CN112285178B - Probe and method for determining ALKBH3 activity based on alkyl removal - Google Patents
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Abstract
The invention discloses a probe and a method for determining ALKBH3 activity based on alkyl removal. When ferrocene (Fc) labeled double-stranded DNA is immobilized on an electrode, a strong electrochemical signal can be obtained. However, in the presence of ALKBH3, since ALKBH3 can remove the alkyl group of the Fc-DNA probe, the 3' -blunt end of the formed DNA can be recognized and degraded by exonuclease III (ExoIII). Therefore, the electrochemical signal generated by Fc is greatly reduced, and the activity of ALKBH3 can be very easily detected by the change of the electrochemical signal. The invention can realize wider detection range and lower detection limit, and also provides a new method for clinically detecting the prostate cancer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a probe and a method for determining ALKBH3 activity based on alkyl removal.
Background
ALKBH3, also known as prostate cancer antigen 1 (PCA-1), is a 37.9kD enzyme that repairs nucleobases by removing alkyl groups from them. It plays a central role in a variety of diseases associated with DNA damage, such as tumors, neurological diseases and developmental defects. Many studies have shown that the expression level of ALKBH3 in prostate cancer is much higher than Benign Prostatic Hyperplasia (BPH) and is closely related to the degree of tumor differentiation and clinical staging of prostate cancer. Therefore, the detection of ALKBH3 is particularly important for enhancing the early diagnosis of prostate cancer.
Currently, quantitative detection methods for ALKBH3 include immunohistochemistry and immunoblotting. However, most of the above methods are cumbersome and time consuming, and cannot be widely used in clinical practice. More importantly, these methods do not detect the activity of ALKBH3, only the amount thereof. To avoid the disadvantages of the above methods, the activity of ALKBH3 in cell lysates has been measured by methods such as radiolabelling and fluorescence. Unfortunately, their inherent disadvantages (e.g., labor, low sensitivity, and potential radiological hazards) limit their widespread use.
Therefore, there is a need to actively search for a highly sensitive, simple, and cost-effective method for determining the activity of ALKBH3.
Disclosure of Invention
Aiming at the problems of low sensitivity and potential radioactive hazard of the determination method for ALKBH3 activity in the prior art, the invention provides a probe and a method for determining ALKBH3 activity based on alkyl removal.
In one aspect, the invention provides a probe for determining ALKBH3 activity based on alkyl group removal,
the sequence of the probe 1 (DNA 1) is 5 '-TACCCCAAACCCGCCACCTTT-SH-3' (SEQ ID NO. 1);
the sequence of probe 2 (DNA 2) is 5'- (Fc) GGGTGGGCGGGTTGGGTA (m 1A) -3' (SEQ ID NO. 2).
In another aspect, the invention provides a method for determining the activity of akbh 3 based on removing alkyl groups, comprising the steps of:
step (1) gold electrode pretreatment:
a. placing a gold electrode in sulfuric acid and H 2 O 2 Sulfuric acid-H in a ratio of 1:3 to 3:7 2 O 2 Soaking in the mixed solution for 10-30 minutes;
b. washing with pure water;
c. and placing the gold electrode in a sulfuric acid solution with the concentration of 1-3mol/L to sweep cyclic voltammetry to obtain the pretreated gold electrode.
Step (2) DNA fixation:
a. modifying a fixing buffer solution containing DNA1 (SEQ ID NO. 1) with the final concentration of 1-3 mu M on the gold electrode obtained in the step (1) through Au-S bonds and incubating; wherein, the 3' end of the DNA1 sequence is modified by Sulfydryl (SH).
b. Washing with 10mM Tris-HCl gold electrode wash buffer pH 8.0 and incubating for 20-40 min with 2mM 6-Mercaptohexanol (MCH) in Tris-HCl buffer;
c. thoroughly rinsing with washing buffer and deionized water;
d. immersing DNA hybridization buffer solution containing DNA2 (SEQ ID NO. 2) with final concentration of 1-3 μ M on the surface of the electrode and incubating at 37 ℃ for 1-3 hours to obtain rigid double-stranded DNA; wherein, the 5 'end of the DNA2 sequence is marked by ferrocene (Fc), and the 3' end is modified by 1-methyl adenosine (m 1A).
