hand, in situ DNA amplification on the surface of MB can be easily achieved to further improve the sensitivity for analyte with lower abundance. In this work, combining single magnetic microprobe with in situ rolling circle amplification (RCA), we have developed a novel single integrated magnetic microprobe-based strategy for Dnmt1 activity assay at the single-cell level. In this strategy, only one single integrated magnetic microprobe is used to recognize Dnmt1 and then trigger target-protected in situ RCA to enrich multiple fluorophores for Dnmt1 activity analysis. As a result, enhanced accumulation of fluorescence signals on a single microprobe was achieved, bringing in much increased detection sensitivity for Dnmt1 with a ultralow detection limit of 0.007 U/mL. Additionally, combined with magnetic separation, which can eliminate interference from sample matrices, the strategy


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was endowed with high tolerance capability towards complex cell lysate, so Dnmt1 activity assays in multiple cell lysates at the single-cell level were successfully performed. These results indicate that this single integrated magnetic microprobe-based strategy is a reliable and sensitive Dnmt1 activity evaluation method for medical research, early clinical diagnostics and therapy. EXPERIMENTAL SECTION

Chemicals and Materials. All DNA oligonucleotides (Table S1) were synthesized and purified by Sangon Biotechnology Co., Ltd. (Shanghai, China). Human DNA (cytosine-5)methyltransferase1 (Dnmt1), AluI, HaeIII, Dam DNA methyltransferase (MTase), BssHII endonuclease, T4 DNA ligase and phi29 DNA polymerase were purchased from New England Biolabs (Ipswich, MA, USA). The 10× Dnmt1 reaction buffer, 10× T4 DNA ligase reaction buffer, 10× Cutsmart buffer, and S-adenosylmethionine (SAM) were also provided by New England Biolabs. The streptavidin magnetic microbeads (high magnetite, 45.0-52.0 µm) were provided by Spherotech, Inc. (Lake Forest, Chicago, USA). RG108 was purchased from Selleck (Houston, USA). All other chemicals were of analytical grade and used as received. All solutions were prepared using the ultrapure water (> 18.25 MΩ cm−1) that was obtained from a Millipore Milli-Q water purification system.

Apparatus. All integrated single microprobe fluorescence images were recorded with an Olympus confocal laser scanning microscope (Model IX81, Japan). The images were obtained using a 40× objective lens and a 488 nm laser. The fluorescence intensity value of each


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fluorescent microprobe was analyzed by counting the integrated fluorescence intensities of images with z-stack scan (Figure S1). In such z-stack scanning mode, each microprobe was divided into 10 slices along the z-direction for laser scanning, and then the integrated fluorescence intensities of these slices were counted together for quantitative analysis. All fluorescence emission spectrum were recorded on a Hitachi F-7000 fluorescence spectrometer (Hitachi, Japan). The excitation wavelength was 497 nm, and the spectra were recorded from 510 nm to 610 nm. Preparation of the Integrated Magnetic Microprobe. The integrated magnetic microprobe is a bioconjugate of magnetic microbead (MB) and functionalized dsDNA. First, biotinylated-R1 (1.0 µM) and methylated-R2 (5.0 µM) were hybridized to form dsDNA for Dnmt1 recognition in Dnmt1 reaction buffer (50 mM Tris-HCl, 1.0 mM EDTA, 1.0 mM DTT, 5% glycerol and 50 mM NaCl, pH 7.8) by heating at 90 °C for 10 min and cooling to room temperature slowly. Then 5.0 µL of as-purchased streptavidin-functionalized MBs solution was pipetted and washed with TBE buffer (50 mM Tris-HCl, 1.0 mM EDTA, 50 mM NaCl, pH 7.5). Subsequently, the formed dsDNA was incubated with the washed MBs for 1.5 h under mild shaking to immobilize dsDNA on the surface of MBs via the streptavidin-biotin interaction. After the reaction was completed, the whole solution was magnetically separated to remove the excess unbound DNA sequences and washed three times with TBE buffer. Finally, the integrated magnetic microprobes were obtained and dispersed in TBE buffer for subsequent use.


