CN113684244A - A Label-Free, Homogeneous Bioluminescence Detection Method for DNA Damage - Google Patents

Abstract

Translated fromChinese

本发明涉及一种无标记、均相生物发光的DNA损伤检测方法。DNA损伤检测与肿瘤、神经退行性疾病的发展具有密切的联系，现有DNA损伤的检测方法往往设计复杂的探针设计和标记方式，检测设备昂贵。本发明提供了一种无标记、均相生物发光的DNA损伤检测方法，基于碱基切除修复(BER)途径和末端转移酶(TDT)启动的无模板等温循环扩增，实现了无标记和均相生物发光法检测基因组DNA中的单碱基损伤和聚集损伤。该检测方法具有良好的灵敏度，检测过程中无需分离及洗涤的步骤，能够显著节约检测程序，用于疾病的诊断或抗肿瘤活性成分的筛选具有重要意义。

The invention relates to a label-free, homogeneous bioluminescence DNA damage detection method. DNA damage detection is closely related to the development of tumors and neurodegenerative diseases. Existing DNA damage detection methods often design complex probe design and labeling methods, and the detection equipment is expensive. The invention provides a label-free, homogeneous bioluminescence DNA damage detection method, which is based on the base excision repair (BER) pathway and the template-free isothermal cyclic amplification initiated by terminal transferase (TDT), and realizes label-free and homogeneous Phase bioluminescence detection of single-base and aggregated lesions in genomic DNA. The detection method has good sensitivity, does not require separation and washing steps in the detection process, can significantly save detection procedures, and is of great significance for disease diagnosis or screening of antitumor active components.

Description

Label-free homogeneous phase bioluminescence DNA damage detection method

Technical Field

The invention belongs to the technical field of DNA damage detection, and particularly relates to a label-free homogeneous bioluminescent DNA damage detection method, a kit based on the detection method, and application of the detection method and the kit in the fields of disease diagnosis and antitumor activity drug screening.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Maintaining genome integrity and stability is of great importance to humans, and cellular DNA is constantly affected by various endogenous factors (such as hydrolysis, reactive oxygen species and nitrogen) and exogenous factors (such as X-rays, UV light and many chemicals), producing 10 per day in a single human cell⁴-10⁶A DNA damage event. Genomic instability plays a key role in cancer, aging and alzheimer's disease. The most common DNA damage is base, ribose and single strand break damage, which is caused by oxidation, alkylation, deamination, spontaneous hydrolysis, etc. When these lesions are converted to mutations by erroneous repair or replication, they will be permanent and continue to trigger a range of effects in progeny cells. An important consequence of mutations is the loss of tumor suppressor genes and the activation of proto-oncogenes, leading to uncontrolled proliferation of cells and thus to the initiation of cancer.

There are two types of DNA damage, including single base DNA damage and aggregated DNA damage. Among these, aggregated DNA damage is difficult to detect because it consists of two or more damage sites, which are closely distributed within one or two helices of DNA. In addition, accurate and specific detection of the damage level of the whole genome DNA is still a great challenge due to low abundance, great diversity and random positions of the damage.

At present, methods for detecting DNA damage include comet method, enzyme-linked immunosorbent assay, mass spectrometry, high performance liquid chromatography-mass spectrometry, single molecule detection method, fluorescence method and the like. Comet assays and enzyme-linked immunosorbent assays are currently the most commonly used methods, but they are time consuming, involve multiple steps of separation, and are costly. Single molecule detection has the advantage of high sensitivity and the ability to image DNA damage in situ, but requires expensive experimental equipment. Fluorescence methods have the advantages of simplicity, rapidity, high sensitivity, etc., but involve complicated probe design and expensive labeling. Compared with fluorescence, bioluminescence has the obvious advantages of low background signal, high flux, self-luminescence without excitation, and the like, and particularly, the signal reading is not interfered by autofluorescence, scattered light and photobleaching. In organisms, cells have multiple repair mechanisms to counteract the deleterious effects of DNA damage, such as Base Excision Repair (BER), Nucleotide Excision Repair (NER), mismatch repair (MMR), Homologous Recombination (HR), and non-homologous end joining (NHEJ), among others. BER is the most common way to maintain DNA stability, and it can repair various DNA damages caused by alkylation, oxidation, and deamination. BER is initiated by DNA glycosylases, which recognize damaged and mismatched bases and create an abasic site by hydrolyzing the N-glycosidic bond between the base and the DNA backbone. Each damaged base corresponds to a unique DNA glycosylase that can distinguish specific nucleobase damage. Inspired by the high specificity of DNA glycosylase, scientists developed a series of methods for detecting DNA specific damage, including nanopore sequencing and single molecule counting. Nanopore sequencing methods based on specific nucleotide labeling and PCR amplification can identify multiple lesions, but require thermal cycling and complex labeling steps; single molecule counting methods can sensitively detect DNA damage but require multiple steps of nucleotide labeling, separation and washing steps.

