Real-time reaction monitoring method and system for DNA (Deoxyribose Nucleic Acid) base removal sites and small molecule toxic compounds

Abstract

The invention discloses a real-time reaction monitoring method and a real-time reaction monitoring system for DNA (Deoxyribose Nucleic Acid) base removal sites and small molecule toxic compounds, and belongs to the technical field of biological analysis. The method comprises the following steps: constructing a compound formed by combining a uracil-containing DNA probe and an electrochemical active marker, and hydrolyzing uracil by using UDG enzyme to form a base removal site. A nanopore single-molecule detection technology is adopted, a compound is driven by voltage to pass through a hole, and a characteristic current signal is generated to establish a baseline map. Hydroxylamine derivatives are introduced to react with base removal sites, and dynamic interaction is analyzed according to time domain change of current signals. The system comprises a labeled compound preparation module, an enzymatic reaction module, a nanopore detection module, a small molecule compound reaction module and a central control and data processing module, all the modules cooperate, full-process automation from probe preparation to dynamic reaction monitoring is achieved, traditional sensitivity limitation is broken through, and a high-precision detection means is provided for studying the DNA damage and toxic compound action mechanism.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological analysis, and particularly relates to a method and system for real-time monitoring of DNA apurinic / apyrimidinic site and small molecule toxic compound reaction. BACKGROUND

[0002] DNA apurinic / apyrimidinic site (AP site) is a damage site with intact sugar-phosphate backbone but missing base, which is formed by spontaneous or environmental-induced loss of purine (A / G) or pyrimidine (C / T) base in DNA molecule. As a key intermediate of DNA damage repair, the accumulation of AP site directly threatens the stability of genome, and its dynamic change is closely related to environmental pollution, drug toxicity and disease mechanism. In the research of environmental and food safety, drug toxicology and disease mechanism, AP site is closely related to various toxic compounds. For example, heavy metal ions, aromatic hydrocarbon mutagenic small molecules or reactive oxygen species (ROS) produced by drug metabolism can specifically induce the generation of AP site through covalent modification or oxidative attack, and then cause secondary damage such as DNA strand break, base mismatch or cross-linking, ultimately leading to cell apoptosis or genetic information transmission error. Therefore, real-time capture of the reaction dynamics of AP site and toxic compounds is of great significance for revealing the mechanism of DNA damage and toxicity, and evaluating the harm level of potential toxic compounds.

[0003] Current AP site analysis mainly relies on population-level detection methods such as fluorescent labeling, gel electrophoresis or liquid chromatography-mass spectrometry (LC-MS), but these detection methods can only detect high-abundance damage and are difficult to capture trace AP sites induced by low-dose toxic compounds; they cannot real-time track the dynamic reaction process of AP site and toxic compounds, losing key mechanism information; the population average data masks the dynamic differences between different DNA molecules or sites, limiting the analysis of selective damage targets of toxic compounds.

[0004] Therefore, it is urgent to develop a new method with high selectivity and high sensitivity, which can real-time monitor the reaction dynamics of DNA apurinic / apyrimidinic site and toxic compounds at single molecule level, so as to reveal the molecular mechanism of toxic compounds and break through the limitations of the prior art. SUMMARY

[0005] In view of the fact that the prior art cannot achieve real-time monitoring of the reaction process of DNA apurinic / apyrimidinic site (AP site) and small molecule toxic compound, especially cannot capture transient intermediates appearing in the reaction, thereby limiting the in-depth study of the reaction mechanism of AP site and its toxicity characteristics, the present application aims to provide a method and system capable of real-time monitoring of the reaction process of DNA apurinic / apyrimidinic site and small molecule toxic compound. To achieve the above object, the present application adopts the following technical solutions: The present application provides a real-time reaction monitoring method for DNA dealkylation sites and small molecule toxic compounds, comprising: Step 1, incubate the electrochemically active label with the DNA probe containing uracil to form a labeled complex; Step 2, hydrolyze the uracil in the labeled complex obtained in step 1 using uracil DNA glycosylase to generate a labeled complex containing a dealkylation site; Step 3, inject the labeled complex into a nanopore detection cell, apply a voltage to drive the complex through the nanopore, and record the initial current signal; Step 4, add a small molecule toxic compound to the nanopore detection cell, continuously record the change in current signal after the complex reacts with the small molecule toxic compound, and analyze the reaction kinetics by signal characteristic difference; the small molecule toxic compound is selected from hydroxylamine derivatives.