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Capture of one Single Integrated Magnetic Microprobe. The catching procedure of the single integrated magnetic microprobe was preformed according to the previously report with a slight modification that a DIY ruler was not needed due to the MBs have uniform size.27 First, the as-prepared integrated magnetic microprobes were spread onto a transparent hydrophobic 96well plate cover. Then the plate cover was loaded on the object stage of a microscope, meanwhile a pipette with a set volume was utilized to catch one single integrated microprobe and transferred to EP tube for further use. Cell Culture and Dnmt1 Extraction. MCF-7 cells and HeLa cells were cultured in 1640 (GIBCO) medium supplemented with 10% fetal calf serum in 37 °C containing 5% CO2. Cell number was determined by a Qiujing cell counter (Shanghai, China). Approximately a certain number of cells were dispensed by trypsinization and washed with ice-cold PBS buffer. Then a nuclear protein extraction kit (Epigentek, New York, USA) was used for cell lysis. Finally, the cell lysate was immediately used for Dnmt1 activity assay or transferred, aliquoted and stored at -80 °C for further use. Dnmt1 Activity Assay Based on A Single Integrated Magnetic Microprobe. For Dnmt1 activity detection, the single integrated magnetic microprobe was first incubated with a series of standard Dnmt1 solutions or cell lysates under shaking at 37 ºC for 2.5 h in Dnmt1 reaction buffer (50 mM Tris-HCl, 1.0 mM EDTA, 1.0 mM DTT, 5% glycerol and 50 mM NaCl, pH 7.8) supplemented with 160 µM SAM and 100 µg/mL BSA. Then, 2000 U/mL BssHII with 1× cutsmart buffer (50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate,


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100 µg/ml BSA, pH 7.9) was added and incubated at 37 °C for 2.0 h. After the methylation and cleavage, the single integrated microprobe was washed by TBE buffer and in situ rolling circle amplification (RCA) on the surface of MB was triggered. Typically, 200 nM padlock probes (PP), 2.5 U T4 DNA ligase and 1× T4 DNA ligase buﬀer (40 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 0.50 mM ATP, pH 7.8) were further treated with the single integrated magnetic microprobe at 37 °C for 1.0 h to form circular template. Next, the in situ RCA was performed at 37 °C for 2.0 h with further addition of 1× cutsmart buffer containing 3.0 U phi29 DNA polymerase and 400 nM dNTPs. After in situ RCA process, the single integrated magnetic microprobe with long repeated sequences was incubated with 600 nM detection probes (DP) at 37 °C for 1.0 h. Finally, after a further cycle of washing and separation steps, the single integrated magnetic microprobe was immediately subjected to fluorescence imaging. Inhibition Study of RG108 to Dnmt1 Activity. To study the inhibition effect of the model inhibitor of RG108 on Dnmt1 activity, all inhibition experiments were performed in conditions similar to those of Dnmt1 activity assays, except for that various concentrations of RG108 were added into the solution before the addition of 10 U/mL Dnmt1 in the methylation step. Then after the BssHII cleavage and in situ RCA process on the surface of MB, the fluorescence images were recorded. RESULTS AND DISCUSSION


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Design of the Single Integrated Magnetic Microprobe-based Strategy for Dnmt1 Activity Assay. As shown in Scheme 1, the construction of the integrated magnetic microprobe and the overall working principle of the proposed single integrated magnetic microprobe-based strategy for Dnmt1 activity assay are illustrated in detail. The integrated magnetic microprobe is a bioconjugate of functionalized dsDNA and streptavidin-modified MB. Here, the functionalized dsDNA is obtained by hybridizing biotinylated DNA1 (R1) and methylated DNA2 (R2) with functions as below. First, biotin-modified dsDNA can ensure it easily immobilized on the surface of MB via the streptavidin-biotin interaction. Additionally, hemimethylated 5′-GC-3′ sites within the recognition sites (5′-GCmGCGC-3′) of BssHII restriction enzyme endow the dsDNA with double recognition functions of Dnmt1 and BssHII. Furthermore, the single-stranded tail in the dsDNA can act as a primer for in situ RCA on the surface of MB to assist multiple fluorescence enrichment. For another, a dT25 sequence is also introduced as a spacer to ensure high accessibility of enzymes to the dsDNA anchored on the surface of MB. Based on the dsDNA with these functions and MB with the capability of enrichment and separation, a single integrated magnetic microprobe is utilized as an entity to sensitively sense the Dnmt1 activity. In the presence of active Dnmt1 either in its purified form or as a component of crude lysate, the methyl group in the molecule of S-adenosylmethionine (SAM) can be transferred to the cytosines in the hemimethylated dsDNA. As a result, fully methylated dsDNA is formed and the cleavage by BssHII will be blocked, because BssHII is a methylation-sensitive restriction enzyme that can cleave both unmethylated (5′-GCGCGC-3′) and hemimethylated (5′-