Disclosure of Invention

Based on the above technical background, maintaining the integrity and stability of DNA is crucial to all organisms, and DNA damage caused by endogenous/exogenous factors can cause various diseases. The level of whole genome DNA damage may become an important biomarker for clinical early diagnosis, risk assessment and therapy monitoring. However, due to the diversity of genomic DNA, label-free, homogeneous detection of genomic DNA damage is difficult to achieve.

In order to overcome the technical difficulties, the invention provides the following technical scheme:

the main technical contribution of the invention is to provide a label-free and homogeneous bioluminescence detection method based on the Base Excision Repair (BER) pathway and template-free isothermal cycle amplification initiated by terminal transferase (TdT), and the detection method can detect single base damage and aggregation damage in genome DNA.

According to the method provided by the invention, all 3' -OH ends in a genome to be detected are blocked, a damaged part of DNA to be detected is identified and cut by DNA glycosylase to expose a new 3' -OH end, a hybrid chain rich in adenine (A) and thymine (T) is constructed at the new 3' -OH end, adenine single-chain in the hybrid chain rich in adenine (A) and thymine (T) is cut by exonuclease to generate adenine nucleotide in a single base form so as to generate adenine ribonucleotide (AMP) molecules, and bioluminescence is realized by the conversion of AMP-ATP-AMP with the assistance of firefly luciferase and luciferin.

In the technical scheme, the shearing action of the Lambda exonuclease on the A-rich probe realizes bioluminescence on one hand and amplifies a DNA damage signal once on the other hand. Proved by verification, the method has good selectivity, can specifically detect single-base DNA damage and aggregated DNA damage, and has the detection limit of the aggregated DNA damage of 8.26 multiplied by 10^-10mol/L。

To further increase the sensitivity of the detection, the present invention also envisages the introduction of an endonuclease (APE1) -induced amplification of the cycle cleavage signal once more. In the method for detecting the circular cleavage induced by the endonuclease (APE1), the invention designs an AP probe with two AP sites, and the probe can be cut by APE1 to generate more 3' -OH primers. The introduction of the AP probe may generate more poly-T structures and more AMPs to generate enhanced bioluminescent signals. The improved method has sensitivity improved by 3 orders of magnitude compared with the original method, and compared with the non-natural nucleoside analogue labeling method (1 × 10)^-10mol/L), high performance liquid chromatography-tandem mass spectrometry (1X 10)^-10mol/L) is improved by 4 orders of magnitude compared with an aptamer fluorescence method (3 multiplied by 10)^-9mol/L) by 5 orders of magnitude. The method can accurately detect uracil in DNA of HeLa cells, A549 cells, HEK-293 cells and MRC-5 cells, and the detection limit is 0.011 ng.

The uracil DNA damage is taken as a model, and the detection limit is 3.26 multiplied by 10^-14mol/L, and can distinguish 0.001% of DNA damage. Importantly, the method can be further applied to the detection of the DNA damage level in a cell sample, the detection limit is 0.011ng, and the method is expected to become the method for detecting various DNA damages by selecting proper DNA glycosylaseA general-purpose platform.

The beneficial effects of one or more technical schemes are as follows:

(1) the detection method provided by the invention obviously compresses the existing detection process, the bioluminescent sensor realizes unmarked and homogeneous detection of genome DNA damage, does not need any separation and washing steps, and is a great innovation in the field of DNA damage detection.

(2) The method of the invention can detect not only single base damage in the genomic DNA, but also aggregation damage in the genomic DNA, and the two damage detections have no interference.

