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On-Chip Sequence-Specific Immunochemical Epigenomic Analysis utilizing Outward Turned Cytosine in a DNA Bulge with Handheld Surface Plasmon Resonance Equipment Ryoji Kurita, Hiroyuki Yanagisawa, Kyoko Yoshioka, and Osamu Niwa Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03520 • Publication Date (Web): 20 Oct 2015 Downloaded from http://pubs.acs.org on November 2, 2015

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Analytical Chemistry

On-Chip Sequence-Specific Immunochemical Epigenomic Analysis utilizing Outward Turned Cytosine in a DNA Bulge with Handheld Surface Plasmon Resonance Equipment Ryoji Kurita*, Hiroyuki Yanagisawa, Kyoko Yoshioka and Osamu Niwa National Institute of Advanced Industrial Science and Technology (AIST) Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566 ABSTRACT: This paper reports a sequence-specific immunoassay chip for DNA methylation assessment by microfluidic-based surface plasmon resonance (SPR) detection. This was achieved by utilizing an affinity measurement involving the target, (methyl-) cytosine, in a single-base bulge region and an anti-methylcytosine antibody in a microchannel, following hybridization with a biotinylated bulge-inducing DNA probe. The probe alters the target cytosine in a looped-out state because of the π-π stacking between flanking bases of the target. The probe design is simple and consists of the elimination of guanine paired with the target cytosine from a fragmented full match sequence. We obtained the single methylation status in 6 attomoles (48 femtograms) of synthesized oligo DNA in 45 min, which is the fastest DNA methylation assessment yet reported, without employing a conventional bisulfite reaction, PCR or sequencing. We also succeeded in the discrimination of the methylation status of single cytosine in genomic lambda DNA and HCT116 human colon cancer cells. The advantages of the proposed method are its small equipment, simple microfluidics design, ease of handling (two injections of DNA and antibody), the lack of a need for a methylation-sensitive enzyme, and a neutral buffer condition.

INTRODUCTION DNA methylation, especially the addition of a methyl group at the fifth position of the cytosine base (5’methlcytosine) in mammalian cells, has received particular attention because it is thought to play a crucial role in controlling genetic expression1, including that in cancer 2-5, genomic imprinting6, cellular differentiation and Alzheimer’s disease7. Now, a growing number of human diseases have been found to be associated with aberrant DNA methylation 5,6. Various methods have been designed for methylcytosine determination in pursuit of various research goals. There are two types of DNA methylation analysis methods, one is the global methylation analysis of genomewide DNA, and the other is the site-specific methylation analysis of a particular DNA region. Global DNA methylation analysis has been undertaken by employing methylated DNA immune-precipitation 8 and the microarray-based integrated analysis of methylation with isoschizomers 9,10. These methods are performed by using digestion with a methylation-sensitive restriction enzyme and a microarray analysis, followed by enrichment with an anti-methylcytosine antibody or a methylated DNA-binding protein. As regards site-specific methylation analysis, the bisulfite-based analytical method is the most widely used approach for distinguishing between cytosine and methylcytosine 11-16. Treatment with bisulfite converts cytosine to uracil, while methylcytosine remains unaffected. Therefore, the methylation status of cytosine in DNA can be obtained by comparing bisulfite treated and untreated samples. For exam-

ple, bisulfite sequencing 17, combined bisulfite restriction analysis (COBRA) 18, methylation-specific PCR 19 and pyrosequencing 20 can provide the methylation status of a specific sequence at a single CpG level. However, the main drawbacks of the bisulfite-based determination methods are the degradation of the sample DNA and the treatment time. More than 99 % of the original DNA is reportedly destroyed after a standard bisulfite treatment lasting 16 hrs 21. Rapid and site-specific methylcytosine assays have been proposed 22-26 that do not require bisulfite treatment, PCR or a sequencer. This is because important CpG sites have been found that significantly affect gene regulation with only a single methylation, or that are closely related to diseases27-29, namely the focus of methylation studies has gradually shifted from the global methylation frequency to the role of each methylation at a single CpG level. However, no method for the rapid DNA methylation assessment of a specific CpG site has yet been established. Although various PCR-free or bisulfitefree techniques such chemical or protein modification have been proposed, they are currently under development for application to long mammalian DNA because they pose problems as regards sensitivity and sequence selectivity. An antimethylcytosine antibody has been used for the immunoprecipitation or concentration of methyl-CpG regions in a DNA sample 30-32. Recently, immunochemical methods for detecting methylcytosine with an anti-methylcytosine antibody have been reported for analyzing the methylation level. The reported methods employ a microtiter plate 33, capillary electrophoresis 34, magnetic particles 35, microspheres 36, a nitrocellulose

