Nanopore-Based, Label-Free, and Real-Time Monitoring Assay for

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A Nanopore-Based, Label-Free, and Real-Time Monitoring Assay for DNA Methyltransferase Activity and Inhibition Sana Rauf, Ling Zhang, Asghar Ali, Jalal Ahmad, Yang Liu, and Jinghong Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03278 • Publication Date (Web): 20 Nov 2017 Downloaded from http://pubs.acs.org on November 21, 2017

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Analytical Chemistry

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A Nanopore-Based, Label-Free, and RealTime Monitoring Assay for DNA Methyltransferase Activity and Inhibition

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Sana Rauf, Ling Zhang, Asghar Ali, Jalal Ahmad, Yang Liu and Jinghong Li*

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Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry &

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Chemical Biology, Tsinghua University, Beijing 100084, China.

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*to whom corresponding should be addressed.

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Phone: 86-10-62795290; Fax: 86-10-62771149

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Email: [email protected]

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ACS Paragon Plus Environment

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Abstract

2

DNA methylation catalyzed by DNA methyltransferase plays an important role in

3

many biological processes. However, conventional assays proposed for DNA

4

methyltransferase activity are laborious and discontinuous. We have proposed a novel

5

method for real-time monitoring of the activity and kinetics of Escherichia coli DNA

6

adenine methyltransferase (Dam) using nanopore technique coupled with enzyme-

7

linkage reactions. A double-stranded DNA probe AB having a recognition sequence

8

5’-GATC-3’ for both Dam and MboI restriction endonuclease was prepared. Dam

9

catalyzed the methylation of substrate probe AB, which blocked the cleavage reaction

10

of MboI. While, the absence of Dam resulted in cleavage of non-methylated probe

11

AB into four ssDNA fragments by MboI. When tested with nanopore, double-

12

stranded methylated probe AB generated long-lived events, distinguished clearly from

13

MboI-cleavage mediated ssDNA fragments that generated only spike-like events. The

14

proposed method has a detection limit of 0.03 U/ml for Dam in a short assay time of

15

about 150 min. This sensing system is easy to perform, simple to design and

16

circumvents the use of radioactive substances, resulting in efficient detection of the

17

activity of Dam even in complex matrixes like human serum sample. Furthermore, it

18

has the potential to screen Dam-targeted inhibitor drugs which may assist in the

19

discovery of new anticancer medicines. This method is general and could be extended

20

easily for monitoring activity of a wide variety of methyltransferases by coupling with

21

their corresponding methylation-sensitive endonucleases.

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Keywords

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Nanopore, Dam activity, methyltransferase, methylation-sensitive cleavage, real-time,

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cleavage fragments, bioassay, Inhibitors, drug analysis

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1

Introduction

2

DNA methylation, catalyzed by DNA methyltransferase, is one of the most

3

extensively studied epigenetic events due to its vital role in prokaryotes and

4

eukaryotes.1,2 Aberrant DNA methylation is related to several genetic disorders and

5

has been considered as a predictive biomarker for various types of cancers in

6

mammals.3 Abnormalities in the DNA methylation process affect pathogen resistance

7

and other vital ecological characteristics of plants.4 DNA methyltransferase (MTase)

8

can recognize specific DNA sequence and results in the covalent addition of methyl

9

group to the target DNA sequence using S-adenosyl methionine (SAM) as a methyl

10

donor. DNA adenine methyltransferase (Dam) found in prokaryotes converts adenine

11

into N6-methyladenine in the recognition sequence 5’-GATC-3’, and plays a pivotal

12

role in many biological processes in bacteria including cell growth, DNA replication,

13

and gene expression.5 In addition, the bacterial virulence also depends on DNA

14

adenine methyltransferase.6,7 Since DNA methylation level is linked to the activity of

15

DNA methyltransferase, therefore monitoring of DNA methyltransferase activity and

16

kinetic parameters is of paramount importance in the drug discovery, clinical

17

diagnostics, and life science research.

18

Conventional assays proposed for the measurement of activity of DNA

19

methyltransferase include high-performance liquid chromatography (HPLC),8

20

polymerase chain reaction (PCR),9 capillary electrophoresis,10 immune reaction,11 and

21

using radioactive labeling of SAM.12 However, the lengthy assay procedures, high

22

cost due to the use of antibodies, and requirement of insecure isotope labeling limit

23

the practical applications of these techniques.

24

In order to address these issues several non-immunological and non-radioactive

25

assays have been introduced such as electrochemical,13,14 chemiluminescence,15

26

colorimetry,16 and bioluminescence based techniques.17 Most of these approaches are

27

discontinuous and laborious. For real-time monitoring of DNA methyltransferase

28

activity fluorescence-based assays based on either fluorescence quenching or

29

fluorescence resonance energy transfer (FRET) have been proposed.18-20 However, the 3 / 32



Page 4 of 32

1

proposed fluorescence sensors require double-labeled oligonucleotide substrates

2

which will not only increase the cost of sensing system but probably also reduce the

3

cleavage efficiency, and these bulky fluorescent groups might interfere with the

4

kinetic behavior of the substrate molecules. Fluorescent assays require the proper

5

design of DNA probes which may limit the versatility of such methods. Moreover,

6

interference by external nonspecific events particularly in complex clinical fluids is

7

always a concern in fluorescence methods.

