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A multi-enzyme cascade bioreactor for ten-minute digestion of genomic DNA into single nucleosides and quantitative detection of structural DNA modifications in cellular genomic DNA Junfa Yin, Shaokun Chen, Ning Zhang, and Hailin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05399 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 9, 2018

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ACS Applied Materials & Interfaces

A multi-enzyme cascade bioreactor for ten-minute digestion of genomic DNA into single nucleosides and quantitative detection of structural DNA modifications in cellular genomic DNA Junfa Yin1, Shaokun Chen1,2, Ning Zhang1 and Hailin Wang1,2* 1

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China, 2 University of Chinese Academy of Sciences, Beijing 100049, China

ABSTRACT: Identification and quantification of chemical DNA modifications provides essential information on genomic DNA changes, e.g., epigenetic modifications and abnormal DNA lesions. In this vein, it requires to digest genomic DNA strands into single nucleosides, facilitating the mass spectrometry analysis. However, rapid digestion of such supramacromolecules DNA of several millions Daltons (M.W.) into single nucleosides remains very challenging. Here we constructed an immobilized benzonase capillary bioreactor, and further tandemly coupled with immobilized snake venom phosphodiesterase (SVP) and alkaline phosphatase (ALPase) capillary bioreactor to form a novel three-enzyme cascade bioreactor (BenzoSAC bioreactor). In these constructions, the chosen enzymes were immobilized onto synthetic porous capillary silica monoliths. With the tailor-made porous structure and high immobilized capacity and high digest rate of benzonase, genomic DNA of > 99.5% can be digested into single nucleosides within only 10 min when passing through the BenoSAC bioreactor by microinjection pump. In contrast, traditional digestion requires 8–24 h. By offline coupling this benzoSAC bioreactor with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we detected 5-hydroxymethyl cytosine (5hmC), a major oxidation product of the epigenetically crucial 5-methylcytosine (5mC), in genomic DNA isolated from ladder cancer (T24) cells. The newly synthesized BenzoSAC bioreactor and the proposed MS detection are promising for fast identification and analysis of structural modifications in DNA.

Keywords: bioreactor, DNA digestion, DNA modification, benzonase, 5-hydroxymethyl cytosine INTRODUCTION Environmental exposure and normal metabolic activities inside the cell can cause DNA damages, occur at a rate of 10,000 to 1,000,000 lesions per cell per day.1 Unrepaired lesions in critical genes (e.g., tumor suppressor genes) can lose or change the normal function and appreciably increase the risk of tumor incidence, becoming a key step towards the onset of cancer.2-4 Damages occurs via structural modifications of the bases, deoxyribose, and phosphate moieties in DNA chains.5 It is difficult to measure these rare but aberrant modifications in cellular DNA since its double strands constitute normal nucleosides of million folds. In this regard, an ideal and demanding technology for predicting the relationship between DNA damages and diseases should be capable of exploring whether and how the covalent interactions modify the nucleotides, probing damaged DNA removal, screening the exact location of damage sites on the DNA and last, being sensitive enough to quantify these lesions. In addition to aberrant DNA damages, there are a few epigenetic modifications, e.g., DNA methylation and hydroxylmethylation.6 Liquid chromatography-tandem mass spectrometry (LCMS/MS) methods have been developed to provide structural information of modified DNA and to size and sequence oligonucleotides (≤ 20 bp).7,8 To determine DNA modifications, it is essential to digest DNA into single nucleosides.5, 9-13, which are easily ionized for more sensitive MS detection compare to