(3) The method of the invention is not affected by different sequences, and can detect the damage of any sequence in the genome.

(4) By utilizing the high efficiency of TdT catalytic polymerization reaction and the inherent high sensitivity of bioluminescence, the method can sensitively detect DNA damage, and the detection sensitivity is superior to that of the previous report. Taking uracil DNA damage model as an example, the detection limit of the method is 3.26 × 10^-14mol/L, the detection limit of tumor cell DNA is 0.011 ng.

(5) In addition, the bioluminescent sensor can detect specific damage by utilizing the high specificity of DNA glycosylase, and can be expanded to detect various damaged bases by properly selecting the DNA glycosylase. It can be a universal platform for detecting other DNA damages.

(6) Besides the A-rich signal probe and the AP probe, no additional signal probe label is needed in the detection process.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a schematic diagram of the detection of DNA damage by a label-free, homogeneous bioluminescence assay;

wherein, (1) TdT mediated polymerization of ddATP to block the 3' -OH of DNA; (2) catalyzing and cutting DNA damaged bases by DNA glycosylase; (3) TdT-mediated template-free isothermal amplification to form a poly-T structure; (4) lambda exonuclease cleavage produces abundant AMPs to produce bioluminescent signals.

FIG. 2 is a schematic diagram of a modified bioluminescence assay for DNA damage;

wherein, (1) TdT mediated polymerization of ddATP to block the 3' -OH of DNA; (2) catalyzing and cutting DNA damaged bases by DNA glycosylase; (3) double-stranded DNA circular cleavage induced by APE1 releases a large amount of 3' -OH terminal primers; (4) TdT-mediated template-free isothermal amplification to form a poly-T structure; (5) lambda exonuclease cleavage produces abundant AMPs to produce bioluminescent signals.

FIG. 3 is a graph showing the effect of detection of uracil damaging DNA in example 1;

(A) gel electrophoresis analysis of ddATP blocking reaction with SYBR Gold as stain;

lane M, DNA marker;lane 1, analysis of U damage DNA reaction products blocking ddATP;lane 2, normal DNA reaction product analysis of blocked ddATP;lane 3, analysis of U damage DNA reaction products;lane 4, normal DNA reaction product analysis;lane 5, U damage DNA;lane 6, normal DNA;

(B) the-dF/dT values of U damage DNA (arrow) and normal DNA after DNA glycosylase excision to remove DNA damage response varied with temperature;

(C) (ii) gel electrophoresis analysis of TdT-mediated template-free amplification products; SYBR Gold as a stain; lane M, DNA marker;lane 1, reaction product of U damage DNA;lane 2, reaction product of normal DNA;

(D) monitoring DNA damage detection by real-time bioluminescence; u damage DNA (arrow), normal DNA. FIG. 4 is the bioluminescent signals generated by U-damaged DNA, U-6 damaged DNA, U-17 damaged DNA and normal DNA;

concentration of each dsDNA was 100 nM; error bars represent standard deviations of three experiments.

FIG. 5 is the bioluminescent signals for U-damaged DNA + UDG + APE1, U-damaged DNA + Fpg + APE1, 8-oxoG-damaged DNA + UDG + APE1, and 8-oxoG-damaged DNA + Fpg +APE 1;

concentration of each dsDNA was 100 nM; the concentration of UDG is 1U/muL, and the concentration of FPG is 1.6U/muL; error bars represent standard deviations of three experiments.

FIG. 6 is a graph showing the relationship between bioluminescent signal measurement and concentration for different concentrations of uracil damage models;

wherein, (A) the real-time bioluminescence curves of the damaged DNA of different concentrations of U-6; (B) measuring bioluminescent signals generated by damaged DNA with different concentrations of U-6; (C) at1X 10^-9～1×10^-7In the mol/L range, the bioluminescence signal and the logarithm of the concentration of the U-6 damaged DNA form a linear relation; error bars represent standard deviations of three experiments.