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GCmGCGC-3′) DNA duplexes, but the cleavage can be blocked by fully methylated DNA duplexes18. And then in situ RCA on microprobe can be triggered by single-stranded tail in intact dsDNA upon further addition of padlock probe, T4 DNA ligase and phi29 DNA polymerase, producing long tandem-repeat sequences. Finally, multiple FAM-labeled detection probes (DP) are hybridized to RCA products, resulting in a greatly amplified enrichment of fluorophores on one single integrated microprobe. In contrast, if the Dnmt1 is absent, the hemimethylated dsDNA will not be fully methylated and can be cleaved into two parts by BssHII. Then the subsequent in situ RCA on microprobe will be prevent due to the separation of the single-stranded tail from the single integrated magnetic microprobe entity, leading to weak fluorescence background signal. As such, the Dnmt1 activity analysis can be achieved by analyzed the fluorescence on the single microprobe with fluorescence microscope. It is noteworthy that the proposed single integrated magnetic microprobe-based strategy has several advantages for Dnmt1 activity assay. Firstly, the concept that one single integrated magnetic microprobe serves as the signal amplification and enrichment platform contributes to the obtaining of highly-concentrated fluorescence signals. Despite small amount of fluorescence signals are produced, these fluorescence signals can be all enriched on one single microprobe to avoid signals loss. Meanwhile, the efficient in situ RCA on microprobe can achieve multiple fluorescence accumulation, thus the single integrated magnetic microprobe-based strategy can offer much increased detection sensitivity. Furthermore, taking advantage of the capacity of magnetic separation, the strategy can possess high tolerance capability towards complex matrix,


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avoiding fluorescence background from the complex clinical biosamples and increasing the detection accuracy.

Scheme 1. Schematic illustration of the single integrated magnetic microprobe-based strategy for Dnmt1 activity analysis. Characterization of the Integrated Magnetic Microprobe. First, we could observe that the obtained streptavidin magnetic microbeads are uniform in size with a mean diameter of 45 µm through the bright-field microscope (Figure S2). Next, non-denaturating polyacrylamide gel electrophoresis (PAGE) experiment was carried out to characterize the hybridization of R1 and R2. Figure S3A showed that R1 could completely hybridize with R2 to form stable dsDNA. Then, the formation of integrated magnetic microprobe was confirmed by fluorescence measurement of double-strand specific dye-Sybr Green I (SGI) and fluorescence image of hybridization between single-stranded tail of dsDNA and complementary FAM-labeled DNA sequence (CP) (Figure S4). As shown in Figure S4, the system of MBs showed very low


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fluorescence intensity, but the system of integrated magnetic microprobe exhibited appreciable enhancement of fluorescence intensity (a bright halo around the MBs surface in the fluorescence image), indicating that the functionalized dsDNA was anchored on the surface of MB and fluoresced via binding with SGI or CP. Meanwhile, the reaction time between functionalized dsDNA and MBs was also optimized to ensure best immobilization effect (Figure S5). Verification of the Single Integrated Magnetic Microprobe-Based Strategy. To confirm the feasibility of the single integrated magnetic microprobe-based strategy for Dnmt1 activity assay, agarose gel electrophoresis and fluorescence images were recorded. There was a distinct band with high molecular weight in lane a (Figure S3B), suggesting that the single-stranded tail in functionalized dsDNA could trigger RCA process. As shown in Figure 1, in the control system without Dnmt1, there was no distinct fluorescence signal (Image D), indicating the fact that in the absence of Dnmt1, the hemimethylated dsDNA in integrated magnetic microprobe was cleaved into two parts by BssHII and the single-stranded tail for in situ RCA was separated from integrated microprobe, thus subsequent in situ RCA and signal binding process on the single microprobe were blocked. However, when Dnmt1 was added into the reaction system, bright halo around the MB surface was observed in both the system of single integrated microprobe (Image A) and multiple integrated microprobes (Image C), suggesting that the hemimethylated dsDNA was specifically recognized and catalyzed to fully methylated dsDNA by Dnmt1 to protect it from the cleavage of BssHII and trigger the subsequent in situ RCA and DP binding process. Notably, when treated with the same concentration of Dnmt1, the fluorescence signals