8

Mainly, most of the reported assays monitor DNA methyltransferase activity by

9

coupling it with their cognate methylation-specific restriction enzyme that scissors the

10

methylated site in DNA sequence. So far, only a small number of methylation-specific

11

endonucleases have been explored in comparison to a large number of methylation-

12

sensitive endonucleases (unable to scissor the methylated DNA sequences). A lot of

13

the methyltransferase and methylation-sensitive restriction endonuclease pairs share

14

the same recognition sequence like both M.SssI methyl transferase (MTase) and HpaII

15

restriction endonuclease recognize the sequence 5’-CCGG-3’. With the addition of

16

M.SssI MTase and cofactor SAM the recognition sequence 5’-CCGG-3’ is methylated

17

to 5’-CCmGG-3’ and the methylated cytosine blocks the cleavage reaction of HpaII.

18

Dam MTase and DpnII recognize the same sequence 5’-GATC-3’. After methylation

19

reaction by Dam MTase, the cleavage reaction of DpnII restriction endonuclease is

20

blocked. Similarly both Dam MTase and Nt.AlwI recognize the sequence 5’-GGATC-

21

3’. Nt.AlwI is a nicking endonuclease that cuts only one strand of DNA on a double-

22

stranded DNA substrate. Subsequently, the strategies based on methylation-sensitive

23

endonuclease cleavage can be generalized for a wide variety of other

24

methyltransferases. Therefore, the development of a simple, highly sensitive,

25

continuous and low-cost method based on the methylation-blocked cleavage of DNA

26

methyltransferase is highly desired.

27

Recently, nanopore technique is studied in the perspective of biosensing.21 α-

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hemolysin (α-HL), a protein nanopore, is spontaneously inserted into a lipid bilayer

29

membrane and forms a nanopore consisting of a vestibule and a β-barrel with an

30

internal diameter of about 1.4 nm. When a charged molecule passes through protein 4 / 32


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nanopore under an applied potential, it induces a transient change in the ionic current

2

and current variation events are recorded electrically. The analyte can be quantified by

3

the frequency of occurrence of current events while characteristic current signatures

4

reveal analyte identity. This nanopore sensor has remarkable advantages such as

5

simplicity, lack of label, high sensitivity, minimal sample preparation, and real-time

6

analysis.22,23 These properties make it a powerful tool in the detection of various

7

analytes including small molecules,24 metal ions,25 DNA,26 RNA,27 and proteins.28 The

8

nanopore stochastic sensing has been used to investigate the structural discrimination

9

of biomolecules at the single-molecule level,29 and is under examination for rapid and

10

low-cost next-generation DNA sequencing technology.30

11

Furthermore, nanopore sensing has also been applied for the detection of

12

enzymatic reactions.23,31 The presence of target enzyme in the sample solution could

13

be identified by monitoring the translocation events of the specific substrate for an

14

enzyme through a nanopore. The presence of target enzyme in the sample is indicated

15

either by the presence of some new types of current events or variation in frequency

16

of signature events in nanopore recording traces.

17

The current ‘‘gold standard’’ methods for methyltransferase activity include

18

bisulfite treatment and gel electrophoresis. In bisulfite treatment, non-methylated

19

cytosine is converted to uracil in the presence of sodium bisulfite, while methylated

20

cytosine leftovers intact. However, bisulfite treatment is a harsh process with high

21

levels of input DNA and one of the major disadvantages of this technique is the

22

incomplete transformation of cytosine to uracil, which results in false positives.32 Gel

23

electrophoresis is used extensively for the detection of DNA methyltransferase

24

activity. In this process, charged biomolecules are driven through the matrix

25

of agarose or polyacrylamide by applying an electric field. Shorter biomolecules

26

migrate rapidly through the pores of the gel than the longer biomolecules, resulting in

27

the separation of molecular species. However, gel electrophoresis is time-intensive,

28

DNA-consuming, laborious process with increased cost. In addition, it is a bulk phase

29

technique involving simultaneous processing of a large number of molecules for less

30

sensitive macroscale optical readout and also structural changes among molecules 5 / 32



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can’t be analyzed using gel electrophoresis.

2

To address these issues, nanopore-based assays have been proposed to detect DNA

3

methylation without the need of PCR amplification and bisulfite conversion. The

4

nanopore analysis has particularly a high throughput, a minimal sample consumption,

5

and reduced footprint. DNA methylation has been analyzed using both biological and

6

synthetic nanopore.33-37 Most DNA sequencing technologies cannot directly

7

distinguish between methylated and non-methylated bases in the native DNA

8

sequence.

9

In the present work, we employed nanopore sensing technology for real-time

10

monitoring of DNA adenine methyltransferases activity and kinetics. Our proposed

11

method involves the real-time analysis of Dam activity by monitoring ionic current

12

variations and ionic current arising from the enzyme-substrate interactions, which is a

13

simple and highly sensitive bioassay using protein nanopore for the real-time analysis

14

of E. coli Dam activity with great selectivity. According to this strategy, we used Dam

15

as a model DNA methyltransferase and coupled this reaction system with MboI

16

restriction endonuclease. The double-stranded DNA (probe AB) used in this assay

17

contained a 5’-GATC-3’ site that was identified by both Dam and MboI restriction

18

endonuclease. In the presence of Dam, this site was methylated and blocked the

19

cleavage reaction of MboI. The double-stranded methylated DNA probe AB, when

20

passed through a nanopore would generate events with prolonged dwell time,

21

recognized as signature signals. However, in the absence of Dam, probe AB could not

22

be methylated and consequently cleaved into four ssDNA fragments after treatment

23

with MboI. The single-stranded DNA cleavage fragments, when passed through a

24

nanopore, would generate spike-like current signals that could be distinguished easily

25

from long-lived signature events generated in the presence of Dam. Using this assay,

26

we have determined the

27

method has an extremely low detection limit of 0.03 U/ml for Dam. This sensing

28

system circumvents the use of radioactive substances, however is efficient in

29

detection of the activity of Dam even in complex matrixes like human serum sample.