DNA polynucleotides or mononucleotides. For instance, during LC-MS/MS analysis of global DNA methylation (e.g. 5methylcytosine, 5mC), hydroxylmethylation (e.g., 5hydroxymethylcytosine, 5hmC), and oxidatively generated DNA damages (e.g., 8-Oxo-7,8-dihydro-2’-deoxyguanosine, 8-oxodG), the genomic DNA samples were first digested into the mixture of for canonic 2’-deoxynucleosides and modified 2’-deoxynucleosides.14-17 Essentially, DNA digestion is time-consuming and is often considered as a bottleneck step in LC-MS/MS-based DNA analysis workflow. This step often requires a set of distinct enzymes, involving deoxyribonuclease, phosphodiesterase and alkaline phosphatase (ALPase).17-22 Deoxyribonuclease (e.g. DNase I) are used to cut DNA polymers into short oligonucleotides. Phosphodiesterases (e.g. snake venom phosphodiesterase (SVP); or bovine spleen phosphodiesterase (BSP)) attack the terminal OH-groups of DNA to release mono-nucleotides. Meanwhile, alkaline phosphatase is responsible for removing phosphate from the released mononucleotides. Traditionally, DNA digestion takes 8 - 24 h.15-17 Recently we constructed a three-enzyme cascade capillary bioreactor that consists of immobilized DNase I, SVP and ALPase, by which genomic DNA strands could be fast digested to nucleosides within 45 min.27 This innovation eliminates ultrafiltration step (for digesting enzyme removal) and reduce possible artificial oxidation of DNA.23-25 Interestingly, single SVP-immobilized capillary bioreactor can be used to cleave oligodeoxynucleotides

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for MS-based sequencing and for identification of damage sites in oligodeoxynucleotides.26 It is known that enzyme-immobilized bioreactors exhibited good performances in on-line fast digestion proteins (typically within 10 minutes) coupled with LC-MS proteomics analysis,28-30 however, up to date, seldom studies reported as for utilizing immobilized enzymes for on-line digestion coupled LC-MS for DNA analysis. This is because, compared to most studies proteins, genomic DNA strands are much larger supramacromolecules (generally, M.W. > 10 million Daltons), thus more difficult to be digested into single nucleosides facilitating the followed MS analysis. On the other hand, more potent bioreactor for ultrafast DNA digestion is essential for accurate screening of chemically unstable and short half-life modifications in genomic DNA. For example, the major DNA adduct of acetaldehyde formed upon reaction with DNA in vitro is N2-ethylidenedeoxyguanosine, which is stable in genomic DNA, but quickly decomposes in the form of single nucleoside (a half-life of 5 -15 min).31-33 Therefore fast digestion protocol and rapid analytical method are meaningful and highly required. The aim of this study is to design a kind of potent cascade bioreactor to further shorten DNA digestion times. For this purpose, a more efficient endonuclease benzonase was selected to replace DNase I for fast cleaving long DNA strands into small oligonucleotides and mononucleotides. The digestion conditions for the cascade bioreactor working including buffer pH were optimized, enabling the ultrafast DNA digestion within 10 min. Consistent with our recent work27, the immobilization of benzonase also improves its stability and makes it reusable. We further developed benzoSAC bioreactor offline coupled LC-MS/MS method for fast and highly sensitive detection of 5-hydroxymethyl cytosine (5hmC), a major oxidation product of DNA 5-methylcytosine (5mC).34-38 EXPERIMENTAL SECTION Chemicals Benzonase endonuclease of grade II purity (> 90%) was purchased from Merck Millipore (Darmstadt, Germany). Snake venom phosphodiesterase (SVP) and calf thymus DNA (ct-DNA) were purchased from Sigma-Aldrich (St. Louis, USA). Deoxyribonuclease I (DNase I) was purchased as lyophilized powder from Sangon Biotech (Shanghai, China). Alkaline phosphatase (ALPase) was purchased fom AppliChem (Darmstadt, Germany). Tetramethoxysilane (TMOS, 98%) and 3-Amino-propyltrimethoxysilane (APTMS, 98%) were purchased from J&K Chemical (Beijing, China). Other chemicals were purchased from Sigma-Aldrich (St. Louis, USA). Preparation of enzyme-immobilized bioreactors Benzonase were immobilized onto capillary silica monolith using glutaraldehyde as a cross linking agent. The preparation of capillary silica monolith and immobilization were performed as reported previously.26,27,39 Briefly,20% glutaraldehyde in phosphate buffer (25 mM, pH 6.8) was pumped into this APTMS–modified monolith for 4 h at 4 ºC. After that, 5 mg/mL of benzonase in 25 mM of phosphate buffer (pH 6.8) was filled into the monolith, allowing for reaction of benzonase with glutaraldehyde modified silica monolith for 4 h. The resulting monolith was then reduced with 2 mg/mL of NaCNBH3 in phosphate buffer for 2 h.