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produced by multiple microprobes system were dispersed on the surface of multiple MBs, leading to weak fluorescence signals diluted on each MB (Image C). Conversely, the same amounts of fluorescence signals produced by single microprobe system were highly concentrated on a single MB, resulting in significant enhancement bright halo on a single MB (Image A). These results demonstrated the single integrated magnetic microprobe-based strategy could achieve more sensitive detection than multiple microprobes-based system. Furthermore, compared to Image B, Image A showed a much enhanced fluorescence intensity, proving the remarkable amplification performance and satisfied fluorescence accumulation effect of in situ RCA on the surface of MB. All these results demonstrated that the proposed single integrated magnetic microprobe-based strategy could be adopted for ultrasensitive detection of Dnmt1 activity.

Figure 1. Fluorescence imaging results for Dnmt1 activity assay under different conditions. (A) Single integrated magnetic microprobe system for detection of 10 U/mL Dnmt1; (B) Single


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integrated magnetic microprobe system without RCA process for detection of 10 U/mL Dnmt1; (C) Multiple microprobes system for detection of 10 U/mL Dnmt1; (D) Control system without Dnmt1. PMT HV for imaging is 450 V. Optimization of Experimental Conditions. Methylation time, BssHII cleavage time and BssHII concentration play key roles in methylation and subsequent cleavage process. Padlock probe concentration and RCA time have important effects on the amplification efficiency of in situ RCA. Additionally, detection probe is used to output signal. Thus, to achieve the best detection performance, the methylation time, BssHII cleavage time, RCA time, BssHII concentration, padlock probe concentration and detection probe concentration were optimized, respectively. As shown in Figure S6, the optimized methylation time, BssHII cleavage time and in situ RCA time were 2.5 h, 2.0 h, and 2.0 h, respectively (Figure S6A–C). The optimized concentrations of BssHII endonuclease, padlock probe and detection probe were 2000 U/mL, 200 nM and 600 nM, respectively (Figure S6D–F).

Detection of Dnmt1 Activity. Under the optimized conditions, the dynamic range and sensitivity of the proposed single integrated magnetic microprobe-based strategy for Dnmt1 activity assay were evaluated. Figure 2A showed the fluorescence responses towards various Dnmt1 concentrations. It could be found that the brightness of bright halo on MB surface gradually enhanced with the Dnmt1 concentration increased from 0 to 10 U/mL. In Figure 2B (inset), it could be observed that the integrated fluorescence intensity (FI) obtained from z-stack


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fluorescence scan had a good linear fit to Dnmt1 concentration ranging from 0.01 to 0.1 U/mL. The linear equation could be evaluated as FI = 1.75 ×105 + 6.65 × 107 CDnmt (U/mL) with a correlation coefficient of 0.994 (R). According to the regulation of three times standard deviation over the blank signal, a detection limit of 0.007 U/mL was evaluated, one or two orders of magnitude lower than that of previous assays for Dnmt1 activity (Table S2). These results indicated again the signal accumulation effect of the proposed single integrated magnetic microprobe-based strategy was indeed realized and provided an ultrasensitive method for Dnmt1 activity assay. Additionally, the intra-assay and inter-assay precision of the strategy were tested under the condition of 0.1 and 10 U/mL of Dnmt1 for three times, respectively. The relative standard deviation (RSD) provided from intra-assay were 4.6% and 3.6% at 0.1 and 10 U/mL Dnmt1, respectively. And RSD from inter-assay were 8.3% and 5.2%. These results verified that the single integrated magnetic microprobe-based strategy had an acceptable reproducibility for the ultrasensitive detection of Dnmt1 activity.


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Figure 2. (A) Fluorescence images towards various concentrations of Dnmt1 from 0 (blank) to 10 U/mL. (B) Changes of integrated fluorescence intensity (FI) versus Dnmt1 concentration. Inset shows the linear relationship of FI with Dnmt1 concentration from 0.01 to 0.10 U/mL. PMT HV for imaging is 450 V. Selectivity of Dnmt1 Assay. To validate the selectivity of the proposed single integrated magnetic microprobe-based strategy for Dnmt1 activity assay, the fluorescence responses towards other DNA methyltransferases including Dam, HaeIII and AluI were tested. As shown in Figure S7, only Dnmt1 gave rise to significant fluorescence response, whereas Dam, HaeIII and AluI gave weak fluorescence responses which were comparable to that in the bank sample.