(SAM and DNA) and

for the Dam. The proposed

6 / 32


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Furthermore, it has the potential to screen Dam-targeted inhibitor drugs which may

2

assist in the discovery of new anticancer and antimicrobial medicines.

3 4

Experimental Section

5

Materials

6

(DPhPC) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). Wild-type α-

7

HL for the formation of nanopore was obtained from Sigma-Aldrich (St. Louis, MO).

8

S-adenosyl methionine (SAM), DNA adenine methyltransferase (Escherichia coli),

9

MboI restriction endonuclease, Hha I methyltransferase, M.SssI methyltransferase,

10

and corresponding buffer solutions were obtained from New England Biolabs, Inc.

11

(Beijing, China). Human serum sample was purchased from Lablead Biotech. Co.,

12

Ltd. (Beijing, China).

and

Reagents

Lipid

1,2-Diphytanoyl-sn-glycero-3-phosphocholine

13

Gentamicin and Ampicillin were purchased from Sigma-Aldrich (Shanghai,

14

China). Ultrapure water was used for the preparation of all solutions. All chemicals

15

utilized throughout the assay were of analytical grade. The DNA samples were

16

custom synthesized and purified by Shanghai Sangon Biological Engineering

17

Technology & Services Co., Ltd. (Shanghai, China). DNA sequences utilized in this

18

nanopore analysis are listed below:

19 20

Probe A: 5’-CCC CCC CCC CCC CCC CCC CCC CCC CCA CCA GAT CGC GAC CCC CCC CCC CCC CCC CCC CCC CCC C-3’

21

Probe B: 5’-GTC GCG ATC TGG TG-3’

22

Fragment A1: 5’-CCC CCC CCC CCC CCC CCC CCC CCC CCA CCA-3’

23

Fragment A2: 5’-GAT CGC GAC CCC CCC CCC CCC CCC CCC CCC CCC

24

C-3’

25

Fragment B1: 5’-GTC GC-3’

26

Fragment B2: 5’-G ATC TGG TG-3’

27

The bolded letters in probe A and probe B represent the recognition sequence of

28

Dam. The black triangles mark the cleavage sites of MboI restriction endonuclease.

29

Fragments A1, A2, B1, and B2 are similar to MboI-mediated cleavage fragments of 7 / 32



Page 8 of 32

1

substrate probe AB.

2

Nanopore Assay for Dam Activity Probe AB was prepared by annealing equal

3

concentrations of probe A and probe B in the hybridization buffer (10 mM Tris, 1.0

4

mM EDTA, 1.0 M NaCl, pH 7.4). The mixture was heated at 95 °C for 5 min, cooled

5

gradually to room temperature, and stored at -20 °C for further use.

6

The methylation reaction was performed by mixing probe AB, SAM and different

7

concentrations of Dam in the presence of 2 µl of 10X Dam buffer (1X Dam buffer: 50

8

mM Tris-HCl, 10 mM EDTA, and 5 mM 2-mercaptoethanol, pH 7.5), at a total

9

volume of 20 µl. The reaction mixture was first incubated at 37 °C for 60 min and

10

then at 65 °C for 20 min to inactivate Dam. Then, cleavage reaction of MboI was

11

performed. In order to minimize the effect of Dam reaction buffer on cleavage

12

reaction of MboI, the reaction volume was increased up to 50 µl by the addition of

13

MboI restriction endonuclease in the presence 5 µl of 10X CutSmart buffer (1X

14

CutSmart buffer: 50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(Ac)2, and 100 µg/ml

15

BSA, pH 7.9). The cleavage reaction was performed at 37 °C for another 60 min.

16

Finally, 950 µl of test buffer containing 1 M KCl buffered with 10 mM Tris (pH

17

= 7.4) was added to 50 µl of the resulting solution mixture, treated by Dam and

18

MboI, respectively, and utilized as the cis test buffer for nanopore experiments. The

19

final concentrations of probe AB, SAM, and MboI tested through nanopore were 200

20

nM, 120 µM, and 20 U/ml, respectively. The tested Dam concentrations were 0, 0.03,

21

0.06, 0.1, 0.5, 1.0, 10.0 and 50.0 U/ml. Trans buffer contained 993 µl of test buffer (1

22

M KCl and 10 mM Tris buffered at pH = 7.4), 2 µl of 10X Dam buffer and 5 µl of

23

10X CutSmart buffer.

24

Real-Time Assay for Dam Activity For real-time analysis of Dam activity,

25

experiments were performed using similar experimental conditions as used for the

26

Dam activity assay, apart from methylation reaction was analyzed at different time

27

points. Briefly, for real-time analysis, probe AB was incubated with Dam (50 U/ml)

28

at 37 °C for t = 0, 1, 2, 5, 7.5, 15, 20, 23, 26, 35, 45, 60 or 84 min. Reactions were

29

stopped by heat denaturation of Dam at 65 °C for 20 min. The time t = 0 min

30

represents the time of addition of Dam. The cleavage reaction was then performed by 8 / 32


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1

the addition of 20 U/ml of MboI. The subsequent increase in signature events

2

frequency due to methylation of substrate probe AB was analyzed by α-hemolysin

3

nanopore.

4

Negative control rates (containing no Dam) were analyzed at the same time

5

points but no increase in signature events frequency was observed in the absence of

6

Dam.