Construction of Benzonase-based cascade (BenzoSAC) bioreactor Benzonase (typically, 20 cm), SVP (15 cm) and ALPase (15 cm) bioreactors were cascaded in order with zero-volume steel or PEEK unions (Valco, Switzerland). If not used immediately, the prepared BenzoSAC bioreactor was stored in phosphate buffer (25 mM, containing 0.01% of NaN3) at 4 ºC. Of note, SVP and ALPase-immobilized bioreactor were prepared as reported previously.27 Enzymatic activity assay Benzonase activity was determined by measuring the increase of UV absorbance at 260 nm (A260) during certain time intervals (0.5 to 2.5 min) utilizing ct-DNA as the substrate. SVP and ALPase activity were determined by measuring concentrations of p-nitrophenyl either obtained from SVPcatalyzed hydrolysis of bis(p-nitrophenyl) phosphate (bisNTP ) or ALPase catalyzed hydrolysis of p-nitrophenyl phosphate (NTP).27 More details are described in Supporting information. Cascade bioreactor coupled LC-MS analysis of 5hmC The LC-MS/MS analysis was conducted on Agilent 1290 UHPLC System coupled with a G6410B triple quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA). A Zorbax Eclipse Plus C18 column (2.1 mm i.d. × 100 mm, 1.8 µm particle size, Aglient) was used for separation of 5hmC and the mobile phase consists of 5% methanol and 95% 2 mM ammonium bicarbonate solution at a flow rate of 0.25 mL/min. Other conditions have been described previously and described in Supporting information.14 RESULTS AND DISCUSSION Immobilization of benzonase on capillary silica monolith and construction of BenzoSAC bioreactor Figure 1a shows the scanning electron microscopy (SEM) image of the porous silica monolith. The macropore size (2.7 ± 1.2 µm) is quite similar to the skeleton size (2.2 ± 0.5 µm), leading to a large macropore/skeleton size ratio and ensuring the good permeability (1.30 × 10-13 m2). BET specific surface area and pore volume were measured by nitrogen adsorption analysis (ASAP 2020M, Micromeritics).26,40,41 Result shows the pore volume of the silica monolith is 0.82 cm3/g and the specific surface area is 136 ± 11 m2/g, which guarantees large amount and uniform distribution of the immobilized enzymes. The immobilization of benzonase was confirmed as stained with a fluorescent SYPRO orange dye. Consistently, as positive controls, SVP, ALPase and DNase I-immobilized bioreactors exhibited bright uniform red-orange fluorescence (Figure 1d-f) compared to no fluorescence of the bare silica monolith (Figure 1b). Benzonase bioreactor also showed the bright uniform red-orange fluorescence, indicating the success of immobilization and even distribution of benzonase. The immobilized capacity of benzonase (estimated by Bradford assay) was 1.44 mg/mL (Table 1), and the bound amount was 0.65µg for 10 cm of bioreactor.


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Figure 1. Characterization of benzonase, DNase I, SVP and ALPase-immobilized bioreactors. (a) Scanning electron microscopic image (4000 X) of the porous silica monolith (the insertion, capillary bioreactor in 1000 X magnification); fluorescence (the upper) and bright filed (the lower) images of (b) the bare silica monolith, (c) benzonase, (d) SVP, (e) ALPase and (f) DNase Iimmobilized bioreactors. The capillaries were stained with SYPRO orange dye and rinsed with Tris-HCl buffer to remove the unbound dyes prior to imaging.

