KR102026299B1 - Electrochemical assay for determining Exonuclease activity with graphene electrode - Google Patents

Abstract

By electrochemical measurement method using a graphene electrode, a method of measuring the activity of exonuclease in real time is disclosed. The method for measuring exonuclease activity includes the steps of reacting a sample expected to include exonuclease and double-stranded DNA labeled with an electrochemical redox label; Contacting the reaction product of the sample and double-stranded DNA with a graphene electrode; And detecting the activity of exonuclease by measuring a reducing peak current of the electrochemical redox label through the graphene electrode.

Description

Electrochemical assay for determining Exonuclease activity with graphene electrode

The present invention relates to an electrochemical measurement method of exonuclease activity, and more particularly, to a method for measuring the activity of exonuclease in real time by an electrochemical measurement method using a graphene electrode.

Exonuclease is an enzyme that sequentially degrades nucleotides from the 3′-end or 5′-end of a polynucleotide chain in a DNA sequence. 3′-5 ′ exonucleases break down phospho-diester bonds at the 3′-end to remove mismatches, modifications, fragments, or base-paired nucleotides, leading to DNA synthesis. Proper 3′-ends are generated and genome stability is maintained.

Lack of 3′-5 ′ exonuclease activity can cause serious disease. The 3′-5 ′ exonuclease TREX 1 is responsible for editing DNA during gap-filling of DNA replication or DNA repair, and mutations of TREX 1 Aicardi-Goutie`res syndrome, Systemic Lupus Erythematosus, Familial Chilblain Lupus, Retinal Vasculopathy and Cerebral Leukodystrophy Is associated with distinct diseases. WRN exonucleases are also involved in DNA repair and are known to be associated with autosomal recessive genetic disorder Werner syndrome.

Thus, quantitative analysis of 3′-5 ′ exonuclease activity is important for disease diagnosis, drug development, and related epidemiological studies. Generally, gel electrophoresis, ³ H or [γ- ³² P] ATP-incorporated DNA substrates were used for the analysis of 3′-5 ′ exonuclease activity. However, these methods are problematic in that they are difficult to control, take a long time, or involve radiation exposure. As a method for analyzing exonuclease activity continuously and in real time, a fluorescence-based assay is also known. However, the fluorescence method is disadvantageous in that the equipment is bulky, fragile and expensive.

Electrochemical methods can be effectively used for the measurement and quantification of biomolecules in a simple and cost-effective manner. The electrochemical method has an advantage of excellent sensitivity and miniaturization compared to the optical method. Recently, methods for measuring exonuclease activity have been reported by electrochemical analysis (R. Miranda-Castro, D. Marchal, B. Limoges, F. Mavre ', Chem. Commun. Camb. Engl. 2012, 48, 8772-8774). In this method, an electrochemical redox probe is used that intercalates double-stranded DNA (dsDNA), and when the substrate is digested, a current signal is generated from a probe that exhibits enzymatic activity. In addition, methods have been reported for the electrochemical detection of redox labels released when redox-labeled DNA substrates are enzymatically hydrolyzed (X. Liu, W. Li, T. Hou, S. Dong, G. Yu. , F. Li, Anal.Chem. 2015, 87, 4030-4036). Meanwhile, a method of using graphene-based homogeneous electrochemical electrodes as an electrochemical biosensor for detecting cancer biomarkers is also known (L. Ge, W. Wang, X. Sun, T. Hou, F. Li, Anal.Chem. 2016, 88, 2212-2219).

It is an object of the present invention to provide an electrochemical measuring method of exonuclease activity that is excellent in sensitivity, easy to manipulate, and capable of quickly and quantitatively detecting exonuclease activity. .

Another object of the present invention is to provide a method of electrochemical measurement of exonuclease activity that can measure the activity of various exonucleases by controlling the structure of the DNA substrate.

In order to achieve the above object, the present invention comprises the steps of reacting a sample expected to include an exonuclease and double-stranded DNA labeled with an electrochemical redox label; Contacting the reaction product of the sample and double-stranded DNA with a graphene electrode; And measuring the reductive peak current of the electrochemical redox label through the graphene electrode to detect the activity of the exonuclease. Here, when the exonuclease is present in the sample, the double-stranded DNA is hydrolyzed by the exonuclease to produce a single-stranded DNA, the single-stranded DNA is fixed to the graphene electrode surface, the electrochemical oxidation Reductive peak currents of reducing labels can be detected.