Step (3) ALKBH3 activity analysis:
a. immersing the gold electrode in ALKBH3 for 10-30 minutes to remove the alkyl group at the 3' end of the DNA2 probe;
b. continuously cleaning the electrode for three times by using a cleaning buffer solution and deionized water;
c. the electrodes were immersed in a solution containing 40mM Tris-HCl, 50mM NaCl, 10mM MgCl 2 And 5-15U/mL Exo III, pH7.9 reaction buffer at 37 degrees C were incubated for 20-40 minutes;
d. heating to 60-90 ℃ for 5-15 minutes to inactivate the ExoIII, and carrying out electrochemical detection on the ALKBH3 activity;
preferably, in step (1) -a, sulfuric acid-H 2 O 2 Sulfuric acid and H in the mixed solution 2 O 2 The ratio of (A) to (B) is 2: 5;
preferably, in the step (1) -a, the soaking time is 20 minutes;
preferably, in the steps (1) -c, the concentration of the sulfuric acid solution is 2mol/L;
preferably, in step (2) -a, the final concentration of DNA1 is 2. Mu.M
Preferably, in steps (2) -b, the incubation time is 30 minutes;
preferably, in steps (2) -d, the final concentration of DNA2 is 2. Mu.M;
preferably, in steps (2) -d, the incubation time is 2 hours;
preferably, in steps (3) -c, the reaction buffer contains 10U/mL Exo III;
preferably, in steps (3) -c, the incubation time is 30 minutes.
Compared with the prior art, the invention has the beneficial effects that:
the present invention provides a new method for detecting ALKBH3 activity in cell lysates based on alkyl removal probes and based on alkyl removal causing degradation of the signaling probe. Has the following advantages:
(1) The method is not only the first report of ALKBH3 activity detection based on biosensing design, but also a brand-new method which is more flexible and reliable than the traditional method;
(2) The use of low cost DNA probes makes the method cost effective;
(3) The existence of ALKBH3 binding sites indicates the thermodynamic feasibility of the method;
(4) The determination method is simple and quick, only needs one-step reaction, and does not need complicated washing and processing steps;
(5) The method can detect ALKBH3 activity in purified samples and complex cell extracts, and has important value for clinical early diagnosis and treatment of prostate cancer.
Drawings
FIG. 1 is a schematic diagram of activity detection of ALKBH 3;
FIG. 2 is a representation of the electrode modification process;
FIG. 3 is a graph showing the optimization results of the measurement conditions;
FIG. 4 is a graph showing the results of electrochemical assay of ALKBH3 activity;
FIG. 5 is a graph showing the results of selectivity of the electrochemical sensor;
FIG. 6 is a graph showing the results of ALKBH3 activity assays for different PC3 cell numbers;
fig. 7 is a comparison of the activity of ALKBH3 detected by the two methods.
Detailed Description
The present invention will be further explained with reference to specific embodiments in order to make the technical means, the original characteristics, the achieved objects and the effects of the present invention easy to understand, but the following embodiments are only preferred embodiments of the present invention, and not all embodiments are possible. Other embodiments obtained by persons skilled in the art without making creative efforts based on the embodiments in the implementation belong to the protection scope of the invention.
Experimental materials:
all oligonucleotide sequences were purified by HPLC and synthesized by Shanghai Biotechnology Ltd, and the basic sequences were as follows: DNA1:5 '-TACCCAACCCGCCACCTTT-SH-3' (SEQ ID No. 1); DNA2:5'- (FC) GGGTGGGCGGGTTGGGTA (m 1A) -3' (SEQ ID No. 2). Purified recombinant human ALKBH3 was purchased from Creative Biomart. Prostate Specific Antigen (PSA), 6-Mercaptohexanol (MCH), exonuclease III, and sarcosine were purchased from Sigma. Tissue samples from prostate cancer patients were obtained from the urology department of prostate cancer at the second hospital, tokyo, and approved by the ethics committee of this hospital.
EXAMPLE 1 pretreatment of gold electrodes
Placing a gold electrode in sulfuric acid and H 2 O 2 Sulfuric acid-H in a ratio of 2:5 2 O 2 Soaking in the mixed solution for 20 minutes; then washing with pure water; and (3) placing the gold electrode in a sulfuric acid solution with the concentration of 2mol/L to sweep cyclic voltammetry to obtain a pretreated gold electrode.
Example 2 DNA fixation:
a fixing buffer containing DNA1 (SEQ ID NO. 1) at a final concentration of 2. Mu.M was modified by Au-S bond on the gold electrode obtained in example 1 and incubated; wherein, the 3' end of the DNA1 sequence is modified by Sulfydryl (SH). Washing with 10mM Tris-HCl gold electrode wash buffer pH 8.0 and incubating for 20-40 min with 2mM 6-Mercaptohexanol (MCH) in Tris-HCl buffer; then thoroughly rinsing with washing buffer and deionized water; immersing a DNA hybridization buffer solution containing DNA2 (SEQ ID NO. 2) at a final concentration of 2. Mu.M on the surface of the above-mentioned electrode and incubating at 37 ℃ for 2 hours to obtain a rigid double-stranded DNA; wherein, the 5 'end of the DNA2 sequence is marked by ferrocene (Fc), and the 3' end is modified by 1-methyl adenosine (m 1A), so that the DNA is fixed on the gold electrode.