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This result suggested that the proposed strategy showed good selectivity towards Dnmt1 against other methyltransferases, which attributes to the specific site recognition of methyltransferases toward their substrate. Additionally, other endogenous biomolecules, especially endogenous biotin and biotin-binding proteins that may compete with biotinylated DNA for the binding site of integrated magnetic microprobe, also make the specific detection of Dnmt1 activity in real biological sample a great challenge. Thus the interference effects of biotin and acetyl-CoA carboxylase were examined. As shown in Figure S7, the integrated fluorescence intensities responding to 1.0 U/mL Dnmt1 with different interference components were comparable to that in the normal sample. This result demonstrated that the interference effects of these endogenous biomolecules was negligible, which may own to the fact that the almost irreversible binding of biotinylated-DNA to streptavidin (Ka = 10-15 M-1)28 makes the as-prepared integrated magnetic microprobe quite stable. In a word, all these results obviously verified the high selectivity of the proposed strategy for Dnmt1 activity assay. Assay of Dnmt1 Activity Inhibition. The regulation of aberrant Dnmt1 activity would convert the aberrant genome DNA methylation pattern or even kill cancer cells, thus Dnmt1 has become a promising target for inhibitor drug development in disease treatment. Here RG108, a novel small molecule, was chosen as a model inhibitor to investigate the capacity of our method in screening of Dnmt1 inhibitor. RG108 is a noncovalent Dnmt inhibitor that can bind at the micromolar range to the Dnmt1 pocket site and effectively block Dnmt1 activity but not cause covalent enzyme trapping in human cell lines. Thus different concentrations of RG108 were


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utilized to evaluate the inhibition effect of RG108 on Dnmt1 activity by comparing the integrated fluorescence intensities. The relative activity (RA) of Dnmt1 was calculated with the equation as following:

where FI0, FIt and FIi are the integrated fluorescence intensities in the absence of Dnmt1, in the presence of Dnmt1, in the presence of both Dnmt1 and RG108, respectively. As shown in Figure 3, the RA of Dnmt1 decreased with the increasing concentration of RG108, indicating that the inhibition effect of RG108 on Dnmt1 was in significant dose-dependent manner. The IC50 value, which expresses the inhibitor concentration required to cause a 50% decrease of Dnmt1 activity, was found to be 110 nM. These results were consistent with the prior report29 and demonstrated that the developed single integrated magnetic microprobe-based strategy could be applied in Dnmt1 inhibitors screening and then serve to quantitatively regulate Dnmt1 activity for anticancer therapeutics.


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Figure 3. Dose-dependent inhibition effect on Dnmt1 activity by RG108. Inset shows the chemical structure of RG108. PMT HV for imaging is 450 V. Detection of Dnmt1 Activity from Cell Lysates. To demonstrate the availability of the proposed single integrated magnetic microprobe-based strategy for cellular Dnmt1 activity profiling, the Dnmt1 activity in cell lysates was further evaluated by this assay. As demonstrated in Figure 4A, no fluorescence signal was observed for the cell lysis buffer. However, the fluorescence signal of MCF-7 cell extracts system (20000 cells) had significant enhancement, showing Dnmt1 activity is highly expressed in MCF-7 cells. To confirm the fluorescence signal was produced by Dnmt1 activity rather than any other component in cell extracts, RG108 was further added into the cell extracts. Not surprisingly, the Dnmt1 activity was suppressed by RG108, and relatively weak fluorescence on MB surface was detected, revealing that the proposed strategy showed high tolerance capability for cellular components and was reliable for evaluate Dnmt1 activity in cellular environment. Moreover, to test the potential of the proposed strategy for different cell lines in clinical diagnostics, the Dnmt1 activity in HeLa cell and normal cell line HL-7702 extracts were further investigated (Figure S8). Results showed that the method could differentiate the cancer cells from normal cells on the basis of the fact that Dnmt1 is overexpressed in human tumors. To further verify the integrated capacity of high sensitivity and high tolerance capability for cellular components of the proposed single integrated magnetic microprobe-based strategy, Dnmt1 activity evaluation in cell samples containing single, 10 and 100 cancer cells were