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membrane 37 and a DNA microarray 38. However, previously reported immunochemical methods for detecting methylcytosine have no sequence specificity and so we can quantify only the total amount of methylcytosine in the analyte DNA. Therefore, there is a real need to be able to detect single methylcytosine and its position in the DNA sequence. Recently, we found that an anti-methylcytosine antibody can recognize mismatched methylcytosine especially in a bulge region, but cannot recognize methylcytosine in a pair. 39,40 This is because methylcytosine at a single-base bulge is dominantly in a looped-out state due to the π-π stacking formation between the flanking bases of bulged methylcytosine. In contrast, methylcytosine paired with guanine is in a stacked state in a duplex. The selective looped-out state of methylcytosine in the bulge was confirmed by an enzyme-linked immunosorbent assay (ELISA) on a microtiter plate, kinetic analysis with BIACORE and a melting curve analysis in a previous study 40. The large difference between the antibody kinetics of the looped-out and stacked states implies the possibility of the sequence-specific immunoassay of methylcytosine in genome DNA. However, the bulge-specific immune recognition in a microfluidic device remains to be explored, and a single CpG methylation analysis in a mammal genome has not been achieved because the detection limit of conventional ELISA is around 1 nM, which is insufficient for application to a mammalian DNA sample. In fact, an immunoassay of methylcytosine cannot be combined with PCR amplification because both cytosine and methylcytosine are amplified as cytosine. Furthermore, several hours are needed to complete a conventional ELISA on a microtiter plate. Therefore, the previous result fell far short of our goal of providing a rapid and on-site DNA methylation analysis on a genome. In this paper, we report the first sequence-specific cytosine methylation assessment from human genomic DNA without PCR, sequencing or electrophoresis. A surface plasmon resonance (SPR)-based microfluidic device has been developed for the immunochemical epigenomic analysis of a single cytosine in a DNA bulge. The bulge-specific immune recognition on the SPR device allows us to achieve the fastest DNA methylation assessment yet reported.

EXPERIMENTAL SECTION Materials Lambda DNA (Promega, D1521), which exhibited neither dam nor dcm methylase activity, was used as an unmethylated DNA. As a methylated DNA, unmethylated lambda DNA was methylated with CpG methyltransferase (New England Biolabs) as follows. 150 µg of unmethylated DNA was methylated with 8 units of CpG methyltransferase in the presence of 0.64 mM S-adenosylmethionine as a methyl donor at 37 °C overnight in a buffer as supplied by the manufacturer (50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9). 15 µL of 3 M sodium acetate (pH 5.2) was mixed with 125 µL of the methylated DNA sample, and then ethanol was added. The mixture was centrifuged at 12000 rpm for 30 min at 4 °C. After the supernatant was discarded, the DNA was rinsed with 70 % ethanol, and centrifuged. After the supernatant was discarded and air dried, distilled water was added and the DNA