[0006] In step 1, the DNA probe containing uracil is an oligonucleotide chain with a 5'-end modified thiol group, and the sequence contains uracil and adenine repeats, wherein the number of adenine repeats is ≥6; the electrochemically active label is vitamin B12 or its derivative, and the electrochemically active label is combined with the DNA probe through thiol-metal coordination or electrostatic interaction.

[0007] Preferably, the electrochemically active label is vitamin B12.

[0008] Preferably, the number of adenine repeats is 8.

[0009] In step 2, the concentration of uracil DNA glycosylase is 4-6 U / mL, and the concentration of the labeled complex is 290-300 nM.

[0010] In step 2, the hydrolysis temperature of the uracil DNA glycosylase is 20-37℃, the hydrolysis time is 10-60 min, the buffer used is potassium chloride phosphate buffer, and the pH is 6.0-8.0.

[0011] In step 3, the nanopore detection voltage is +100 mV to +150 mV, and the electrophoresis buffer is potassium chloride phosphate buffer.

[0012] The hydroxylamine derivative is at least one of O-methoxyamine (MX), O-isobutylhydroxylamine (IBX), O-(2-aminoethyl)hydroxylamine (AEX), and O-carboxymethylhydroxylamine (CMX).

[0013] Preferably, the hydroxylamine derivative is O-isobutylhydroxylamine (IBX).

[0014] The concentration of the hydroxylamine derivative is 1-20 mM, and the continuous detection time is greater than or equal to 5 min.

[0015] The nanopore is a solid nanopore or a biological nanopore, and the pore size is 0.5-3 nm.

[0016] The real-time reaction monitoring method of the DNA dealkylation site and the small molecule toxic compound also includes the steps of regulating the salt concentration and the pH value of the electrophoresis buffer.

[0017] Preferably, the salt concentration is 0.5-1.5 M, and the buffer pH is 6.0-8.0.

[0018] The present application provides a system for realizing the real-time reaction monitoring method of the DNA dealkylation site and the small molecule toxic compound, comprising: A labeled complex preparation module is used to complete the incubation and combination of the electrochemically active label and the DNA probe; An enzymatic reaction module is used to hydrolyze the uracil in the labeled complex by using UDG enzyme to generate a complex containing a dealkylation site; A nanopore detection module is used to drive the labeled complex to pass through the nanopore and collect the current signal; A small molecule compound reaction module is used to add a hydroxylamine derivative to the detection pool and monitor the reaction kinetics; A central control and data processing module is used for process control and data storage and output.

[0019] Compared with the prior art, the present application has the following beneficial effects: The real-time reaction monitoring method of the DNA dealkylation site and the small molecule toxic compound provided by the present application constructs a specific labeled DNA probe system, generates a controllable dealkylation site model through enzymatic reaction, and realizes dynamic tracking of the reaction by using nanopore single-molecule detection technology. The DNA probe containing uracil is combined with the electrochemically active label, the thiol group is used for directional modification to ensure the stability of the probe structure, and the complex is endowed with electrochemical signal characteristics; the uracil DNA glycosylase specifically hydrolyzes the uracil to form a standardized dealkylation site on the DNA chain, providing a uniform detection template for subsequent reactions; the nanopore electrochemical detection technology is used to establish the baseline signal spectrum of the dealkylation site by using the characteristic current signal generated when the labeled complex is driven to pass through the nanopore by voltage; the hydroxylamine derivative is introduced to specifically react with the dealkylation site, and based on the time-domain variation characteristics of the nanopore detection current signal, the dynamic interaction process between a single DNA molecule and a toxic compound is analyzed. The selection of the hydroxylamine derivative is based on its strong nucleophilic reactivity with the sugar ring of the dealkylation site, which can induce DNA chain rupture or structural rearrangement, thereby generating distinguishable current signal patterns.