The electrochemical measurement method of exonuclease activity according to the present invention is excellent in sensitivity, easy to operate, and fast and quantitatively exonuclease activity, compared with conventional assays such as gel electrophoresis and fluorescence. As it is detectable, it is useful as a novel assay in drug screening, basic research, and the like involving enzymes. In addition, the methods of the present invention can be applied to measure the activity of various exonucleases by modulating the structure of a DNA substrate.

1 is a schematic diagram illustrating a method for measuring exonuclease activity according to the present invention.
2A and 2B show (a) AFM images, cross sections, and (b) Raman spectra of CVD-grown monolayer graphene, respectively.
3A is a differential pulse voltage-current measurement graph obtained from a graphene electrode, with or without Exo III.
Figure 3b is a graph showing the degree of degradation of hsDNA substrate over time by the catalysis of Exo III.
3C is a graph showing the Exo III catalyst concentration and the degree of degradation of the hsDNA substrate.
4 is a graph showing the inhibition of Exo III and IC ₅₀ values for the doses of (a) ATA and (b) EDTA.
5 is a surface Michaelis-Menten graph and Langmuir graph for hsDNA substrate concentration.

Hereinafter, with reference to the accompanying drawings, the present invention will be described in detail.

The present invention measures exonuclease activity by a homogeneous electrochemical assay using a graphene monolayer electrode.

1 is a conceptual diagram illustrating a method for measuring exonuclease activity according to the present invention. As shown in FIG. 1, in order to electrochemically measure exonuclease activity according to the present invention, first, an electrochemical redox label (4) and a sample expected to contain exonuclease (2) Labeled, tagged double-stranded DNA (6, double-stranded DNA: dsDNA) is reacted (see A and B in FIG. 1).

The double stranded DNA 6 is a DNA substrate in which a pair of polynucleotide chains are bonded in base pairs, preferably a hairpin-structured DNA substrate (hsDNA). Specifically, the double-stranded DNA 6 may include a stem structure in which a pair of polynucleotide chains are coupled by base pairs, and a loop structure connecting the polynucleotide chains of the stem structure. For example, the double-stranded DNA (6) may have a sequence of SEQ ID NO: 1, the GGCCGAGC and GCTCGGCC sequence across the double-stranded DNA (6) is combined in base pairs to form a stem structure, the intermediate poly T ( The TTTTTT loop sequence is back-folded to impart stability of the stem-loop structure. The hairpin structure of the double stranded DNA 6 prevents the DNA substrate from adsorbing onto the graphene surface.

[SEQ ID NO: 1]

5′-GGCCGAGC TTTTTT GCTCGGCC-3 ′

The electrochemical redox probe is a redox probe that is oxidized or reduced when attached to or detached from an electrode to generate an electrical signal. Specific examples of such a label include methylene blue (MB) and ferrocene (ferrocene). ), Anthraquinone, and the like. The electrochemical redox label may be covalently attached to the 5′-end of the double stranded DNA 6 and labeled on the double stranded DNA 6.

If exonuclease (2) is present in the sample, double-stranded DNA (6) and exonuclease (2) react, and the double-stranded DNA (6) substrate is hydrolyzed by exonuclease (2) To generate single-stranded DNA (ssDNA). The reaction between the double-stranded DNA (6) and the exonuclease (2) is carried out by catalysis of the enzyme exonuclease (2), resulting in the nucleotide from the 3'-end of the double-stranded DNA (6). By sequentially degrading and digesting, the double stranded DNA (6) portion is lost and a single stranded DNA (8) fragment is generated (see FIG. 1B).

Next, the reaction product of the sample expected to contain the exonuclease 2 and the double-stranded DNA is contacted with the graphene electrode 10. When the double stranded DNA (6) substrate is hydrolyzed by the exonuclease (2) by reaction of the sample with the double stranded DNA, the enzymatic product generated by hydrolysis, i.e., the single stranded DNA (8) It is fixed to the surface of the graphene electrode 10 (see C of FIG. 1). This is based on the fact that, compared to the double stranded DNA 6, the single stranded DNA 8 is more preferentially adsorbed to the surface of the graphene electrode 10. Although the function of the present invention is not limited to the following theory, the reason why the single-stranded DNA 8 is more preferentially adsorbed on the surface of the graphene electrode 10 is because the exposed nucleic acid base of the single-stranded DNA 8 ) And the hexagonal structure of the graphene electrode 10 may be explained by the π-π stacking interaction. On the other hand, in the double-stranded DNA 6, since the nucleic acid base is located inside the DNA double helix, the double-stranded DNA 6 is relatively difficult to be adsorbed on the graphene electrode 10 surface. The graphene electrode 10 used in the present invention is an electrode capable of fixing a single-stranded DNA 8, and a graphene monolayer 10 is attached to an electrode, that is, a working electrode. It may have been. The graphene monolayer or graphene sheet used for the graphene electrode 10 may be used as it is manufactured without further chemical treatment such as adding chemical functional groups.