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concentration was measured with a NanoDrop 2000 (ThermoScientific). Methylated and unmethylated genomic human DNAs were obtained from Takara-Bio Inc. (Shiga, Japan). Methylated DNA was derived from human HCT116 cells, and enzymatically methylated with CpG Methylase and purified with phenol/chloroform. Unmethylated DNA was purified from a DNMT double knock-out human HCT116 cell line, which is genetically lacking in both methyltransferases DNMT1 and DNMT3B. The bulge-inducing probe DNA was synthesized and purified with high performance liquid chromatography by GeneDesign Inc. Pretreatment of genomic DNA Before injecting a sample DNA into our microchip, genomic DNA was fragmented by a restriction enzyme. Lambda DNA was fragmented by AluI (Takara Bio) in a buffer as supplied by the manufacturer. Then, 14.4 µg (32 ng/µL in 450 µL) of the fragmented lambda DNA and 10 nM biotinylated bulge-inducing DNA were mixed. Finally, the mixture solution was heated at 95 °C for 5 min, and then cooled to room temperature for hybridization. Human DNA was fragmented with Xsp (Takara Bio). 10 µg of human DNA was fragmented with 100 units of XspI at 37°C for 30 min. The fragmented DNA was then mixed with 10 nM of probe DNA. The mixture solution was heated at 95 °C for 5 min, and then cooled to room temperature for hybridization. The solution was mixed with 50U of Exonuclease I, and was incubated at 37°C for 30 min. This treatment is effective in reducing the background level because the unhybridized DNA was digested. Microchip fabrication procedure A microchip consists of a poly-dimethylsiloxane (PDMS) plate with a microchannel and a glass plate with two thin gold films. The PDMS plate was formed using previously reported procedures with a minor modification. The master pattern was fabricated on a glass wafer with a conventional photolithographic technique. Then dimethylsiloxane oligomer (Dow Corning Asia, Japan) was poured on the glass wafer. After polymerization for 60 min at 60 °C, the PDMS layer was peeled from the master. As regards the glass plate, we first sealed it using an adhesive sheet with two holes 2 mm in diameter. We then deposited a thin titanium layer on the glass plate with the adhesive sheet using RF sputtering equipment, and formed a gold film without breaking the vacuum. After removing the adhesive sheet, we dipped the glass plate in 1 mM carboxydecanethiol to modify both gold film surfaces by gold-thiol binding. Then, we modified one of the films with streptavidin using a carbodiimide coupling reaction provided by Nhydroxysulfosuccinimide (sulfo-NHS) and N, N’- diisopropylcarbodiimide (DIC). The carboxyl group was activated by a mixture solution consisting of 5 mM sulfo-NHS and 40 mM DIC in MES buffer (pH 6.0) for 30 min, and then 0.1 mg/mL streptavidin in PBS (pH7.4) was reacted for 2 hrs. After washing with PBS, 0.1 M ethanolamine was reacted for 15 min to

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deactivate the unreacted esters. Finally, a PDMS plate with a microchannel was put on the glass plate. Designs of microchip and measurement system Bulge-specific immune recognition allows the microchip design to be very simple in that it contains just a straight microchannel with two gold films (figure 1). The microchip (18×18 mm) was constructed from PDMS with a 20µm-deep flow channel and a BK7 glass plate with 50-nm-thick gold films for SPR measurements. One of the gold films was modified with streptavidin to allow it to accumulate a biotinylated DNA duplex. The other was modified with carboxydecanethiol, which was used as a reference for monitoring nonspecific absorption. Detailed dimensions of the microchip are shown in figure 1S in the supporting information. Figure 2 is a schematic of the measurement procedure. First, genomic DNA was fragmented by using a restriction enzyme above mentioned, and then mixed with a biotinylated probe DNA to form a bulge region once it had hybridized with the fragmented target sequence. Next, the mixture solution was heated, and then cooled to room temperature for hybridization with the probe. The mixture solution was injected into the microchip for 30 min at a flow rate of 2µL/min by a syringe pump so that the biotinylated duplex accumulated on the streptavidin surface. After the DNA duplex accumulation was complete, we started to measure the SPR angle. An anti-methylcytosine antibody was injected for 15 min, and the shift in the SPR angle caused by the immunoreaction between the antibody and duplex was measured. The shift in the SPR angle was measured with a hand-held SPR system, which was developed in collaboration with NTT-AT Corp. The SPR system is 15 cm wide, 7 cm deep and 10 cm high, and weighs about 740 g.

Figure 1 Photographs of a) a microchip for assessing the DNA methylation, and b) hand-held SPR equipment. c) Schematic of antibody binding with target methylcytosine in a DNA bulge region.

Figure 2 Schematic procedure for pretreatment and measurement of sequence-selective immunochemical epigenetic analysis on a microchip. a) Fragmentation by restriction enzyme b) Mixing with a biotinylated bulge-inducing probe DNA. The probe has a complementary sequence to hybridize with the fragmented sequence of interest, however it lacks guanine paired with the target cytosine. 3) Heat denaturation at 95°C for 5 min followed by cooling to room temperature. 4) Injection of the mixture solution into a microchip for 30 min, and anti-methylcytosine antibody for 15 min.