FIG. 7 is a graph showing the relationship between bioluminescence signal and concentration in the APE 1-induced cyclic lysis optimized detection method;

(A) real-time bioluminescence curves of damaged DNA with different concentrations of U-6 measured by an improved method; (B) the bioluminescence intensity changes with the concentration of the U-6 damaged DNA at 25 min; (C) at1X 10^-13～1×10^-8In the mol/L range, the bioluminescence intensity and the logarithm of the concentration of the U-6 damaged DNA are in a linear relation; (D) in the mixture of U-6 damaged DNA and normal DNA, a linear correlation with the measured level of U-6 damaged DNA was actually added; the total concentration of U-6 damaged DNA and normal DNA was 10 nM; error bars represent standard deviations of three experiments.

FIG. 8 shows the signal intensity of the APE 1-induced optimal detection method for circulating lysis in detecting DNA damage in different tumor cells;

(A) detecting uracil lesions in genomic DNA extracted from HeLa cells, A549 cells, HEK-293 cells and MRC-5 cells using an improved method; the genomic DNA content per cell was 100 ng; (B) the linear relation between the bioluminescence intensity and the genomic DNA of the HeLa cells with different qualities; error bars represent standard deviations of three experiments.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

Interpretation of terms

poly-T: in this document, a nucleotide sequence in which a plurality of thymines are linked to each other at the end of a damaged DNA is shown, and the poly-T single strand, the poly-T sequence, the thymine-rich single strand and the like in this document are referred to as the same meaning;

a-rich probe: or poly-A signal probe, represents a single nucleotide strand having multiple adenine bases;

AP probe: in the present document, the AP probe is an a-rich probe containing an endonuclease recognition site, and the AP site is an abasic site in a DNA double strand;

AP site: represents a site free of base in the double strand of DNA for recognition by an endonuclease;

TdT: terminal transferase, or terminal deoxynucleotidyl transferase (a DNA polymerase that is template independent, capable of catalyzing the binding of deoxynucleotides to the 3' -OH end of a DNA molecule;

ddATP: dideoxy adenosine triphosphate;

APE1 enzyme: an endonuclease;

dTTPs: deoxynucleotide triphosphate of thymine;

rSAP: shrimp alkaline phosphatase;

conversion of AMP-ATP-AMP: in the firefly luminescence reaction, ATP + fluorescein + O₂AMP + oxyfluorescein + PPi + light; released AMPs can be converted to ATPs by a combination of enzymatic reactions of Adenylate Kinase (AK) and Pyruvate Kinase (PK) in the presence of phosphoenolpyruvate monosodium salt hydrate (PEP) and dCTP, and the conversion of ATPs to AMPs with the aid of firefly luciferase and luciferin generates a strong bioluminescent signal;

AMP: adenosine phosphate;

AP Probe/poly-T double-stranded DNA: represents a double strand formed by hybridization of an AP probe strand and poly-T;

UDG: uracil DNA glycosylase, which is used for identifying uracil base modification in the sequence and shearing the uracil base modification;

aggregated DNA damage: clustered damage, aggregated DNA damage "means that the DNA damage sites are tightly distributed within one or two helices of DNA.

As introduced in the background art, the DNA damage detection method in the prior art usually needs more biomarker and washing separation processes, and in order to solve the technical problems, the invention provides a label-free and homogeneous bioluminescence method for detecting DNA damage in a genome based on a Base Excision Repair (BER) pathway and template-free isothermal cycle amplification started by terminal transferase (TdT).

In a first aspect of the present invention, a label-free, homogeneous bioluminescent DNA damage detection method is provided, which comprises: blocking all 3' -OH tail ends in a genome to be detected, identifying and cutting damaged DNA parts of the genome to be detected through DNA glycosylase to expose new 3' -OH tail ends, constructing an adenine (A) -rich hybrid chain at the new 3' -OH tail ends, adding exonuclease to cut adenine (A) -rich single chains in the hybrid chain to generate adenine single bases so as to generate adenine ribonucleotide (AMP) molecules, catalyzing fluorescein to realize bioluminescence, and detecting the strength of damaged DNA through detecting light signals.

In a preferred embodiment of the first aspect, the 3' -OH is blocked by adding dideoxy adenosine triphosphate (ddATP) to the sample to be detected.

Further, the dideoxy adenosine triphosphate (ddATP) labels the 3' -OH terminus catalyzed by terminal transferase (TdT).

In some embodiments of the above preferred embodiments, in order to avoid the influence of the excess ddATP on the subsequent reaction, the blocking reaction further comprises adding shrimp alkaline phosphatase (rSAP) to the system after the reaction for hydrolyzing the excess ddATP.