When the digested enzyme product, ie, the single stranded DNA (8) fragment, is bound to the graphene electrode 10 surface by π-π stacking, it is fixed at the end of the single stranded DNA (8) ( Anchoring) The redox tag (4, redox tag) is electrochemically reduced (reduction) to increase the reduction current and generate a voltammetric current signal. Thus, through the graphene electrode 10, by measuring the reducing peak current of the redox label 4, such as methylene blue, at a low negative potential (for example, about-0.1 V vs Ag / AgCl) , Exonuclease (2) activity can be directly quantified. This method of measurement is simple and easy to implement, as it uses the graphene sheet as it is prepared, without further chemical treatment or sophisticated probe immobilization process. Thus, the method of the present invention can be used as a new and highly sensitive platform for exonuclease (2) activity assays.

Hereinafter, the present invention will be described in more detail with reference to specific examples. The following examples are intended to illustrate the present invention, and the present invention is not limited to the following examples. In the examples below, hairpin-structured DNA substrates labeled with methylene blue (MB) label (hsDNA, 5′-MB-GGCCGAGC TTTTTT GCTCGGCC-3 ′) were purchased from Bioneer, Korea, and magnesium chloride (MgCl) ₂ ), arintricarboxylic acid (ATA), β-mercaptoethanol and β-mercaptoethanol and exonuclease III (Exo III, 100,000 U / μL) were obtained from Sigma-Aldrich. , Korea). Ethylenediaminetetra acetic acid disodium salt (Na ₂ EDTA) was purchased from Duksan Pure Chemical CO., LTD.

Preparation Example 1 Preparation and Characterization of Single Layer Graphene

Using chemical vapor deposition (CVD), monolayer graphene was formed on Cu foil having a thickness of 25 μm (X. Li et al., Science 2009, 324, 1312-1314), and the characteristics of the prepared single layer graphene Was confirmed by atomic force microscopy (AFM) and Raman spectroscopy. Graphene substrates were used with or without further chemical treatment.

2A and 2B are (a) AFM images, cross sections, and (b) Raman spectra of CVD-grown monolayer graphene, respectively. From the AFM image of FIG. 2A, it can be seen that a single layer graphene having a 2.5 nm-high micron-sized ripples / wrinkles shape is formed, which is a CVD-grown graphene. Is an essential characteristic of. In the Raman spectrum shown in FIG. 2B, characteristic peaks are observed at about 1580 cm ⁻¹ and about 2670 cm ⁻¹ , due to G and 2D bands. G bands originate from the in-plane vibrations of sp2 carbon atoms and are doubly degenerated (TO and LO) phonon modes (E2g symmetry) at the Brillouin zone center. to be. In this spectrum, a strong 2D band resulting from double resonance Raman scattering was also observed. The peak intensity ratio of the 2D and G bands (I _2D / I _G ) was about 2, indicating the formation of high quality monolayer graphene. The absence of the D band in the Raman spectrum means that the graphene layer is free of defects.

Preparation Example 2 Preparation of Graphene Electrode for Exo III Activity Analysis

Monolayer graphene on Cu foil (area 1 cm ² ) in the form of rectangular pieces was wired to the working electrode using conductive copper tape. The graphene electrode surface was washed with deionized water and exonuclease in varying amounts (1-100 U / μL) in 10 mM Tris-HCl buffer (pH 7.5, 10 mM MgCl ₂ , 10 mM β-mercaptoethanol) It was completely immersed in 2 mL enzyme reaction mixture containing III (Exo III) enzyme.

Experimental Example 1 Electrochemical Analysis of Exo III Activity

Differential pulse voltammetry (DPV) was performed using a CHI 660D electrochemical analyzer (CH Instruments, Inc. USA). Measurement conditions were scan range 0.1-0.5V, amplitude 0.05V, pulse width 0.2 second, and pulse time 0.5 second. All measurements were made at room temperature (25 ° C.) against a graphene monolayer using Ag / AgCl (3M NaCl) as reference electrode in 10 mM Tris-HCl buffer (pH 7.5) and Pt wire as counter electrode. Was performed.