[0020] Further, the 5'-end thiol-modified oligonucleotide chain can form a stable chemical bond with a metal electrode or a nanomaterial surface through a thiol group, thereby enhancing the fixing efficiency of the probe on the detection interface; the alternating arrangement of uracil and adenine repeats, especially the design of adenine repeats of 6 or more, not only provides a specific recognition site for uracil DNA glycosylase, but also promotes the formation of a double-stranded structure through the stacking effect of adenine, thereby ensuring efficient enzyme cutting; vitamin B12 and its derivatives are selected as electrochemically active markers, and the redox activity generated by the unique cobalt ion coordination center can produce a characteristic current signal in nanopore electrochemical detection; the binding of the marker and the DNA probe is achieved through thiol-metal coordination or electrostatic interaction, which not only ensures the structural stability of the marker complex, but also avoids the influence of covalent modification on the DNA conformation and enzyme cutting efficiency, thereby providing a precise molecular recognition basis for the generation of abasic sites and small molecule compound reactions.

[0021] Further, the use of solid-state nanopores can improve mechanical stability and detection reproducibility, and is suitable for high-throughput continuous detection; biological nanopores can maintain the molecular recognition characteristics of natural protein channels and enhance the sensitivity to conformational changes of the complex. By controlling the pore size in the range of 0.5-3 nm, the DNA complex containing electrochemically active markers can be effectively perforated, and the interaction between the marker and the inner wall of the channel is enhanced through the steric hindrance effect, thereby amplifying the current signal difference caused by the structural changes of the complex during the reaction.

[0022] The system provided by the application includes a marker complex preparation module, an enzyme reaction module, a nanopore detection module, and a small molecule compound reaction module. The marker complex preparation module constructs a detection probe with specific signal response by the directional binding of electrochemically active markers and DNA probes, thereby providing identifiable electrochemically active markers for subsequent nanopore detection. The enzyme reaction module generates an abasic site by specific hydrolysis of uracil by UDG enzyme, thereby simulating a real DNA damage environment and ensuring the biological relevance of the detection target. The nanopore detection module drives the complex to pass through the nanopore by applying a voltage, and directly recognizes and quantifies the abasic site by using the current signal difference generated by the single-molecule perforation event, thereby breaking through the sensitivity limitation of traditional group detection methods. The small molecule compound reaction module controls the addition time and concentration of hydroxylamine derivatives, and combines with the continuous acquisition of nanopore current signals, thereby real-time capturing the dynamic processes such as the binding and modification of the abasic site and toxic compounds, and revealing the reaction kinetics characteristics. The central control and data processing module coordinates the running time sequence of each module, synchronously records the current signal changes and reaction condition parameters, realizes the automatic control of the whole process and the high-throughput data correlation analysis, eliminates the manual operation errors, and improves the reliability of the detection results. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1The strategy and current trace diagram for monitoring the AP site generated by UDG enzyme hydrolysis of uracil based on nanopore technology, wherein (a) is a schematic diagram of the strategy for monitoring the AP site generated by UDG enzyme hydrolysis of uracil based on nanopore; (b) is V B12 The current trace diagram obtained by adding (dU)(dA)8 complex to the detection pool and applying +140 mV voltage at the Cis end.

[0024] Figure 2 The V B12 The current signal feature diagram of (dU)(dA)8 product in nanopore detection, wherein (a) is V B12 The current trace diagram obtained by adding (AP)(dA)8 to the detection pool and applying +140 mV voltage at the Cis end; (b) is V B12 The V B12 The histogram of blocking current (ΔI / I0) of (AP)(dA)8; (c) is V B12 The V B12 The scatter plot of blocking events of (AP)(dA)8.

[0025] Figure 3 The current trace diagram at a specific time point during the real-time recording of the process of UDG enzyme hydrolysis to generate AP site when the salt concentration of the buffer is 1 M.

[0026] Figure 4 The current trace diagram at a specific time point during the real-time recording of the process of UDG enzyme hydrolysis to generate AP site when the salt concentration of the buffer is 0.75 M Figure 5 The V B12 The schematic diagram of the reaction between (AP)(dA)8 and four hydroxylamine derivatives.

[0027] Figure 6 The V B12 The blocking current signal diagram at a specific time point during the dynamic reaction monitoring of (AP)(dA)8 and O-isobutyl hydroxylamine (IBX).