Example 3 ALKBH3 Activity assay
The gold electrodes obtained in example 1 and example 2, on which the DNA immobilization was completed, were used for the subsequent assay of the ALKBH3 activity. The method comprises the following steps: immersing the gold electrode in ALKBH3 for 10-30 minutes to remove the alkyl group at the 3' end of the DNA2 probe; continuously cleaning the electrode for three times by using a cleaning buffer solution and deionized water; the electrodes were immersed in a solution containing 40mM Tris-HCl, 50mM NaCl, 10mM MgCl 2 And 10U/mL Exo III, pH7.9 reaction buffer at 37 degrees C were incubated for 30 minutes; heating to 60-90 ℃ for 5-15 minutes to inactivate the ExoIII, and carrying out electrochemical detection on the ALKBH3 activity.
Experimental example 1 characterization of the modification of the sensing platform
As is clear from FIG. 1, when double-stranded DNA was immobilized on the electrode, DNA2 could obtain a higher electrochemical signal due to the labeling with Fc. Without ALKBH3, 3' DNA blunt ends could not be formed and further could not be digested by Exo Il1, and the electrochemical signal generated from DNA2 would not change. However, the presence of ALKBH3 allows the alkyl group at the 2-terminal end of 3' DNA to be removed and then recognized and digested by Exo Ill, resulting in a rapid drop in electrochemical signal, enabling quantification of ALKBH3 activity.
The experimental example uses an electrochemical alternating current impedance method (EIS) to verify the stepwise modification process of the electrode. As shown in fig. 2, the EIS of the bare electrode is almost a straight line (curve a), indicating that the electron transfer resistance is very low. Then, a small semicircle (curve b) appeared after the self-assembly of DNA1, indicating that DNA1 is immobilized on the electrode surface. Thereafter, a large semicircle was observed after addition of DNA2 due to hybridization of DNA2 and DNA1 (curve c). However, when ALKBH3 and ExoIII were added to DNA 2/DNA 1, the diameter of the semicircle showed a significant decrease (curve d) due to the DNA cleavage by ExoIII. The above results demonstrate the success of stepwise modification of the electrode.
Experimental example 2 optimization of measurement conditions
In order to obtain the best sensing performance, the experimental example optimizes important conditions influencing the experiment. Several key conditions including the reaction time of ALKBH3, the concentration of Exo III and the reaction time were optimized separately. The following experimental conditions were found to give the best results: (a) The optimal reaction time for ALKBH3 was about 20 minutes (FIG. 3A). (b) The optimal concentration and reaction time for Exo III were 10U/ml (FIG. 3B) and 30 minutes (FIG. 3C), respectively.
Experimental example 3 electrochemical detection of ALKBH3 Activity
The activity of ALKBH3 was determined by DPV under the optimized conditions of Experimental example 2. As can be seen from fig. 4A, the peak current decreased significantly as the ALKBH3 concentration increased between the range of 0.1 and 20ng/mL, because the alkyl group of the signaling probe (DNA 2) was eluted from the electrode after being removed by the ALKBH3 and cleaved by Exo III. As shown in FIG. 4B, the linear relationship contained between the logarithm of the ALKBH3 concentration and the peak current is y = -1.70x +2.406 (R = -1.70x + 2 = 0.997), where "y" is the peak current, "x" is the logarithm of the ALKBH3 concentration, with a detection limit of 0.04ng/mL (S/N = 3). The results show that the biosensor can effectively detect the activity of ALKBH3.
Experimental example 4 Selectivity of sensing System
To assess the selectivity of the biosensor, its response to potential interference from other proteins (e.g., BSA, PSA, and sarcosine) was assessed. Due to the substrate specificity of ALKBH3, the present invention allows the identification of ALKBH3 from BSA, sarcosine and PSA. As shown in fig. 5, in the presence of ALKBH3, only a small peak current was observed. In contrast, significant peak currents were obtained in the presence of BSA, PSA and sarcosine. These results indicate that the detection method for ALKBH3 activity shows good selectivity.