The immobilization parameters of SVP and ALPase in the used bioreactors were also listed in Table 1. The benzonase based cascade bioreactor (BenzoSAC) was constructed by tandem connecting the micro-reactors in an order of benzonase-SVP-ALPase with zero-volume steel unions. In this case, putting SVP bioreactor in front of benzonase bioreactor would lead to a decreased digestive efficiency of 74.2 ± 5.8%. This is probably because benzonase is adept in digesting doublestrands DNA into small DNA fragments (2-7 nucleotides) whereas SVP tends to hydrolyze short oligonucleotides into mononucleotides.22,23 The reproducibility for benzonase-SVP-ALPase bioreactor was 6.2% RSD (n = 5) for batch-to-batch analysis and 3.4% RSD (n = 5) for ru n-to-run analysis, implying that the cascade bioreactor is suitable for the daily digestion and analysis. The longevity of BenzoSAC bioreactor was examined by storing it with Tris-HCl buffer (20 mM, pH 8.4) containing BSA (0.1 mg/mL) and 0.01% NaN3 at 4 ºC. Less than 15% variation of enzyme activity was found during the bioreactor was reused for 20-times or stored at 4 ºC for 30 days (Figure S2, Supporting information). However, a mixture of benzonase, SVP and ALPase in solution stored at 4 ºC would entirely lose its activity within two days. This implied that the stability of the enzymes in the bioreactor was evidently enhanced by immobilization on the porous silica monolith. Enhanced enzymatic activities of the BenzoSAC bioreactor As seen in Table 1, the enzymatic activity for each single bioreactor is 1174 U/mL for benzonase, 0.036 U/mL for SVP and 5.96 U/mL for ALPase bioreactor, whereas it is 20, 0.007 and 0.062 U/mL for benzonase, SVP and ALPase in the

Figure 2. Digestive performance of benzonase and DNase I in free solution and enzyme-immobilized bioreactors. (a) AGE image of ct-DNA (lane 1) and its digested products after 5-min incubation with 5.0, 1.0, and 0.2 pmol DNase I (lane 2-4) or benzonase (lane 5-7) in solution; (b) AGE image of ct-DNA (lane 1) and its digested products at incubation time of 5, 10, and 15 min by benzonase bioreactor (lane 2-4), or DNase I bioreactor (lane 57), MALDI-TOF-MS analysis of (c) pristine ct-DNA, (d) products of ct-DNA digested by benzonase-immobilized bioreactor for 10 min, and (e) products of ct-DNA digested by DNase Iimmobilized bioreactor for 10 min.

conventional solution protocol. Essentially, the immobilization of the enzymes in the bioreactor enhanced their enzymatic activities by 58.7-fold for benzonase, 5.1-fold for SVP and 96.2-fold for ALPase over those in free solution protocol.



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Table 1. The immobilized capacity and the relative enzymatic activity of the bound enzymes in the cascade bioreactor and free enzymes in solution formula. a Relative enzymatic activity (U/mL)

Capacity (mg/mL)

Bioreactor

Solution

Enhanced

Benzonase

1.46 ± 0.41

1174

20

58.7

SVP

1.28 ± 0.29

0.036

0.007

5.14

ALPase

2.09 ± 0.79

5.960

0.062

96.2

DNase I

1.55 ± 0.52

1240

130

9.54

a

The activity of immobilized and free enzymes was measured with UV-Vis spectrophotometer. More details are described in the Supporting information.