7

Selectivity The selectivity experiments were conducted with 50 U/ml of Hha I or

8

M.SssI MTase similar to Dam activity assay, apart from Dam was substituted by Hha

9

I or M.SssI methyl transferase in the methylation step.

10

Detection of Dam in Human Serum Sample Human serum sample was centrifuged

11

at 15000 rpm for 12 min and supernatants were obtained. The test for the detection of

12

Dam in the human serum sample was performed under similar experimental

13

conditions to those used for the Dam activity assay, excepting the addition of 10 µl of

14

human serum sample supernatants.

15

Effect of Drugs on Dam Activity All the drug inhibition experiments were

16

performed using similar experimental conditions to those used for the Dam activity

17

assay, only with the addition of ampicillin or gentamycin in the methylation step. The

18

final concentrations of ampicillin and gentamycin used were 20 µM and 2 µM,

19

respectively.

20

The relationship between the gentamycin concentration and Dam activity was

21

also investigated for different concentrations of gentamycin at fixed concentration of

22

Dam (50 U/ml) in the methylation step, and with all the ensuing steps being similar as

23

mentioned above. The final concentrations of gentamycin tested were 0 (control), 0.5,

24

1.0, 1.5 and 2.0 µM.

25

Single-Channel Recording The vertical chamber setup was used for electrical

26

current recordings. For the generation of a lipid bilayer, 30 mg/ml of DPhPC in

27

decane was applied to 150 µm aperture in a derlin cup which separated the cis and

28

trans chambers (Warner Instruments, Hamden, CT, USA). Each side of cuvette

29

aperture was pretreated by 0.5 mg/ml of DPhPC in hexane prior to the formation of

30

the bilayer. The analyte was added to the cis chamber which was connected to ground. 9 / 32



Page 10 of 32

1

The α-HL was applied adjacent to the cuvette aperture in the cis chamber. Once

2

insertion of single protein nanopore was confirmed, then potential was applied at

3

+150 mV from the cis side by Ag/AgCl electrodes.

4

Single-channel current values were recorded with a patch-clamp amplifier

5

(HEKA EPC10; HEKA Elektronik, Lambrecht/Pfalz, Germany), while the signals

6

were filtered with a low-pass Bessel filter set to 3 kHz at a sampling rate of 20 kHz by

7

using an LIH 1600 A/D converter (HEKA Elektronik). All the nanopore experiments

8

were carried out at room temperature.

9

Data Analysis Data were analyzed using MATLAB (R2011b, MathWorks) software

10

and OriginLab 8.0 (OriginLab Corporation, Northampton, MA, USA). I/I0 is the

11

normalized current blockage, where I0 is the ionic current of the open nanopore and I

12

is the blockage current for translocation of the analyte through a nanopore. The mean

13

dwell time and the mean value of I/I0 were acquired from the dwell-time histogram

14

and blockage histogram, respectively, by fitting the distributions to Gaussian function.

15

The frequency of the signature events was calculated from at least 5 min recorded

16

data, and all the data sets were based on the mean ± standard deviation (s.d.) of three

17

separate experiments.

18 19

Results and Discussion

20

Nanopore Bioassay Principle The principle of a label-free nanopore-based assay for

21

the activity of Dam is shown in Scheme 1. To clear our strategy, it is important to note

22

that the cleavage reaction of MboI is blocked after the methylation of recognition

23

sequence 5’-GATC-3’. Considering this fact, the DNA probe AB containing a

24

recognition site for both Dam and MboI is designed. When probe AB was mixed with

25

MboI it would be cleaved into four pieces of ssDNA. The released single-stranded

26

DNA fragments generated nanopore recording traces with short dwell time when

27

passed through nanopore.

28

However, in the presence of Dam, the recognition sequence 5’-GATC-3’ in the

29

DNA probe AB would be methylated at the adenine base, which blocked probe AB 10 / 32


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1

scissoring by MboI. Since, MboI was disabled to cleave methylated probe AB, when

2

passed through nanopore this double-stranded DNA would generate signals with

3

prolonged dwell time, which we identified as signature signals. The activity of Dam

4

could be analyzed by calculating the frequency of methylated probe AB signature

5

signals since, MboI-cleavage mediated spike-like short current signals of ssDNA

6

fragments were dynamically blocked by Dam-catalyzed DNA methylation. The site-

7

specific methylation by Dam results in high selectivity.

8 9

Differentiation of Probes In order to accomplish differentiation of methylated probe

10

AB from other probes, the nanopore current signals of different sample solutions were

11

analyzed. The methylated duplex probe AB, and 1:1:1:1 solution mixture of four

12

single-stranded DNA fragments (A1, A2, B1, and B2) were tested with nanopore using

13

similar experimental conditions (Figure 1). The nanopore recording traces of probe

14

AB in the presence of 50 U/ml of Dam generated events with prolonged mean dwell

15

time (~21 ms in Figure 1Ac), and mean current blockade I/I0 was 0.89 ± 0.04 (Figure

16

1Ae).

17

Then, four single-stranded DNA fragments A1, A2, B1, and B2 (similar to cleavage

18

fragments of probe AB, mediated by MboI) in 1:1:1:1 solution mixture were tested

19

through nanopore technique as the control. As documented in Figure 1B, these short

20

ssDNA fragments generated events with a much shorter average dwell time of 0.30 ±

21

0.10 ms and comparatively smaller value of I/I0 to 0.81 ± 0.03 (Figure 1Be).