Melting curve and melting temperature Melting curves were obtained by measuring the changes in absorption at 260 nm as a function of temperature with a UV-Vis spectrophotometer (Shimadzu, Model UV1800). The melting temperature (Tm) was determined as the temperature corresponding to the maximum value in the firstderivation profile of the melting curves with Tm analysis software (Shimadzu, TMSPC-8). Combined Bisulfite Restriction Analysis (COBRA) COBRA was performed to assess the DNA methylation of the target cytosine for comparison with our microchip. Bisulfite treatment was performed using a commercial kit (Xceed Rapid DNA Bisulfite Modification Kit, catalogue no. GR004, Takara Bio) according to the manufacturer’s manual. 2 µg of DNA was used for each bisulfite modification. PCR amplification was performed in a 50 µL volume solution comprising 40 ng (2 µL in 20 ng/µL) of bisulfite treated DNA, 1.25 Units of DNA polymerase (Takara Bio), 5 µL of 10 x Epi PCR buffer (Takara Bio), 2.5 mM MgCl2, 0.3 mM dNTP mixture and 1 µM of each primer. The forward and reverse primer sequences were 5’-GTG ATT TAT TGA ATT TGT AG-3’, and 5’-ATA ACC TCT TCC AAC CAA CA-3’, respectively. PCR amplification consisted of 30 cycles at 94 °C for 30 sec, at 55 °C for 30 sec, and at 72 °C for 30 sec. 1 µL of PCR products was digested with 10 units of restriction enzyme

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HpyCH4IV (New England Labs) for 1 hour according to the manufacturer's instructions. HpyCH4IV had one site (no sites) in the bisulfite-converted sequence (5’-ACGT-3’) when the original sequence was methylated (unmethylated). The digested PCR products were separated using electrophoresis equipment (Bioanalyzer 2100, Agilent Technology).

RESULTS AND DISCUSSION Characterization with oligo-DNAs First, we used the synthesized oligo-DNAs listed in table 1S to investigate the performance of our microchip as follows. The concentration of the probe DNA was optimized by changing the concentration of Probe-2 for 1nM Analyte-1. The SPR response increased as the probe DNA concentration increased as shown in figure 2S in the supporting information. This is reasonable because the amount of hybridized and captured DNA duplex on a streptavidin-modified SPR surface increased. However, the SPR response became saturated around 100 nM since the amount of accumulable biotin on the surface was limited. Moreover, the SPR response decreased under a large excess probe DNA concentration. This is because the ratio of the unhybridized probe DNA increased, and this DNA was largely captured on the surface. To obtain a sensitive and stable SPR response, 10 nM probe DNA was used in this study. The antibody was injected at 0.5 ~ 100 µg/mL to optimize its concentration as shown in figure 3S (a). 10 µg/mL was used in this study because of the saturation of the SPR response around this concentration. Figure 3S (b) shows typical SPR sensorgrams when 10 µg/mL antibody was injected into our microchip. The SPR angle gradually increased and reached a steady state as a result of the mass transfer limitation after 10 min. The SPR sensorgram reached 90 % of the steady state after about 430 sec, which is longer than that (220 sec) taken by the BIACORE system in our previous report 40. In our previous report, the antibody was injected into a microchannel (0.5 mm wide, 40 µm deep) at a flow rate of 10 µL/min, therefore the linear flow rate was 500 mm/min. In this report, the linear flow rate was 50 mm/min (2 mm wide, 20 µm deep, 2 µL/min). We expect to achieve a more rapid assay with our microchip at a high flow rate. Unfortunately, the flow became unstable above 2 µL/min because air bubbles formed in the microchannel, which was made of gas permeable PDMS. Therefore, 2 µL/min was adopted with a view to realizing a stable and sensitive DNA methylation assessment in the following experiments. Next, the oligo-DNA containing methylcytosine was hybridized with 1, 3, 5 or 7 mismatched bases to form a bulge region or a full match in a DNA duplex, and then each calibration curve was obtained as shown in figure 4S. The SPR angle increased as the analyte DNA concentration increased when the target cytosine was methylated. This is reasonable because the amount of hybridized and trapped analyte DNA in a microchannel increases. In contrast, no increase was observed when the target cytosine was unmethylated up to the µM range. Moreover, no increase was observed even when the target cytosine was methylated in a full match sequence. These results indicate that the methylcytosine in the bulge was selectively detected with our microchip, whereas the methylcytosine in a pair was not. This is because an anti-methylcytosine