[0028] Figure 7 The V B12 The blocking current signal diagram at a specific time point during the dynamic reaction monitoring of (AP)(dA)8 and O-methoxyamine (MX), O-(2-aminoethyl)hydroxylamine (AEX) and O-carboxymethyl hydroxylamine (CMX); wherein (a) is MX, (b) is CMX, and (c) is AEX. Figure 8 The V B12-(AP)(dA)8 is the blocked current signal diagram at a specific time point during the monitoring process of the dynamic reaction of O-isobutylhydroxylamine (IBX).

[0029] Figure 9 V is 0.5 V when pH is 6.5 B12 -(AP)(dA)8 is the blocked current signal diagram at a specific time point during the monitoring process of the dynamic reaction of O-isobutylhydroxylamine (IBX). DETAILED DESCRIPTION

[0030] In order to better understand the technical scheme of the present application, the technical scheme in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should fall within the scope of protection of the present application.

[0031] It should be noted that the terms "first", "second" and the like used in the specification and claims are only used to distinguish similar objects, and do not necessarily describe a specific order or sequence; it should be understood that these terms can be interchanged under appropriate circumstances. In addition, the terms "include", "have" and any variations thereof are intended to cover non-exclusive inclusion; for example, a process, method, system, product or device that includes a series of steps or units does not necessarily limit to the explicitly listed steps or units, but can include other steps or units not explicitly listed or inherent thereto.

[0032] The technical scheme of the present application will be described below in conjunction with the drawings in the embodiments of the present application. Figures 1-9 The present application will be described in further detail; The present application aims to provide a method for real-time monitoring of DNA dealkylation sites reacting with small molecule toxic compounds by using nanopores.

[0033] (1) Dynamic detection of AP sites produced by UDG enzyme hydrolysis of uracil First step: 20 μL V B12 (5 mM) and 10 μL 5'-SH-(dU)(dA)8 (incubated at 37°C for 30 min, concentration 100 μM) to obtain a complex V B12 -(dU)(dA)8; Second step: take 15 μL V B12(dU)(dA)8complex (final concentration 0.33 μΜ) was added to the Cis side of the nanopore detection chamber, and a +140 mV voltage was applied, and the current signal was recorded. The electrophoresis buffer was 1 M KCl, 20 mM Tris, pH 7.4.

[0034] Step 3: Take 30 μL of V B12 (dU)(dA)8was added to the UDG enzyme (final concentration 5.3 U / mL) to generate complex V B12 (dU)(dA)8was added to the UDG enzyme (final concentration 5.3 U / mL) to generate complex V B12 (dU)(dA)8was added to the UDG enzyme (final concentration 5.3 U / mL) to generate complex V Step 4: Take 15 μL of V B12 (dU)(dA)8was added to the UDG enzyme (final concentration 5.3 U / mL) to generate complex V

[0035] Step 5: To explore the effect of salt concentration on the UDG enzyme hydrolysis reaction, 15 μL of V B12 (dU)(dA)8was added to the UDG enzyme (final concentration 5.3 U / mL) to generate complex V

[0036] (2) Real-time monitoring of the reaction of AP sites with small molecule toxic compounds Step 1: Take 15 μL of V B12 (dU)(dA)8was added to the UDG enzyme (final concentration 5.3 U / mL) to generate complex V 8加入 (dU)(dA)8was added to the UDG enzyme (final concentration 5.3 U / mL) to generate complex V

[0037] Step 2: To explore the effect of different pH values on VB12-(AP)(dA)8and four kinds of hydroxylamine derivatives, the electrophoresis buffer was 1 M KCl, 20 mM Tris, pH=6.5, and the monitoring was carried out according to the method of step one.