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performed. As shown in Figure 4B, there was no fluorescence signal on MB in blank samples (cell lysis buffer). In contrast, positive fluorescence signal that could be differentiated from the blank was observed in the system containing single, 10 and 100 MCF-7 or HeLa cells, respectively. Additionally, the brightness of the bright halo enhanced with the increasing of cell number, in accordance with the fact that Dnmt1 activity increases with cell number increased. Subsequently, to verify the possibility for highly concentrated cell extracts that contains higher levels of other interferants, the Dnmt1 assays in mixture cell extracts of cancer cells doped in normal cells were performed. As shown in Figure 4C, there was no fluorescence signal in the system of 100 HL-7702 cells, while obviously enhanced fluorescence signal could be observed from the system of 10 MCF-7 cells as well as the systems of 10 MCF-7 cells doped in 10, 100 HL-7702 cells, respectively. All these results indicate that our proposed strategy is a reliable candidate tool for Dnmt1 activity analysis from cancer cells even at the single-cell level or doped within a large background of Dnmt1-negtive cells, showing great potential for Dnmt1 activity assay in clinical samples.


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Figure 4. (A) Fluorescence signal response to the Dnmt1 activity in MCF-7 cells extracts (20000 cells), and the inhibitory effect of 1000 nM RG108 on the Dnmt1 activity in the MCF-7 cells extracts. PMT HV for imaging is 450 V. (B) Fluorescence images towards different cell extracts samples containing single, 10, and 100 MCF-7 or HeLa cells. PMT HV for imaging is 550 V. (C) Fluorescence images towards mixture cell extracts samples of 10 MCF-7 cells doped in 0, 10, 100 normal HL-7702 cells. PMT HV for imaging is 550 V.


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CONCLUSIONS In summary, we have developed an ultrasensitive and accurate single integrated magnetic microprobe-based strategy for Dnmt1 activity detection at the single-cell level. The single integrated microprobe serves as an entity to enrich fluorescence signals by integrating Dnmt1catalyzed DNA methylation, site-specific cleavage by BssHII and methylation protect in situ RCA on microprobe. Due to the cooperative fluorescence signal accumulation effect of single microprobe and in situ RCA, excellent analytical performance is obtained for the strategy with an ultralow detection limit of 0.007 U/mL Dnmt1. More importantly, combined with the high tolerance capability for cellular components owed to magnetic separation, the single integrated magnetic microprobe-based strategy is successfully applied to detection Dnmt1 activity in multiple cell lysate even at the single-cell level. Furthermore, the inhibition effect of RG108 can be evaluated conveniently. Therefore, this robust single integrated magnetic microprobe-based strategy shows potential application in sensitive quantitatively monitoring of Dnmt1 activity and screening of anticancer drugs that may serve to DNA methylation-related clinical diagnosis and therapy. Furthermore, the single integrated magnetic microprobe-based strategy proposed here has great potential in assays of many other biological analytes by the change of functionalized DNA on the surface of microbead.


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ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. DNA sequences (Table S1), illustration of z-stack confocal fluorescence scanning mode by cutting a single MB into 10 slices along the z-direction (Figure S1), size of obtained streptavidin magnetic microbeads (Figure S2), gel electrophoresis characterization of functional dsDNA and RCA on MB surface (Figure S3), principle and characterization of the formation of integrated magnetic microprobe (Figure S4), optimization the reaction time of functional dsDNA and MBs (Figure S5), optimization of the experimental conditions of single integrated magnetic microprobe-based sensing system (Figure S6), the selectivity of the proposed single integrated magnetic microprobe-based strategy for Dnmt1 activity assay (Figure S7), Dnmt1 activity detection in HeLa cell and normal cell line HL-7702 extracts (Figure S8) and comparison of analytical performance (Table S2) (PDF).

AUTHOR INFORMATION

Corresponding Author

* E-mail: [email protected]. Tel: +86-531-88363888. Fax: +86-531-88564464.

Notes

The authors declare no competing financial interest.


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ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21375078, 21475077, 21675100, 21675101).

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Table of Contents Graphic


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Ultrasensitive and Accurate Assay of Human Methyltransferase Activity

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