It is well known in the art that glycosylases specifically recognize the type of damage in a DNA strand and cleave it leaving a vacancy in the original DNA strand, with the cleaved portion exposing the 3' -OH terminus. In order to ensure that the damaged DNA base can be cut by the glycosylase to expose 3' -OH after the glycosylase recognizes the damaged DNA base, in a preferred embodiment of the invention, an endonuclease (APE1) is added to recognize and cut the damaged site together with the glycosylase.

Since the type of DNA damage in the genome to be tested is unknown in most cases, in order to determine the type of DNA damage, a person skilled in the art can divide the sample to be tested into multiple parts, and add different types of glycosylases and subsequent reagents to the multiple parts respectively to determine the type of DNA damage in the genome to be tested.

Preferably, the adenine (A) and thymine (T) rich hybrid is constructed as follows: deoxynucleotide triphosphate of thymine is added to the 3' -OH end of the damaged DNA by terminal transferase catalysis to form a thymine (T) -rich DNA single strand, and an adenine (A) -rich probe is correspondingly added, so that the thymine (T) -rich DNA single strand and the adenine (A) -rich probe are hybridized to form a hybridized strand rich in adenine (A) and thymine (T).

Further, the 5' end of the probe rich in adenine (A) has phosphate group modification.

Preferably, the exonuclease used in the present invention should be capable of cleaving the a-rich strand in the hybrid strand and obtaining adenine single base to achieve bioluminescence, so that the exonuclease capable of achieving the cleavage effect can be used in the detection method of the present invention, and in a specific example, the exonuclease is Lambda exonuclease. The Lambda exonuclease cleaves the A-rich single strand of the hybrid strand into single bases, while the remaining T-rich single strand can continue to bind to the probe to initiate the next cycle.

In a specific implementation manner of the above preferred technical solution, the detection method is as follows: all 3' -OH in the genomic DNA is blocked by catalyzing ddATP through TdT, and rSAP is added to stop the reaction; DNA glycosylase cleaves the damaged base to generate a new 3' -OH terminus; then, repeatedly adding dTTPs into the 3' -OH tail end of the damaged DNA under the catalysis of TdT to generate rich poly-T structure single chains, and adding an A-rich signal probe for hybridization to form a hybrid chain; the hybrid chains are hydrolyzed by Lambda exonuclease to produce a large number of AMP molecules, which are converted to AMP-ATP-AMP with the aid of firefly luciferase and luciferin, resulting in bioluminescence.

In another preferred embodiment of the first aspect, in order to further improve the detection sensitivity, the present invention further provides a detection method for amplifying the detection signal by endonuclease (APE1) -induced cyclic cleavage, wherein the detection method for the ap 1-induced cyclic cleavage is as follows: the method comprises the steps of closing the 3' -OH end in a genome to be detected, adding glycosylase to cut damaged bases and generate new 3' -OH, expanding a single chain rich in T bases at the new 3' -OH end, hybridizing the single chain with an AP probe containing a poly-A sequence and an AP site to form a double chain, recognizing the AP site by APE1 enzyme and catalyzing the hybrid double chain to be circularly cracked, releasing a new 3' -OH sequence, and repeatedly amplifying the 3' -OH end of the newly released primer sequence to form a hybrid double chain rich in adenine and thymine so as to realize signal amplification again.

Further, in the above embodiment, the AP site is at least one abasic site; in a specific example, the AP probe contains a poly-A sequence and two abasic sites (AP sites) of the AP probe, whose 3' end is NH-bonded₂Modified to prevent non-specific amplification.

In a specific embodiment, the steps of the detection method for the amplification of the APE 1-induced cyclic cleavage signal are as follows: ddATP was introduced via TdT catalysis, blocking all free 3' -OH ends; subsequently, glycosylase is added to recognize and cleave the damaged base to generate a new 3' -OH; the cleaved damaged DNA initiates TdT-catalyzed amplification to produce a poly-T single strand; the AP probe is hybridized with a poly-T single strand to form an AP probe/poly-T double-stranded DNA, and then APE1 enzyme starts the cycle cleavage of the double-stranded DNA to release a large amount of 3' -OH primers; TdT catalyzes dTTPs to be repeatedly added to the 3' -OH end of the newly generated primer to form a long-chain poly-T sequence; the generated poly-T is hybridized with a poly-A signal probe to form a poly-T/signal probe double chain; the signaling probe in the duplex is specifically degraded by Lambda exonuclease from its 5' -phosphorylated end to release a large amount of AMPs, and the remaining poly-T sequence can hybridize with the new signaling probe to form a new poly-T/signaling probe duplex, initiating cyclic cleavage of the signaling probe and the release of more and more AMPs.