[0038] See the attachedFigure 1 To address this issue, 5'-SH-(dU)(dA)8 was designed because the sulfur atom (S) in the 5'-terminal modified thiol group (-SH) can react with V... B12 Cobalt ions (Co) 3+ Coordination and binding ultimately generate V B12 -(dU)(dA)8. This is because using wild-type hemolysin alone to measure VB12 and ssDNA produces irregular current signals with very short retention times and chaotic amplitudes. However, through this coordination binding mechanism, because the molecular structure of VB12 is larger than the contraction site of wild-type hemolysin, it can pull and hang ssDNA in the pores of wild-type hemolysin, increasing the ssDNA transit time; V B12 -(dU)(dA)8 at a final concentration of 0.33 μM was added to the Cis terminal of the nanopore detection cell. A +140 mV voltage was applied, and detection was performed in an electrophoresis buffer of 1 M KCl, 20 mM Tris, and pH 7.4. The applied voltage produced a blocking current signal with a long retention time and uniform amplitude. This is attributed to V... B12 The binding mechanism with the DNA probe slows down the velocity of DNA as it passes through the nanopore, thus increasing its residence time within the nanopore. This longer residence time allows for more accurate capture and analysis of current changes as DNA passes through the nanopore, providing a more stable and observable signal basis for subsequent studies on processes such as the hydrolysis of uracil by UDG enzymes to generate AP sites. The uniform amplitude indicates the formation of V... B12 The (dU)(dA)8 complex exhibits a relatively stable structure as it passes through nanopores, with a consistent degree of current blockage each time it passes through. This facilitates quantitative signal analysis and serves as a reference for subsequent studies on the interactions of other molecules with this complex.

[0039] See appendix Figure 2 In order to study the effect of UDG enzyme on V B12 Hydrolysis of uracil (dU) in (dU)(dA)8 and hydrolysis product V B12 - (AP)(dA)8 current signal characteristics in nanopore detection, towards V B12 After adding UDG enzyme to (dU)(dA)8 to a final concentration of 5.3 U / mL and incubating at room temperature for 30 min, the UDG enzyme specifically hydrolyzes uracil (dU), removing it from the DNA strand to produce product V containing an abase (AP) site. B12 - (AP)(dA)8; The product V generated by hydrolysis B12- (AP)(dA)8 was added to the Cis terminal of the detection cell, and a +140mV voltage was applied. Detection was performed under the same electrophoresis buffer conditions (1 MKCl, 20 mM Tris, pH = 7.4), and the current signal was recorded. Simultaneously, V B12 - (dU)(dA)8 uses the current signal under the same conditions as a reference.

[0040] From the appendix Figure 2 The data shows that each event corresponds to a V. B12 The current signals from the -(AP)(dA)8 complex passing through nanopores exhibit a certain regularity, but are significantly different from the signals of VB12 -(dU)(dA)8. Under the same conditions, V B12 -(AP)(dA)8 produced a value greater than V B12 The small amplitude and distinct oscillation of the -(dU)(dA)8 current signal indicate that the formation of AP sites alters the structure or charge distribution of the complex, leading to a reduced and more varied degree of current blockage during its passage through the nanopore. This difference is due to the lack of bases at the AP sites, which changes the spatial structure and electrical properties of the complex, thus affecting its interaction with the nanopore. UDG enzymes can hydrolyze uracil (dT) to generate AP sites. By comparing V... B12 - (dU)(dA)8 and V B12 The current signal characteristics of - (AP)(dA)8 clearly show the influence of the process of UDG enzyme hydrolysis of uracil to generate AP sites on the detection signal of the complex nanopore, providing important experimental evidence for studying the properties of DNA abase sites and related biological processes.

[0041] In order to monitor the production of V at the AP site by UDG enzyme hydrolysis of uracil (dT) B12 The dynamic process of -(AP)(dA)8 will V B12 -(dU)(dA)8 was added to the Cis terminal of the detection cell (final concentration 0.33 μM), a voltage of +140 mV was applied, and current signal recording began. After 2 min, UDG enzyme (final concentration 5.3 U / mL) was added to the Cis terminal and stirred thoroughly. At this point, the current signal was continuously recorded. After 10 min, the current signal completely transformed into an oscillating VB12-(AP)(dA)8 current signal. Figure 3 This represents the blocking current signal at a specific time point during the hydrolysis of UDG enzyme. The electrophoresis buffer consisted of 1 M KCl, 20 mM Tris, and pH 7.4.