Given that the average size of ct-DNA is 20 Kb and the complete digestion of 0.6 µg DNA for producing single nucleosides by DNase I/SVP/ALPase in solution requires 12 h (in solution), it is estimated that the average digestive rate of ctDNA in solution is about 0.3 bp/s. Interestingly, it is enhanced to approximately 10 bp/s by DNaseSAC bioreactor (in Ref. 27) and 45 bp/s by benzoSAC bioreactor (in this study). The enhancement of enzymatic efficiency and digestive rate are probably attributed to the high local concentration and improved stability of enzymes in the cascade bioreactors. On the one hand, the high intrinsic activity and broad substrate tolerance of benzonase make itself an ideal endonuclease in fast DNA digestion. Benzonase is a nonspecific endonuclease with high activity, capable of completely digesting either single-strand or double-stranded DNA and RNA into 5’monophosphate-terminated oligonucleotides of 3-7 bases in length. It works between pH 6 and 10 (optimal at 8.0-8.5).42-44 We compared the enzymatic activity of the benzonase/SVP/ALPase set and the DNase I/SVP/ALPase set in solution (the conventional protocol) by using ct-DNA as the substrate. As can be seen from agarose gel electrophoresis (AGE) image (Figure 2a), after the incubation of ct-DNA with enzymes mixture for 5 min, shorter oligonucleotides fragments were obtained by benzonase/SVP/ALPase than those by DNase I/SVP/ALPase system. For example, there were no strips observed in lane 5, implied that ct-DNA was digested into fragments less than 100 bp by application of 5 pmol of benzonase. Nevertheless, the strip size digested by 5 pmol DNase I in lane 2 was among 100-300 bp. The measured enzyme activity of 0.1 pmol of benzonase is comparable to that of 12 pmol DNase I in solution (Table S1, Supporting information). We further examined the digestion of ct-DNA samples using benzonase and DNase I bioreactors. The products were tested by 1% agarose gel electrophoresis. As seen from Figure 2b, lanes 2-4 represent digestive products of ct-DNA produced by benzonase bioreactor at incubation time of 5, 10, and 15 min, and lanes 5-7 represent which of DNase I bioreactors correspondingly. Both bioreactors had same length (4 cm). Consistently, given the same digestion time, the digested fragments of ct-DNA by benzonase bioreactor were much smaller than that of DNase I. For example, there was no strip can be seen in the AGE image after digested by benzonase bioreactor for 15 min (lane 4), but the fragments digested by DNase I bioreactor for 15 min were more than 100 bp (100 5000 bp). In another word, benzonase bioreactor showed better enzymatic performance than DNase I one.

Figure 3. The compatibility of the optimal buffers required for each bioreactor including pH and divalent cations. (a) Illustration of the optimization of pH values in DNA digestion buffer, (b) pH value and (c) divalent cation additives in digestion buffer influence the digestive performance of benzonase and DNase I based bioreactors. On the other hand, the compatibility of the optimal buffers required for each bioreactor, including pH and divalent cations, was greatly improved by using benzoSAC bioreactor. The optimal pH values in DNA digestion buffer are recommended as 8.0-8.5 for benzonase, 7.2-7.6 for DNase I, 8.2-9.2 for SVP and 8.2 -10 for ALPase, respectively. 42,45-47Benzonase possesses a stronger alkaline optimal pH (8.0-8.5) than that of DNase I (7.2-7.6).42The optimized pH 8.4 is favorable for all of the three enzymes, i.e. benzonase, SVP and ALPase (Figure 3a). The optimal pH of DNaseⅠis 7.6,48 which is incompetent to boost the other two enzymes work, and that would hinder enhancement of the overall enzymatic efficiency. Cleaving oligonucleotides into mononucleotides by SVP is a bottleneck in this process, low pH tends to result in low activity of SVP, makes the degradation oligonucleotides cannot keep up with long DNA strands cleaving (DNase) and phosphate removal (ALPase). As an example, a 20-mer oligodeoxynucleotides probe (5’-CCCATTACCA GCCAGCTAAT-3’) was applied to SVP-bioreactor digestion. The influence of pH in digestion buffer on digestive performance of SVP-immobilized bioreactor was examined by its ability in generating mass ladder from the digestion of 20-mer oligodeoxynucleotides. At pH 8.4, it generated shorter fragments predominated with a length of 4 nucleotides (nts) within 10 min (SI Figure S3). In contrast, at pH 7.6, longer fragment (13-14 nucleotides) predominated. It is evident that pH 8.4 promotes better enzymatic digestion performance of SVP-bioreactor. When pH was adjusted to 8.4 in the digestion buffer, the digestive efficiency of ct-DNA could reach more than 100% (Referring to 12h solution digestion) by BenzoSAC bioreactor digestion, whereas DNaseSAC bioreactor shows relatively lower efficiency (86.4 ± 9.3%) than that at pH 7.0 - 7.6 of the buffer (Figure 3b). Divalent cations in digestion buffer, preferably Mg2+, are required for benzonase enzyme activity. Commonly, 1- 2 mM of MgCl2 was recommended as the reaction-promoting additive in solution. As to DNase I, however, it requires both Mg2+ and Ca2+ divalent cations at approximate-