22

Consequently, the recording traces of the methylated probe AB can be identified

23

clearly from the cleavage fragments on the basis of the dwell time. The obvious

24

difference in dwell time of two different sample solutions is evident in histogram

25

distributions as well (Figure 1Ad and 1Bd). The corresponding 3D plots of the

26

number of events vs dwell time vs I/I0 and scatter plot of the events (I/I0 vs dwell

27

time) generated by methylated probe AB and the mixture of cleavage fragments are

28

presented in Figure S1 (see supporting information).

29

The mixture of Dam (50 U/ml), SAM (120 µM) and MboI (20 U/ml) without

30

addition of substrate probe AB was also analyzed using nanopore technique, resulting 11 / 32



Page 12 of 32

1

in only noise-like short blocks, which would not interfere with identification of

2

methylated probe AB signature signals (Figure S2A).

3

Analysis of Dam A double-stranded DNA probe AB containing the recognition

4

sequence for both Dam and MboI was designed for monitoring of Dam activity.

5

Current signals with blockage larger than 0.70 and dwell time greater than 1 ms

6

generated by methylated probe AB translocation through nanopore were defined as

7

signature events. These signatures could be distinguished clearly from cleavage

8

fragments of non-methylated probe AB which showed signals with translocation time

9

< 1 ms (due to scissoring of probe AB into short ssDNA by MboI).

10

The control experiment (without the addition of Dam) with translocation of

11

substrate probe AB in the presence of MboI through protein nanopore, generated

12

small background signals at a frequency of 16.9 ± 1.5 min-1 (n = 3). This background

13

was probably due to unscissored non-methylated probe AB, translocation through a

14

nanopore. Dwell-time histogram of this control demonstrates that majority of the

15

signals were situated in the region of less than 1 ms dwell time (Figure 2Bb). The

16

mean current blockage I/I0 for this control was at 0.82 ± 0.03, similar to that of the

17

mixture of ssDNA fragments (Figure 2Bc). However, the addition of target Dam (50

18

U/ml) to substrate probe AB (before MboI treatment), generated large signature

19

signals in nanopore current traces at a frequency of 91.3 ± 4.2 min-1. Dwell-time

20

histogram also showed a clear indication of an increase in characteristic signature

21

signals with enhanced dwell time, namely larger than 1 ms (Figure 2Ab). A slight

22

increase in mean current blockage I/I0 was noticed at 0.86 ± 0.04, similar to

23

methylated probe AB (Figure 2Ac). These results clearly suggest that methylated

24

probe AB was able to maintain its sequence completely and blocked the cleavage of

25

MboI, thus would generate events with prolonged dwell time, which were used as the

26

signature signals.

27

Quantification of Dam Activity To obtain the best analytical performance, the effect

28

of the concentrations of MboI on the assay was investigated. The MboI concentration

29

was optimized to 20 U/ml for the following experiments (Figure S3). The SAM

30

concentration was optimized to 120 µM for this sensing system (Data not shown). 12 / 32


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1

Under aforementioned optimized experimental conditions, the analytical

2

performance of the proposed bioassay was evaluated with various concentrations of

3

Dam. Figure S4 shows the signature events frequency dependence to different Dam

4

concentrations. As Dam concentration varied from 0 to 50 U/ml, a gradual increase in

5

signature events frequency was observed. It is reasonable as a higher concentration of

6

Dam would methylate more probe AB, which blocked the cleavage of MboI.

7

According to Figure 3A, it is found that signature events frequency was proportional

8

to the logarithmic concentration of Dam in the range from 0 U/ml to 50 U/ml. The

9

linear relationship can be described as Y = 56.10 + 19.85X, with the correlation

10

coefficient of R2 = 0.985, where Y and X are the frequency of signature events (in

11

min-1) and logarithmic concentration of Dam (in U/ml), respectively. The detection

12

limit for Dam was determined to be 0.03 U/ml (S/N=3), which was lower than most

13

of previously reported assays.18,38-40 For comparison with previous studies, we have

14

summarized the detection limit, detection time, type of DNA used and the real sample

15

application of other reported methods for detection of Dam in Table S1.

16

Dam activity was evaluated using gel electrophoresis as a comparison and results

17

are presented in Figure S5. In lane 1 there is only one band for probe AB in the

18

absence of Dam and MboI. The new band of cleavage fragment appeared in lane 2

19

when probe AB was treated with MboI only, suggesting the cleavage reaction of

20

probe AB in the absence of Dam. In lane 3, only one band of methylated probe AB

21

appeared due to methylation of probe AB by Dam which blocked the cleavage

22

reaction of MboI. These results are in accordance with the nanopore results.

23

Nanopore-based sensing system took 150 min to complete, while gel electrophoresis

24

required more than 6 h to complete. Contrary to traditional gel electrophoresis the

25

proposed nanopore-based assay can be monitored in real time and is homogeneous.

26

Nanopore Real-Time Monitoring of DNA Methylation The single-molecule

27

nanopore technique gives a lot of information about dynamic enzyme-substrate

28

interaction, and individual binding events between an enzyme and specific substrate

29

can be analyzed in real-time using this technique. In our proposed assay we have

30

monitored DNA methylation process in real-time by coupling it with an enzyme13 / 32



Page 14 of 32

1

linkage reaction. Figure 3B shows the real-time nanopore analysis of probe AB,

2

SAM, and MboI in the presence of Dam and in the absence of Dam. There was no

3

significant increase in signature events frequency in the absence of Dam,

4

demonstrating that the MboI restriction endonuclease would scissor the non-

5

methylated probe AB in the absence of Dam. Conversely, a rapid increase in signature

6

events frequency was noticed after the addition of Dam. These results clearly showed

7

that the increase in signature events frequency was due to methylation of substrate

8

probe AB. The slope of the curve decreased gradually with the extension of time,

9

indicated that Dam based methylation was slowed shown the substrates were used up.