[0042] From the appendix Figure 3 The data shows that in the initial stage (0~2 min): without the addition of UDG enzyme, the current signal mainly presents as VB12 - (dU)(dA)8, with relatively stable amplitude and certain blocking pattern. Reaction proceeding stage (2~10 min): After adding UDG enzyme, the current signal gradually changed over time, V B12 - (AP)(dA)8, with relatively small and oscillating amplitude, and V B12 - (dU)(dA)8, with obvious difference. With the reaction proceeding, V B12 - (AP)(dA)8, indicating that the hydrolysis reaction of UDG enzyme was continuously going on, constantly converting V B12 - (dU)(dA)8 into V B12 - (AP)(dA)8. Reaction completion stage (after 10 min): After 10 min, the current signal was completely converted into oscillating V B12 - (AP)(dA)8. This indicates that at this time, V B12 - (dU)(dA)8 was almost completely hydrolyzed by UDG enzyme, and the reaction reached a relatively stable state, with the product mainly being V B12 - (AP)(dA)8.

[0043] To investigate the effect of different salt concentrations on the hydrolysis rate of UDG, the UDG enzyme hydrolysis process was monitored using 0.75 M KCl, 20 mM Tris, pH=7.4 electrophoresis buffer in the same way as above and the current signal was recorded. Figure 4 The blocking current signal at a specific time point selected for the UDG enzyme hydrolysis process. With the passage of time (0~6 min), the blocking current signal changed significantly; at 0 min, the signal mainly corresponded to V B12 - (dU)(dA)8, showing specific current blocking characteristics; with the proceeding of UDG enzyme hydrolysis reaction, V B12 - (AP)(dA)8, while V B12 - (dU)(dA)8 signal correspondingly decreased. This intuitively demonstrated that UDG enzyme hydrolyzed V B12 - (dU)(dA)8, i.e. uracil (dU) was gradually hydrolyzed to produce product V B12 - (AP)(dA)8. Under low salt concentration (0.75 M KCl) conditions, it can be seen from the signal change that UDG enzyme completely or mostly hydrolyzed V B12 - (dU)(dA)8 into V B12The time taken for -(AP)(dA)8 is relatively short. This indicates that UDG enzymes can catalyze hydrolysis reactions more efficiently and at a faster rate under low salt concentrations. Lower salt concentrations reduce the adverse effects of ions on the electrostatic interactions between the enzyme and substrate, making UDG enzymes more readily react with V... B12 -(dU)(dA)8 binding promotes the hydrolysis reaction; changes in salt concentration affect the enzyme conformation, making its active site more conducive to catalytic reaction, thereby increasing the activity of UDG enzyme and shortening the hydrolysis time.

[0044] Experimental results show that under low salt concentration conditions of 0.75 M, UDG enzyme will increase V B12 -(dU)(dA)8 hydrolyzes to V B12 The short time required for -(AP)(dA)8 indicates that the UDG enzyme activity is stronger under low salt concentration conditions.

[0045] Figure 5 For V B12 The diagram illustrates the reaction between -(AP)(dA)8 and four hydroxylamine derivatives. The core of the reaction is the interaction between the debasement site (AP) and the hydroxylamine derivative, leading to a change in the state of the AP site. MX: The R group is methyl (CH3). MX reacts with the AP site to form a new chemical bond, changing the AP site from a closed state to an open state. AEX (aminoethyl hydroxylamine): The R group is aminoethyl (NH2-CH2-CH2-). The reaction mechanism of AEX with the AP site is similar to MX, changing the state of the AP site through the formation of a new chemical bond. CMX (carboxymethyl hydroxylamine): The R group is carboxymethyl (COOH-CH2-). CMX reacts with the AP site, also causing a change in the state of the AP site. IBX (isobutyl hydroxylamine): The R group is isobutyl. The reaction of IBX with the AP site also follows the same pattern, changing the structure of the AP site through the formation of chemical bonds.