Experimental example 5 analysis of ALKBH3 Activity in biological samples
This experimental example used the biosensor to evaluate the activity of ALKBH3 in cell lysates. As shown in fig. 6, the DPV peak current continued to decrease with the increase in PC3 cells. As a result, it was found that: with the increase of PC3 cells, the alkyl group at the end of DNA 23' is removed by more and more ALKBH3, and subsequent cleavage by Eox III results in a weakened peak current.
The exponential phase cells were collected and resuspended in 10mM PBS (pH 7.4). Typically, the intracellular components are obtained by repeated freeze-thawing. After centrifugation at 2000 rpm for 20 minutes at 4 ℃, the supernatant was collected for further use. The activity of ALKBH3 in tissue samples is detected by ELISA according to the instructions of ALKBH3 ELISA kit (Nanjing PunuoEn Biotechnology Co., ltd.). As shown in fig. 7, the results indicate that this method is consistent with the ELISA method results, and that the activity of ALKBH3 is increased in highly differentiated tissues.
In general, the present invention constitutes a novel and efficient biosensor that utilizes Exo III to degrade Fc-labeled DNA to detect the activity of ALKBH3 in eukaryotic cells, which has several significant advantages over conventional methods. First, the method is flexible and economical, it is based on the design of DNA probes, avoiding the use of expensive antibodies. Secondly, the detection limit of the method for detecting ALKBH3 activity can be as low as 0.04ng/ml. In addition, the method has been successfully applied to actual biological samples. In view of the above advantages, the biosensor constructed by the present invention is expected to be a powerful tool for early detection and treatment of prostate cancer.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> second Hospital of Nanjing City
NANJING GAOCHUN PEOPLE'S Hospital
NANJING DRUM TOWER Hospital
<120> probe and method for determining ALKBH3 activity based on alkyl removal
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tacccaaccc gcccaccctt t 21
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Claims (3)
1. A method for determining the activity of ALKBH3 based on removal of alkyl groups, characterized in that: the method comprises the following steps:
(1) Pretreatment of a gold electrode:
a. placing a gold electrode in sulfuric acid-H 2 O 2 Soaking in the mixed solution;
b. washing with pure water;
c. placing the gold electrode in a sulfuric acid solution to sweep cyclic voltammetry to obtain a pretreated gold electrode;
(2) DNA fixation:
a. modifying a fixing buffer solution containing DNA1 (SEQ ID NO. 1) on the gold electrode obtained in the step (1) through an Au-S bond, and incubating, wherein the final concentration of the DNA1 is 1-3 mu M;
b. washing with gold electrode washing buffer solution, and incubating for 20-40 min with Tris-HCL buffer solution;
c. thoroughly rinsing with washing buffer and deionized water;
d. immersing DNA hybridization buffer solution containing DNA2 (SEQ ID NO. 2) into the surface of the electrode and incubating to obtain rigid double-stranded DNA, wherein the final concentration of the DNA2 is 1-3 mu M;
(3) ALKBH3 activity assay:
a. immersing the gold electrode in ALKBH3 for 10-30 minutes to remove the alkyl group at the 3' end of the DNA2 probe;
b. continuously cleaning the electrode three times by using a cleaning buffer solution and deionized water;
c. immersing the electrode in a reaction buffer solution to incubate at 37 ℃;
d. heating to 60-90 ℃ for 5-15 minutes to inactivate the ExoIII, and carrying out electrochemical detection on the ALKBH3 activity;
wherein the DNA1 sequence is 5 '-TACCAAACCCGCCACCTTT-SH-3' (SEQ ID NO. 1);
the DNA2 sequence is 5'- (Fc) GGGTGGGCGGGTTGGGTA (m 1A) -3' (SEQ ID NO. 2).
2. The method of claim 1, wherein:
in step (1) -a, sulfuric acid-H 2 O 2 Sulfuric acid and H in the mixed solution 2 O 2 The ratio of (1);
in the step (1) -b, the soaking time is 10-30 minutes;
in the steps (1) to (c), the concentration of the sulfuric acid solution is 1 to 3mol/L.
3. The method of claim 1, wherein:
in the steps (2) -b, the gold electrode washing buffer solution is 10mM Tris-HCL with the pH value of 8.0;
in steps (2) -b, the Tris-HCL buffer contained 2mM 6-mercaptohexanol.
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| CN104833712A (en) * | 2015-03-30 | 2015-08-12 | 南昌大学 | Double electrical signal and DNA circulating amplification technique-based Dam methyltransferase activity detection method |
| WO2018010681A1 (en) * | 2016-07-14 | 2018-01-18 | 青岛大学 | Electrochemical biosensor based on aptamer/nano-silver probes and exo i enzyme |
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