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ACS Applied Materials & Interfaces ly 5 mM and 0.5 mM respectively for the maximal digestion activity, and reduced to 1 - 2 mM Mg2+ and 0.2 - 0.5 mM Ca2+ for DNase I-SVP-ALPase digestion, at an optimum pH of 7.6 (Figure 3c). The combination of the above two aspects enables benzonase bioreactor a faster speed and much higher efficiency of DNA digestion than DNase I bioreactor. We then tested a 10cm-length benzonase bioreactor for digestion of 3 µL of 200 ng/µL. No product strips were found in 12% polyacrylamide gel at digestive time of 5 min, which seemed that ct-DNA was digested into short fragments less than 10 bp, which were undetectable on PAGE. These small-size fragments were then examined by MALDI-TOF/MS. As shown in Figure 2 compared with no signals of small fragments from m/z 900 to 4000 of the pristine ct-DNA (Figure 2c), there are many mass ladder from m/z 600 to 4000 of ct-DNA which were digested by benzonase (Figure 2d) and DNase I bioreactors (Figure 2e) or for 10 min. However, the fragments generated by benzonase bioreactor are generally smaller than those produced by DNase I bioreactor. The mass ladder of the former were more focused on small fragments from m/z 600 to 1200 (~ 98%), which suggested that benzonase bioreactors could cleave ct-DNA into smaller-size fragment (less than 4 nt) rapidly, and the next SVP bioreactors would digest these smaller-size fragment into mononucleotides easier. Thus the enzymatic efficiency of BenzoSAC bioreactors was greatly enhanced. Fast digestion of genomic DNA into single nucleosides using BenzoSAC bioreactor The utilization ofBenzoSAC bioreactor facilitates the more efficient and ultrafast digestion of genomic DNA in short time (10 min). In this experiment, 3 µL of ct-DNA (200 ng/µL) was pumped through BenzoSAC bioreactors using a digestive buffer containing 10 mM Tris-HCl (pH 8.4), 0.2 mM MgCl2, 1% trehalose and 0.01% Triton, and digested for 5, 10, 30, and 40 min, until 30 µL of products were collected. The digestion products were tested by 12% PAGE gel (Figure 4a). The pristine ct-DNA (lane 1), and its cleavage products produced by using BenzoSAC bioreactor (lane 2-4), or digested by DNaseSAC bioreactor for 10, 20, 30, and 40 min (lane 5-7) were shown in the agarose gel image. By application of BenzoSAC bioreactor, ct-DNA was fast digested and no strips were found after incubated for 5 min (lane 2). Incubating ctDNA in the bioreactor for 10 and 45 min, ct-DNA was digested into small-size fragments less than 10 bp, and the products were applied to PAGE analysis and showed no DNA strips. This indicated that almost all of ct-DNA was degraded into nucleosides within 10 min (lane 3). As for the other cascade bioreactor, i.e. DNaseSAC bioreactor, the products obtained in 10 or 20 min contains fragments whose sizes were between 10 to 30 bp. This result confirms that newly constructed BenzoSAC bioreactor is more efficiency than DNaseSAC bioreactors in the digestion of ct-DNA. More sensitive and accurate results were obtained by LCMS/MS determination. dG was chosen as the marker for evaluation of the digestive efficiency. The digestive efficiency at 10 min is more than 99.5 ± 1.2 % (Figure 4b, c). This confirmed that BenzoSAC bioreactor can be employed for genomic DNA digestion and to release single nucleosides effectively as fast as 10 min.

Figure 4. The enzymatic efficiency of the cascade bioreactors to ct-DNA. (a) PAGE image of ct-DNA (lane 1), and its products digested by BenzoSAC (benzonase-SVP-ALPase) bioreactor for 10, 20, 30, and 40 min (lane 2-4), or digested by DNase I-SVPALPase for 10, 20, 30, and 40 min (lane 5-7). (b) Digestion efficiency of the cascade three-enzyme bioreactors in term of dG nucleosides (n = 3). The chromatogram of dG in cleavage products by LC-MS analysis which was generated (c) from BenzoSVC bioreactor digestion of 10, 20, 30, and 40 min (red trace), and from solution digestion for 12 h (black trace), and (d) from DNaseSAC (DNase I-SVP-ALPase) bioreactor digestion of 10, 20, 30, and 40 min (green trace).