10

Under the conditions of the assay, as methylated probe AB would not be cleaved by

11

MboI providing a direct relationship between each single methylation event and

12

signature signals frequency increase. The continuous real-time data provide

13

information about the dynamics of the DNA methylation process and may be

14

extended further to real-time drug testing technology. Therefore, this nanopore-based

15

sensing system could be successfully applied for real-time monitoring of Dam

16

activity. It is anticipated that rapid and accurate DNA methylation quantification will

17

proliferate the research and eventually improve health care.

18

Kinetic Analysis of E. coli Dam using Nanopore Assay The nanopore assay was

19

used to determine the Michaelis-Menten kinetic constants

20

turnover number (

21

the linear regression of the initial parts of the real-time curve from t = 0 min to t = 20

22

min. At methylation time t = 0 min the solution contained only cleavage fragments of

23

probe AB after treatment with MboI, therefore the concentration of methylated probe

24

AB produced in the 20 min methylation period, namely the rate of the reaction, would

25

be proportional to the change in the frequency,

26

(SAM and DNA) and

) for E. coli Dam. The initial rate of reaction was calculated by

of the signature events, where

. The frequency of the signature events at t = 20 min (

)

27

was utilized in this nanopore assay to construct the Lineweaver-Burk plot, while

28

is the frequency of signature events at t = 0 min. The data were plotted after 14 / 32


Page 15 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

deducting the background (without Dam).

2

In a series of identical reactions containing 50 U/ml (corresponding to 0.31 nM)

3

of Dam and 120 µM of SAM, the concentration of substrate probe AB was varied in

4

the range of 50-500 nM. A double-reciprocal plot of the

5

AB concentration allowed the determination of

6

double-reciprocal plot of the

7

calculation of

8

at fixed concentration of probe AB to 500 nM (Figure S6B).

9

The

versus substrate probe (Figure S6A). Similarly, a

versus SAM concentration was constructed for the

. The SAM concentration was varied in the range of 10-120 µM

values determined from y-intercepts of Lineweaver-Burk plots of both

10

substrates were close to each other. The value of

11

frequency of the signature events. The value of

12

the correlation of the frequency of signature events and concentration of DNA probe

13

AB, as the concentration of the substrate (probe AB) consumed in a given period of

14

time would be equal to that of the product formed (methylated probe AB) after

15

treatment with Dam and MboI. The data were plotted after deducting a negative

16

control, which contained MboI but lacked Dam (Figure S7). The value of

17

calculated by dividing

18

obtained was actually the

in nM/s could be calculated from

could be

with the enzyme concentration used.

Values of important kinetic parameters for substrate probe AB were obtained: , 10.0 ± 2.5 µM for

and 0.10 ± 0.04 s-1 for

19

50.0 ± 4.5 nM for

20

Michaelis-Menten kinetic constants

21

reported to vary from 3.6 to 55.0 nM and 3.0 to 12.0 µM, respectively.4,41,42 This

22

variation comes from a variety of assay conditions and different substrates employed

23

for DNA methyltransferase analysis.

and

.

for E. coli Dam have been

24 25

Quantification of Dam in Complex Biological Sample To validate the practical

26

applications of our proposed nanopore-based sensing system, Dam was tested in the 15 / 32



Page 16 of 32

1

presence of human serum sample. The nanopore current traces for the human serum

2

sample (10 µl) without an addition of substrate probe AB were recorded, which

3

generated only noise-like spikes in recording traces (Figure S2B). A control

4

experiment (without Dam) for the translocation of substrate probe AB and MboI in

5

the presence of human serum sample was carried out which produced only a few

6

signature signals. Conversely, after the addition of Dam in the mixture of substrate

7

probe AB, MboI, and human serum sample, a distinct increase in signature signals

8

frequency was observed as presented in Figure 4A and 4B. The accuracy of the

9

proposed assay in the real sample was evaluated through observing the human serum

10

sample spiked with three different Dam concentrations (0.1, 1.0, and 50.0 U/ml) and

11

the results are given in Table 1. The average recovery ratios of samples were found to

12

be in the range of 91-102%. These results suggest that this method has great potential

13

for the accurate detection of Dam in clinical samples like the human serum.

14 15

Effect of Drugs on Dam Activity Due to key role of Dam in prokaryotes and

16

eukaryotes, the study of inhibition of Dam activity has provided an effective tool for

17

antibacterial therapeutic applications. The potential applications of the proposed

18

technique were extended further to screen the inhibitors of Dam by using penicillin

19

and gentamycin (antibacterial drugs) as model inhibitors. Considering the

20

involvement of MboI in this sensing method, it was essential to investigate whether

21

these inhibitors have an influence on its activity. Control experiment was performed

22

and the results suggest that these drugs (ampicillin and gentamycin) have no

23

inhibition effect on the MboI activity (Figure S8). The nanopore current traces for

24

ampicillin (20 µM) and gentamycin (2 µM) without an addition of substrate probe AB

25

were also recorded, which did not generate any signals (Figure S2C and S2D).

26

Subsequently, the inhibition effect of these drugs on Dam activity was investigated.