In 10 mM Tris-HCl buffer, pH 7.5, 100 nM of hsDNA, 10 mM MgCl ₂ , 10 mM beta-mercaptoethanol and various amounts (1-100 U / μL) of Exo III and denatured Exo An enzyme reaction mixture was prepared in which III enzyme was dissolved. The enzyme was denatured by heating at 65 ° C. for 15 minutes. After incubation at room temperature for a predetermined time, the graphene electrode was immersed in the reaction mixture and DPV measurements were performed.

FIG. 3A is a differential pulse voltammogram obtained from graphene electrodes when Exo III is absent (black) or present (red). As shown in FIG. 3A, after the addition of Exo III, by electrochemical reduction of the methylene blue (MB) label, a strong reducing peak current appears at -0.14 V, whereas without the Exo III enzyme, a small cathodic The peak appears at -0.06 V. Thus, it can be seen that the hsDNA substrate is hydrolyzed by enzyme catalysis and the MB-labeled single stranded DNA (ssDNA) fragment is adsorbed on the graphene surface.

To confirm this phenomenon, further DPV measurements of MB-labeled ssDNA (msDNA), not MB molecules and products digested with Exo III, showed MB with one reduction peak at about -0.15 V. , msDNA was reduced at a more positive potential (-0.06 V), but shifted to a more negative value (-0.14 V) over time. This is because msDNA is adsorbed to graphene, causing a change in the polarizability of the DNA surrounding environment and inducing a negative shift of reduction potential. The voltage characteristics of these msDNAs are very consistent with the trend of hsDNAs in the presence of Exo III.

3b is a graph showing the degree of digestion of hsDNA substrate with time due to the catalysis of Exo III, and FIG. 3c is a graph showing the concentration of Exo III catalyst and the degree of degradation of hsDNA substrate. 3B and 3C, the relative peak current is a value obtained by comparing the peak current obtained from the graphene electrode with the peak current obtained from the graphene electrode in the absence of Exo III. As shown in FIG. 3B, when 1, 10 U / μL Exo III and 10 U / μL modified Exo III were used, enzymatic hydrolysis increased until stabilization at 100 min standing time. However, in the case of denatured Exo III, very small current changes appear even at 10 times higher concentrations of 1 U / μL Exo III, whereas 10 U / μL Exo III leads to higher current changes. As shown in Figure 3b, after leaving for 100 minutes at various concentrations of Exo III, the peak current was measured, the higher the Exo III concentration, it can be seen that the larger the amount of hsDNA substrate is degraded. The current signal gradually increased until the Exo III concentration reached 100 U / μL, and 20 U / μL concentration was appropriate for performing the inhibition analysis of Exo III.

Experimental Example 2 Exo III Inhibition Assay

As two types of Exo III inhibitors, aurintricarboxylic acid (ATA) and ethylenediaminetetraacetic acid (EDTA) were used, and inhibition of Exo III activity was measured according to the method of the present invention. Specifically, for EDTA-Exo III inhibition assay, various concentrations of EDTA solution were mixed with reaction buffer containing 100 nM hsDNA and then 1 U of Exo III was added. After standing at room temperature for 30 minutes, the electrode was immersed in the reaction mixture, and the redox current signal was measured by DPV method. For ATA-Exo III, various concentrations of ATA solution were mixed with enzyme reaction buffer containing 100 nM hsDNA followed by the addition of 1 U of Exo III. After 1 hour at room temperature, electrochemical measurements were performed, similar to the EDTA inhibition assay. 50% inhibitory concentration (IC ₅₀ ) was calculated with OriginPro software using a sigmoidal dose-response function.

FIG. 4 is a graph showing inhibition of Exo III and IC ₅₀ values for the doses of (a) ATA and (b) EDTA, showing the percentage value (% Activity) for inhibitor content. Here,% Activity was obtained by normalizing the measured current sensitivity values (I) to the measured maximum sensitivity values (I _max ) (% Activity = (II ₀ ) / ( I _max -I ₀ ) x 100, I ₀ is the reference sensitivity value. IC ₅₀ values of ATA and EDTA were 28.8 pM and 20.1 nM, respectively. As shown in FIG. 4, according to the method of the present invention, the inhibition of Exo III activity can be effectively measured.