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antibody cannot recognize methylcytosine in a stacked state in a DNA duplex owing to the large antibody size 40. However, the methylcytosine in the bulge can rotate freely since deoxyribose and phosphate groups in a DNA chain are connected with a single bond as shown in figure 1(c). A large increase was observed in the SPR angle at a single-base bulge, and no significant increase was observed as the bulge size was increased to seven bases as shown in figure 4S. Detection in a single-base bulge is advantageous for methylation analysis at a single-base level. Furthermore, a duplex is thermally stable as shown in figure 5S. The thermally stable duplex with a singlebase bulge would be useful in terms of collecting an analyte sequence from fragmented genomic DNA when later performing an epigenome analysis. The detection limit of our microchip, which is defined as three times the standard deviation of the negative control, was 0.1 pM as shown by a calibration curve in a low concentration range (figure 6S). Also, 0.1 pM of bulged methylcytosine can be clearly detected without any crossover of error bars of 0.1 pM of methylcytosine in a pair. The detection limit of our microchip is about seven orders of magnitude better than that of chemical modification methods 23,25 (around 1 µM) with respect to methylcytosine combined with electrophoretic analysis or direct electrochemical detection. Furthermore, it is four orders of magnitude better than our previous result on a conventional microtiter plate 40. This is due to the large surface-to-volume ratio in a microchannel of our microchip, which we obtained by a flow measurement. A trace level of biotinylated duplex can be effectively accumulated on a streptavidin surface thanks to the 20-µm-thick layer channel and the high affinity of avidin-biotin interaction (KD is approximately 10-15). Since we injected 0.1 pM oligoDNA for 30 min at a flow rate of 2 µL/min, we were able to distinguish a methylation status of about 6 attomoles (48 fg) of DNA with two simple injections (sample DNA and an antibody) into our microchip.

Figure 3 a) Schematics of DNA duplex forms between Probe-2 and Analyte-1~4. The sequences of Analyte-1~4 are the same,

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however the methylation position is different. The filled red star indicates methylcytosine, and the open black star indicates nonmethylated cytosine. b) Results for sequence specificity in the same sequence.

sistent with that obtained with 1 nM of synthesized oligo DNA in figure 7S.

The selectivity for methylcytosine in a bulge against non-target methylcytosine in the same duplex was further investigated as follows. Figure 3 (a) shows schematics of a DNA duplex, and the sequence is shown in table 2S. The sequences of the analyte DNAs are the same, however, the sequence of the methylation region is different. The SPR angle increased for Duplex-1 and 3, however no increase was observed for Duplex-2 and 4 as seen in figure 3 (b). This is because the target cytosines in the bulges in Duplex-1 and 3 are methylated, whereas those in the bulges in Duplex-2 and 4 are not. The important point is that no SPR angle increase was observed in Duplex-2, which contains one methylcytosine in the non-target region. Moreover, no significant difference was observed between Duplex-1 and Duplex-3. These results clearly demonstrate that a highly selective methylation status can be obtained with our microchip for a target cytosine without being affected by the non-target methylation status, even though a fragmented analyte DNA from a genome contains a non-target methylcytosine. Lambda genomic DNA measurement Next, we obtained calibration curves for methylated and unmethylated genomic lambda DNAs. The genomic DNAs were fragmented with a methylation-insensitive restriction enzyme (AluI), and then mixed with a biotinylated probe DNA to form a single-base bulge at a target cytosine where it hybridizes with the fragmented target sequence. Figure 4 (a) shows calibration curves for methylated and unmethylated lambda DNAs with our microchip. The SPR angle increased as the concentration of methylated lambda DNA increased. The relative standard deviations (RSD) for the calibration curve of the methylated lambda DNA in figure 4(a) were 6.3 % (1 pM) and 6.9 % (10 pM). In contrast, there was little increase for the unmethylated lambda DNA. The detection limit of lambda genomic DNA is around 0.1 pM (0.19 ng), which is the same as that of oligo-DNA described in figure 6S. This suggests that the biotinylated probe DNA hybridizes sufficiently with a target sequence from the fragmented genomic DNA, and an intended bulge is formed at the target cytosine. We can also estimate the methylation ratio at a target cytosine with our microchip as follows. Methylated and unmethylated lambda DNAs were blended to form a model DNA containing 0, 25, 50, 75 and 100 % methylcytosine. The methylation ratio of the target cytosine in the DNA was confirmed from a conventional site-specific methylation analysis by COBRA. As shown in figure 4(b), we successfully obtained a high correlation between the methylation ratio of a target cytosine in lambda DNA and the SPR signal (r2= 0.9942) without losing the correlation when measuring the methylation ratio of a blended negative and the positive control of synthesized oligo-DNAs (r2= 0.9904) (figure 7S). Furthermore, the 14.4 µg of lambda DNA in figure 4(b) (final concentration of 32 ng/µL) is assumed to contain around 1 nM of the target sequence, i.e. the SPR angle shift value in figure 4 (b) is con-