27

As presented in Figure 4C, the relative activity of Dam decreased with the addition of

28

these inhibitors, presenting inhibition of Dam. In the presence of inhibitor, the methyl

29

group transfer to the target DNA sequence was blocked, which resulted in scissoring

30

of substrate probe AB. The concentration-dependent inhibition of Dam activity was 16 / 32


Page 17 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

investigated further for different concentrations of gentamycin at fixed concentration

2

of Dam (Figure 4D). It was observed, with the increase in the concentration of

3

gentamycin, the inhibitory effect increased. The relative activity (RA) of Dam can be

4

calculated by using the following equation:

(1)

5 6

where

,

, and

were the signature events frequencies without Dam, with Dam,

7

and with both Dam and inhibitor (gentamycin or ampicillin), respectively. The relative

8

activity of Dam decreased with the increase in the concentration of gentamycin. A plot

9

of relative activity (RA) versus gentamycin concentration allowed the determination

10

of IC50 value (the amount of inhibitor which decreases enzyme activity by 50%) of

11

gentamycin to 1.8 ± 0.15 µM (Figure 4D). The result was consistent with the reported

12

IC50 value of gentamycin determined by other techniques.14,43 Therefore, the proposed

13

assay seems reliable for the screening of Dam-targeted inhibitor drugs.

14

Selectivity Selectivity is an important factor to judge the analytical performance of a

15

sensing system. The selectivity of the proposed assay for Dam was evaluated by

16

selecting two other methyltransferases (Hha I and M.SssI) as interference enzymes.

17

M.SssI methylates the recognition sequence 5’-CG-3’ and Hha I target the sequence

18

5’-GC-3’.

19

These enzymes were analyzed by this nanopore assay using the similar

20

experimental conditions as described for Dam activity assay. The nanopore current

21

traces for interfering enzymes Hha I and M.SssI (50 U/ml each) without the addition

22

of substrate probe AB were recorded (Figure S2E and S2F). Figure 5A shows the

23

representative current signals for the nonspecific enzymes or Dam incubated with

24

substrate probe AB and MboI. Experimental results show that signature event

25

frequency increase was observed only in the presence of Dam, however, no distinct

26

increase in signature events frequency was noticed in the presence of either M.SssI or

27

Hha I methyltransferase (Figure 5B). The selectivity obviously comes from the fact

28

that Dam recognition sequence 5’-GATC-3’ could not be methylated by nonspecific 17 / 32



Page 18 of 32

1

methyltransferases (M.SssI and Hha I). Therefore, the proposed sensing system

2

exhibits good selectivity toward Dam assay.

3

In combination with the portable sensor chip44-46 that can be used in various

4

locations, the proposed sensing system may help in rapid sensing of Dam for clinical

5

applications such as early diagnosis of cancer and can be extended for point-of-care

6

testing of other methyltransferases. Bulk sensing methods often require excessive

7

sample consumption but the low copy number of DNA can be obtained from

8

mammalian samples. Therefore, for clinical application of a sensing system, nanopore

9

analysis is a promising approach as it works with minimal sample consumption. The

10

conceptual and practical simplicity, combined with high sensitivity, robust data

11

interpretation, speed, and minimal sample preparation support conversion of this

12

strategy into an industrial platform.

13

The inherent instability of lipid bilayer membrane (in which protein pore is

14

inserted) is one of the major hurdles to circumvent the use of protein nanopore for

15

routine real-time clinical analysis. Development of strategies by using a robust protein

16

pore devices that stabilize lipid bilayer membrane have therefore been a focus of

17

continuous research efforts in recent years.

18 19

Concluding Remarks

20

In conclusion, we have developed a unique nanopore assay for the simple,

21

sensitive, real-time, and label-free analysis of E. coli DNA adenine methyltransferase

22

activity and kinetics. This method is based on signature events frequency

23

enhancement due to methylation of substrate probe AB, which blocked cleavage by

24

the MboI restriction endonuclease. The proposed assay is simple to design and can

25

rapidly detect Dam activity. The detection limit of E.coli Dam was calculated to be

26

0.03 U/ml in total detection time of 150 min and low cost. In addition, the method has

27

been successfully applied in human serum samples. The assay presented herein

28

obviates the need of any chemical modification of DNA probe, which makes it more

29

convenient and cost-effective with reduced sample consumption. Finally, the protocol 18 / 32


Page 19 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

provides an ideal system for screening of broad spectrum of Dam inhibitors. Given

2

attractive analytical features, we expect that the proposed strategy will have important

3

applications in a wide range of antimicrobial therapeutics.

4 5

Acknowledgement

6

This work was financially supported by National Natural Science Foundation of

7

China (No. 21621003, No. 21235004, No. 21327806), National Key Research and

8

Development Program of China (No. 2016YFA0203101) and Tsinghua University

9

Initiative Scientific Research Program. Moreover, Ms. S. Rauf highly acknowledges

10

the Chinese Scholarship Council for the financial assistance of my PhD program in

11

the Tsinghua University of China.

12 13 14 15

Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.

16 17

19 / 32



Page 20 of 32

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1 2

Figure Legends

3

Scheme 1. Schematic illustration of the proposed nanopore assay for the analysis of

4

Dam activity. In the presence of target Dam, substrate probe AB is methylated. The

5

methylated probe AB blocks the cleavage reaction of MboI and when tested with

6

nanopore double-stranded methylated probe AB, will generate signature signals with

7

prolonged dwell time. In the absence of the target (Dam), probe AB is cleaved by

8

MboI. These ssDNA cleavage fragments, when tested with a nanopore, will generate

9

short spike-like signals, distinguished clearly from signature signals. The frequency of

10

the signature signals is used to quantify the target Dam.