[Example 3] Michael less of Exo III - menten constant determined

Using the method of the present invention, the Michaelis-Menten parameter of Exo III was determined. In surface-enzyme action, enzymatic reactions are explained by Langmuir adsorption and Michaels-Menten kinetics (Langmuir 2005, 21, 4050-4057 and Surf. Colloids 2006, 22, 5241-5250). Surface enzyme kinetics can be evaluated by the following equation.

[Equation 1]

은 "surface" Michaelis-Menten 상수이고, 여기서는 Langmuir 흡착 상수(

)의 역수일 수 있다.

는 효소-기질(enzyme-substrate: ES)의 중간 형태의 표면 점유율(coverage)이고, [S]는 hsDNA 기질의 벌크(bulk) 농도이다. 상기 수학식 1은, 아래 수학식 2와 같이,

를

로 교체하여 계산될 수 있다(RSC Adv. 2015, 5, 46617-46623).here,

Is the "surface" Michaelis-Menten constant, where Langmuir adsorption constant (

May be the inverse of

Is the surface coverage of the intermediate form of the enzyme-substrate (ES), and [S] is the bulk concentration of the hsDNA substrate. Equation 1, as shown in Equation 2 below,

To

(RSC Adv. 2015, 5, 46617-46623).

[Equation 2]

:

:

Where I is the peak current after enzyme addition and I _max is the maximum peak current measured in the saturated enzyme state.

을 계산한 결과 90 nM였고, 이는 Exo III의 보고된 값에 근접한 것이다. 따라서, 본 발명의 전기화학적 그래핀 전극을 사용하는 방법은, Exo III 활성의 심각한 손실을 유발하지 않고, 다른 통상적인 방법과 유사한 효용성이 있음을 알 수 있다.Enzymes were added to the reaction mixture containing various concentrations of dsDNA substrate (20 U / mL), and allowed to stand at room temperature for 100 minutes, after which peak currents were measured to obtain surface Michaelis-Menten and Langmuir graphs. 5 is shown respectively. As shown in a of FIG. 5, the current reaches a saturation value in a high concentration of hsDNA substrate, showing the characteristics of Michaelis-Menten kinetics. From b of FIG. 5

Was calculated to be 90 nM, which is close to the reported value of Exo III. Thus, it can be seen that the method using the electrochemical graphene electrode of the present invention has similar utility as other conventional methods without causing serious loss of Exo III activity.

According to the present invention, an electrochemical measuring method using a graphene electrode for measuring Exo III activity is proposed. The method of the present invention can directly and uniformly and quantitatively measure enzyme activity using graphene electrochemical measurements, simply, cost-effectively and time-efficiently. Given the important role of exonucleases in biochemical processes, the methods of the present invention can be used as alternative measurement tools in drug screening and basic research involving enzymes.

<110> Soonchunhyang University Industry Academy Cooperation Foundation <120> Electrochemical assay for determining Exonuclease activity with          graphene electrode <130> PD10838 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GGCCGAGC and GCTCGGCC sequences bind and form a stem structure,          and TTTTTT sequence forms a loop structure. <400> 1 ggccgagctt ttttgctcgg cc 22

Claims (6)

Reacting a sample expected to contain an exonuclease with a double stranded DNA labeled with an electrochemical redox label;
Contacting the reaction product of the sample and double-stranded DNA with a graphene electrode; And
Detecting the activity of the exonuclease by measuring the reducing peak current of the electrochemical redox label through the graphene electrode,
The electrochemical redox label is covalently bound to the 5′-end of the double stranded DNA and labeled on the double stranded DNA (6),
When the exonuclease is present in the sample, the double-stranded DNA is hydrolyzed from the 3′-end by the exonuclease to generate single-stranded DNA, and the single-stranded DNA is immobilized on the graphene electrode surface. A reducing peak current of the electrochemical redox label is detected,
The graphene electrode is a method for measuring exonuclease activity that a graphene monolayer is attached to the electrode. delete According to claim 1, wherein the double-stranded DNA is a exo-based stem structure (stem) is a pair (stem) structure comprising a loop (link) structure (link) to connect the polynucleotide chain of the stem structure, exo Method for Measuring Nuclease Activity. The method of measuring exonuclease activity according to claim 1, wherein the double-stranded DNA has a sequence of 5'-GGCCGAGC TTTTTT GCTCGGCC-3 '. The method of claim 1, wherein the electrochemical redox label is selected from the group consisting of methylene blue, ferrocene, anthraquinone, and mixtures thereof. delete

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