[0046] Furthermore, real-time monitoring of the interaction between the AP site and inhibitors such as O-methoxyamine (MX), O-isobutylhydroxylamine (IBX), O-(2-aminoethyl)hydroxylamine (AEX), and O-carboxymethylhydroxylamine (CMX) is valuable for assessing their mechanisms of action and therapeutic potential. B12 Add UDG enzyme (final concentration 5.3 U / mL) to -(dU)(dA)8 and incubate at room temperature for 30 min to allow complete reaction to generate VB12-(AP)(dA)8. B12 -(AP)(dA)8 was added to the Cis terminal of the detection cell (final concentration 0.33 μM), a voltage of +120 mV was applied, and current signal recording began. After 2 minutes, IBX (final concentration 10 mM) was added to the Cis terminal and stirred until homogeneous. The current signal was then continuously recorded.B12 -(AP)(dA)8 reacts with IBX to produce V B12 -(IBX)(dA)8, it can produce more than V B12 -(AP)(dA)8 is a blocking current signal with a large amplitude and no oscillation. The proportion of this newly generated signal increases over time. Figure 6 This represents the blocking current signal at a specific time point selected during real-time monitoring of the AP site and IBX reaction. The electrophoresis buffer consisted of 1 M KCl, 100 mM Tris, and pH 7.4. The same method was used to monitor MX (final concentration 10 mM), AEX (final concentration 10 mM), and CMX (final concentration 10 mM) in real time with V. B12 The reaction of -(AP)(dA)8 (final concentration 0.33 μM). The three and V B12 The blocking current signals generated by the reaction products of -(AP)(dA)8 are significantly different, such as Figure 7 .

[0047] From the appendix Figure 7 Data shows that "V" B12 The signal point "-(AP-MX)(dA)8" appears, and its corresponding blocking current signal characteristics are similar to the original V. B12 The -(AP)(dA)8 signal shows a clear difference, indicating that MX and V B12 -(AP)(dA)8 underwent a specific reaction, altering the structure of the complex and thus affecting its current signal as it passed through the nanopore; similarly, CMX and V B12 The reaction of -(AP)(dA)8 produces products with different electrical properties, V B12 The signal point -(AP-AEX)(dA)8 also exhibits a different current signal pattern than the previous two, indicating that AEX and V B12 The reaction of -(AP)(dA)8 has its own characteristics. These different current signal characteristics can be used to distinguish different reagents from V. B12 The reaction product of -(AP)(dA)8 provides experimental evidence for further research on the interaction between the AP site and different reagents. To investigate the effects of different pH values ​​on the interactions between the AP site and inhibitors of O-methoxyamine (MX), O-isobutylhydroxylamine (IBX), O-(2-aminoethyl)hydroxylamine (AEX), and O-carboxymethylhydroxylamine (CMX), a low-pH electrophoresis buffer (1 MKCl, 100 mM Tris, pH=6.5) was used. Real-time monitoring was performed using the same method described above. See Appendix for details. Figures 8-9 As shown.

[0048] From the appendix Figures 8-9The data shows that, over time, under pH 6.5 conditions, V B12 During the reaction of -(AP)(dA)8 with O-isobutylhydroxylamine (IBX), the blocking current signal changed significantly. In the initial stage of the reaction, V was the primary current. B12 -(AP)(dA)8's own current signal, as the reaction proceeds, V B12 The signal of -(AP-IBX)(dA)8 gradually appeared and increased, and a significant signal change was observed in a short period of time, indicating that the AP site and IBX could react quickly at pH 6.5, and the reaction rate was fast. At pH 6.5, V... B12 The signal of -(AP)(dA)8 gradually decreases, while V B12 The signal of -(AP-MX)(dA)8 gradually increases, and the signal transition rate is faster at pH 6.5 compared to the reaction under higher pH conditions, indicating that low pH promotes the reaction between AP sites and MX, thus increasing the reaction rate; similarly, a signal transition from V... B12 -(AP)(dA)8 signal to V B12 The transition of the -(AP-AEX)(dA)8 signal is observed, and this transition is more rapid under low pH conditions. This indicates that the interaction between AEX and AP sites is also affected by pH; a low pH environment favors the reaction, altering the protonation state of AEX or the reactivity of the AP site, making the two more likely to bind. At pH 6.5, CMX and V... B12 -(AP)(dA)8 also reacts relatively quickly, V B12 The signal of -(AP-CMX)(dA)8 increased significantly within a short period of time. This indicates that low pH also promotes the response of CMX and AP sites.