11 12

Figure 1. Differentiation of methylated probe AB from the mixture of cleavage

13

fragments A1, A2, B1, and B2. (a) Schematic illustration of methylated probe AB (A)

14

and the mixture of cleavage fragments (B) interacting with or translocating through

15

the α-HL nanopore. (b) Representative single-channel nanopore recording traces for

16

the experiments of methylated probe AB (A) and the mixture of cleavage fragments

17

(B). (c) Expanded view of the events indicated in the traces by the red stars. (d)

18

Histograms of dwell times for methylated probe AB (A) and the mixture of cleavage

19

fragments (B). (e) Histograms of normalized current blockade I/I0 for methylated

20

probe AB (A) and the mixture of cleavage fragments (B). Each histogram was ﬁt to a

21

Gaussian distribution. Test for methylated probe AB was performed by incubating

22

probe AB, SAM with target Dam for 60 min before adding to cis chamber. The final

23

concentrations of probe AB, SAM, and Dam used were 200 nM, 120 µM, and 50

24

U/ml, respectively. The cleavage fragments A1, A2, B1, and B2 (100 nM each) were

25

mixed in 1:1:1:1. All the nanopore experiments were performed in a solution

26

containing 1 M KCl and 10 mM Tris (pH = 7.4), with a transmembrane potential of

27

+150 mV. Each experiment was repeated three times.

28 29

23 / 32



Page 24 of 32

1

Figure 2. Nanopore analysis of target Dam. (a) Representative single-channel current

2

traces of the signals generated by substrate probe AB, SAM, and MboI with (A) and

3

without (B) target Dam. Red triangles mark the signature signals in the recording

4

traces. (b) Histograms of dwell time for the methylated probe AB, SAM, and MboI

5

with (A) and without (B) target Dam. Blue boxes represent the signature signals

6

regions. (c) Histograms of normalized current blockade I/I0 for substrate probe AB,

7

SAM, and MboI with (A) and without (B) target Dam. Each histogram was fit to a

8

Gaussian distribution. Before nanopore analysis, probe AB was methylated by Dam

9

for 60 min using SAM as a methyl donor and then treated with MboI for another 60

10

min. The nanopore tests were performed with 200 nM probe AB, 120 µM SAM, 20

11

U/ml of MboI and 50 U/ml of Dam. All the nanopore experiments were performed in

12

a solution containing 1 M KCl and 10 mM Tris (pH = 7.4), with a transmembrane

13

potential of +150 mV. Each experiment was repeated three times.

14 15

Figure 3. Nanopore based detection of target Dam. (A) The variance of the signature

16

signals frequency in response to different concentrations of target Dam in the range of

17

0 U/ml to 50 U/ml. Inset: The curve shows a linear response from 0 U/ml to 50 U/ml

18

with the concentration of target Dam in the logarithmic co-ordinate. The nanopore

19

tests were performed with 200 nM probe AB, 120 µM SAM, 20 U/ml of MboI and

20

varying concentrations of Dam. (B) Time course of real-time monitoring of

21

methylation of substrate probe AB (200 nM), SAM (120 µM) and MboI (20 U/ml)

22

with and without addition of 50 U/ml of target Dam.

23 24

24 / 32


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1

Figure 4. Detection of Dam in complex biological sample. (A) Representative single-

2

channel current traces for the mixture of substrate probe AB, MboI, and human serum

3

sample without (a) and with (b) 50 U/ml of target Dam. (B) Comparison of Dam (50

4

U/ml) detection capability without and with the addition of human serum samples. (C)

5

Influence of ampicillin (20 µM) and gentamycin (2 µM) on the relative activity of

6

Dam. (D) Inhibition effect of different concentrations of gentamycin on Dam activity.

7

The gentamycin concentrations tested were 0, 0.5, 1.0, 1.5, and 2.0 µM, respectively.

8

Before nanopore analysis, substrate probe AB was methylated for 60 min by Dam at

9

different concentrations of ampicillin or gentamycin and then cleaved by MboI for

10

another 60 min. The nanopore tests were performed with 200 nM probe AB, 120 µM

11

SAM, 20 U/ml of MboI and 50 U/ml of Dam. The nanopore analysis was performed

12

in the test buffer, 1 M KCl, and 10 mM Tris (pH = 7.4), with a transmembrane

13

potential of +150 mV.

14 15

Figure 5. Selectivity of the proposed assay. (A) Representative single-channel current

16

traces of substrate probe AB, SAM, and MboI in presence of Dam (a) or interfering

17

enzymes, Hha I (b) and M.SssI (c). In the blank (d), all experimental conditions were

18

the same as (a), except that there was no Dam. (B) Comparison of the signature

19

signals frequency of substrate probe AB with target Dam and other nonspecific

20

enzymes. The nanopore tests were performed with 200 nM probe AB, 120 µM SAM,

21

20 U/ml of MboI. The concentration of Dam was 50 U/ml. The concentration of each

22

Hha I or M.SssI was 50 U/ml. Before nanopore analysis, the probe AB was treated

23

with Dam (60 min) or interfering enzymes Hha I, M.SssI then cleaved by MboI for 60

24

min. The nanopore analysis was performed in the test buffer, 1 M KCl, and 10 mM

25

Tris (pH = 7.4), with a transmembrane potential of +150 mV. Red triangles mark the

26

signature signals in current traces. The error bars were based on three repetitive

27

experiments performed.

25 / 32



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Table 1. Recovery of Dam tested in human serum sample Spiked Dam (U/ml)

Detected Dam (U/ml)

Recovery (%) n=3

0.1

0.091 ± 0.004

91.0

1.0

1.02 ± 0.03

102.0

50.0

46.10 ± 2.15

92.2

4 5

Scheme 1

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Figure 5

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For TOC Only

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Nanopore-Based, Label-Free, and Real-Time Monitoring Assay for

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