As a contrast, the digestion reaction also conducted in solution. The digestion efficiency reached 97.2% until 8 h in solution enzymatic system (Figure S4), whereas a relatively higher enzymatic efficiency (99.5 ± 1.2%) was obtained within 10 min by using BenzoSAC bioreactor. This result indicates that, in respect of ct-DNA digestion, BenzoSAC bioreactor appears to be more potent than conventional solution digestion protocol.



that our newly constructed cascade bioreactor is successful in the release of modified cytosine in genomic DNA. By application of BenzoSAC bioreactor off-line coupled LC-MS/MS method, only a minute (less than 1.0 µg) of genomic DNA sample were required for a single run. Moreover, TBBQ exposure obviously increased the level of 5hmC by 2.3-fold compared to DMSO control (from 17.4/106 dC to 40.2/106 dC). The increase of 5hmC level in genomic DNA from T24 cells was TBBQ-dose-dependent (1 - 20 µM). CONCLUSION We demonstrate the successful immobilization of benzonase on capillary silica monolith, enabling the construction of benzonase-based cascade bioreactor and the fast DNA digestion (~10 min). This newly prepared BenzoSAC bioreactor made a great progress as to our previous DNaseSAC (DNase I-SVP-ALPase) bioreactor, shortening the digestion time of genomic DNA from 45 min to 10 min. The proposed off-line BenzoSAC coupled LC-MS/MS detection is promising for fast identification and analysis of other structural modifications in cellular DNA.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XX.XXXX/ acsami.XXXXXXX. Figure 5. (a) LC-MS/MS analysis of 5hmC in genomic DNA isolated from TBBQ-exposed T24 cells (0 - 20 µmol/L) using BenzoSAC bioreactor for DNA digestion; (b) comparison of the frequency of 5hmC between the free enzymatic digestion (12h, white column) and the BenzoSAC bioreactor digestion (10 min, black column) (n=3).

Enzymatic activity assay, preparation capillary silica monolith, cell culture and exposure, genomic DNA enzymatic hydrolysis, LC-MS/MS analysis of 5hmC, gel electrophoresis and MALDITOF MS analysis (PDF)

Identification and quantification of 5-hydroxymethyl cytosine in genomic DNA We further demonstrated the applicability of BenzoSAC bioreactor to determine structural DNA modifications in genomic DNA using 5hmC as an example. 5hmC is a major oxidation product of the epigenetically crucial 5mC., Decreased 5hmC levels are suspected to be associated with various tumors, thus 5hmC is an emerging biomarker for cancer diagnosis and treatment.14 We have reported that halobenzoquinones (HBQs), an emerging class of halogenated disinfection byproducts (DBPs) in drinking water, 48-50 not only can induce oxidative damages, but also alters DNA demethylation process through indirectly enhancing Tet oxidation activity.51,52 In this experiment, DNA samples (0.6 µg each) isolated from terabromo-1,4-benzoquinone (TBBQ)-exposed T24 cells were subjected to BenzoSAC bioreactor-mediated digestion for 10 min followed by LC-MS/MS analysis. As shown in Figure 5a, LC-MS/MS analysis of 5hmC could be accomplished within 5 min. Together with the digestion process in cascade bioreactor (10 min), a single run can be accomplished as fast as 15 min. This is more time-saving than that of free solution digestion coupled HPLC-MS/MS method (8 -24 h). Evidently, 5hmC in genomic DNA could be released effectively using BenzoSAC bioreactor (Figure 5b). It demonstrates

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ACKNOWLEDGMENT This work was supported by the Ministry of Science and Technology of China (2016YFA0203102 and 2014CB932003 to J.Y. and 2016YFC0900300 to H. W.), and the National Natural Science Foundation of China (21375142 to J.Y., and 21435008, 21527901 to H.W.), and the Strategic Priority Research Program of the Chinese Academy of Sciences, China (XDB14030302 to J. Y. and XDB14030200 to H.W.).

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