[0049] In summary, under low pH conditions (pH 6.5), the reaction rate between the AP site and MX, IBX, AEX, and CMX is faster. This not only contributes to a deeper understanding of the interaction mechanism between the AP site and different inhibitors, but also provides experimental evidence for utilizing pH-regulated reactions in fields such as biochemistry and molecular biology. When developing biosensors or therapeutic methods based on AP site detection or regulation, optimizing reaction conditions by adjusting pH can be considered.

[0050] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for real-time monitoring of the reaction between DNA abase sites and small molecule toxic compounds, characterized in that, include: Step 1: Incubate the electrochemically active label with a DNA probe containing uracil to form a labeling complex; Step 2: Use uracil DNA glycosylation enzyme to hydrolyze uracil in the labeling complex obtained in Step 1 to generate a labeling complex containing a base-depleting sites. Step 3: Inject the labeled complex into the nanopore detection cell, apply a voltage to drive the complex through the nanopore, and record the initial current signal; Step 4: Add a small molecule toxic compound to the nanopore detection cell, continuously record the changes in current signal after the complex reacts with the small molecule toxic compound, and analyze the reaction kinetics by the difference in signal characteristics; the small molecule toxic compound is selected from hydroxylamine derivatives.

2. The method for real-time monitoring of the reaction between DNA abase sites and small molecule toxic compounds according to claim 1, characterized in that, In step 1, the uracil-containing DNA probe is an oligonucleotide chain with a 5'-end modified with a thiol group, the sequence of which contains uracil and adenine repeat sequences, wherein the number of adenine repeats is ≥6; the electrochemically active label is vitamin B12 or its derivative, and the electrochemically active label binds to the DNA probe through thiol-metal coordination or electrostatic interaction.

3. The method for real-time monitoring of the reaction between DNA abase sites and small molecule toxic compounds according to claim 1, characterized in that, In step 2, the concentration of the uracil DNA glycosylation enzyme is 4-6 U / mL, and the concentration of the labeling complex is 290-300 nM.

4. The method for real-time monitoring of the reaction between DNA abase sites and small molecule toxic compounds according to claim 1, characterized in that, In step 2, the hydrolysis temperature of the uracil DNA glycosylation enzyme is 20~37℃, the hydrolysis time is 10~60min, and the buffer solution used is potassium chloride phosphate buffer with pH=6.0~8.

0.

5. The method for real-time monitoring of the reaction between DNA abase sites and small molecule toxic compounds according to claim 1, characterized in that, In step 3, the nanopore detection voltage is +100 mV to +150 mV, and the electrophoresis buffer is potassium chloride phosphate buffer.

6. The method for real-time monitoring of the reaction between DNA abase sites and small molecule toxic compounds according to claim 1, characterized in that, The hydroxylamine derivative is at least one of O-methoxyamine, O-isobutylhydroxylamine, O-(2-aminoethyl)hydroxylamine, and O-carboxymethylhydroxylamine.

7. The method for real-time monitoring of the reaction between DNA abase sites and small molecule toxic compounds according to claim 1, characterized in that, The concentration of the hydroxylamine derivative is 1~20mM, and the continuous detection time is ≥5min.

8. The method for real-time monitoring of the reaction between DNA abase sites and small molecule toxic compounds according to claim 1, characterized in that, The nanopores are solid nanopores or biological nanopores with a pore size of 0.5~3 nm.

9. A method for real-time monitoring of the reaction between DNA abase sites and small molecule toxic compounds according to any one of claims 1 to 8, characterized in that, It also includes steps for adjusting the salt concentration and pH value in the electrophoresis buffer.

10. A system for implementing the real-time reaction monitoring method for DNA abase sites and small molecule toxic compounds as described in any one of claims 1 to 9, characterized in that, include: The labeling complex preparation module is used to complete the incubation and binding of electrochemically active labels with DNA probes; The enzyme-catalyzed reaction module utilizes UDG enzyme to hydrolyze uracil in the labeled complex, generating a complex containing a base-depleting sites; Nanopore detection module, used to drive the labeled complex through the nanopore and acquire the current signal; Small molecule compound reaction module, used to add hydroxylamine derivatives to the detection cell and monitor reaction kinetics; The central control and data processing module is used for process control and data storage and output.

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