Figure 4. a) Calibration curves for methylated and unmethylated lambda DNAs. b) Results of a methylation ratio assessment of a target cytosine from genomic DNA. Methylated and unmethylated lambda DNAs were mixed to form a model DNA.

Methylation assessment in human genome Finally, we assessed methylated and unmethylated genomic DNAs of HCT116 human colon cancer cells with our microchip. The single methylation status of the target cytosine was detected without any crossover of the error bar shown in figure 5. It is difficult to compare the SPR angle shift value with the synthesized oligo and lambda DNA results in figures 3 and 4, respectively. However, we believe that the value in figure 5 is consistent in consideration of the fact that the full genome lengths are different and target LINE-1 is found in large numbers in the human genome. Our microchip has advantages over conventional site-specific methylation analysis in terms of its short assay time, small equipment, ease of handling, simple probe design, a mild buffer condition, and no need for a methylation-sensitive enzyme. Our microchip does not require bisulfite treatment, which often involves an over-

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night reaction. The measurement time for our microchip is 45 min; 30 min is needed for the accumulation of a biotinylated duplex and 15 min is needed for measuring the affinity between the duplex and antibody. For a genomic DNA measurement, one additional hour was required for fragmentation by a restriction enzyme. Even so, we achieved the fastest genomic DNA methylation assessment yet reported. Furthermore, the bisulfite treatment is prone to variability due to DNA degradation under the required acidic condition. More than 99 % of the original DNA is reportedly destroyed by the bisulfite treatment 21. With our microchip, the methylation status can be obtained under a biologically neutral solution throughout the assay. This means that the degradation can be minimized. Moreover, the bisulfite treatment makes the analyte DNA thymine-rich since unmethylated cytosine is converted to thymine, and this complicates and limits the design of specific probes for PCR amplification. The probe design for our microchip is quite simple, because it requires solely the elimination of guanine paired with the target cytosine from a complementary sequence for the fragmented analyte DNA. Furthermore, a methylation-insensitive restriction enzyme such AluI and XspI is used to fragment the genomic DNA, i.e. a wide variety of methylation-insensitive enzymes are applicable. A conventional site-specific methylation assay with a methylation-sensitive restriction enzyme is limited because it depends on the available methylation-sensitive restriction enzyme. In addition, the DNA methylation status can be obtained by performing two simple injections with hand-held SPR equipment. This would contribute to the future realization of a fast-working research tool for clinical DNA methylation assessment because some significant methylations have recently been discovered that are closely related to diseases with a single CpG level.

Figure 5. Results of a methylation assessment of human genome DNAs

CONCLUSIONS We report a microfluidic chip for the sequenceselective immunochemical discrimination of the cytosine methylation status in a genomic DNA. The sequence-specific discrimination with an anti-methylcytosine antibody is realized by utilizing the fact that the target methylcytosine in the

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bulge region is predominantly in a looped-out state from a DNA duplex. We achieved the fastest DNA methylation assessment yet reported using the microfluidic based surface plasmon resonance detection. This was mainly because there was no need for conventional bisulfite treatment, which generally requires an overnight reaction in an acidic condition. Other advantages of the proposed method are its small equipment, ease of handling (two injections of DNA and antibody), simple probe and microfluidics designs, the lack of a need for a methylation-sensitive enzyme, and a neutral buffer condition through the assay.

ACKNOWLEDGEMENTS Part of this project was supported by JSPS KAKENHI, Grant No. 26410168. We thank Mr. D. Meacock for revising the language of the manuscript.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * [email protected]

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