In a second aspect of the present invention, there is provided a kit for DNA damage detection, comprising at least adenine dideoxynucleotides (ddATPs), deoxynucleotides triphosphate of thymine (dTTPs), fluorescein, terminal transferase (TdT), shrimp alkaline phosphatase, DNA glycosylase, Lambda exonuclease, luciferase, Adenylate Kinase (AK) and Pyruvate Kinase (PK).

Preferably, the kit further comprises an endonuclease (APE1) and an AP probe.

In a third aspect of the present invention, there is provided the use of the label-free, homogeneous bioluminescent DNA damage detection method of the first aspect or the DNA damage detection kit of the second aspect in the fields of disease diagnosis, screening of anti-tumor active ingredients, and the like.

In the above application fields, the applications in the disease diagnosis field include the diagnosis of DNA damage-related diseases such as tumors, neurodegenerative diseases or other DNA damage-related diseases, and specific examples of the other DNA damage-related diseases include xeroderma pigmentosum, warfarin syndrome, huntington's disease, and the like.

The detection methods provided by the present invention can be applied to diseases that have DNA damage or modification as a diagnostic criterion, either now or as may be disclosed in the future.

In addition, as is known in the art, the tumor cells have strong capability of repairing damaged DNA, and the capability of inhibiting the tumor cells from repairing DNA damage is also an antitumor drug development idea, so that the DNA damage detection method provided by the invention can also be used for evaluating antitumor active ingredients.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

1. Feasibility experiment of detecting DNA damage by bioluminescence method.

In this example, the feasibility of the method was verified using uracil bases (U) in DNA as targets (fig. 3). Uracil is a common DNA damage that causes cytosine deamination and, in the event of dUTP misinterpretation, a mutagenic U/G mismatch or genotoxic U/A pair. The blocking effect of ddATP on 3' -OH was first verified by gel electrophoresis (FIG. 1). In the presence of ddATP and rSAP, only a 39bp band was observed in normal DNA (FIG. 3A, lane 1) or U-damaged DNA (FIG. 3A, lane 2), and no amplified band was detected, since dsDNA first underwent a TDT-mediated ddATP labeling reaction, and then rSAP removed free excess ddATP. The dsDNAs are blocked by ddATP when TDT catalyzes repeated extension of dTTPs to the 3' -OH terminus of the DNA. Thus, neither normal DNA (FIG. 3A, lane 1) nor damaged DNA (FIG. 3A, lane 2) form an amplified band of poly-T structure catalyzed by TdT, at the same position as the untreated double-stranded DNA (FIG. 3A,lane 5 and lane 6). In contrast, in the absence of ddATP and rSAP, large bands of more than 500bp were observed for both normal DNA (FIG. 3A, lane 3) and U-damaged DNA (FIG. 3A, lane 4), indicating that TdT-catalyzed template-free isothermal amplification produced large molecular weight DNA fragments. These results indicate that ddATP can effectively block the 3' -OH end of dsDNA.

To verify whether DNA glycosylase can remove damaged DNA bases, the present invention measures the melting curves of normal and damaged double-stranded DNA (FIG. 3B). UDG can specifically recognize and excise uracil by catalyzing the cleavage of N-glycosidic bonds between uracil and the DNA backbone, releasing damaged bases and creating AP sites. APE1 cleaves the phosphodiester backbone at the AP site, yielding a backbone with 3'-OH and 5' -PO₄The DNA single strand of (1) is broken. After UDG and APE1 treatment, the melting temperature of normal dsDNA was 78 ℃ (fig. 3B) and that of damaged DNA was 65 ℃ (fig. 3B). The Tm of damaged dsDNA is much lower than that of normal dsDNA. The decrease in melt temperature indicates that the DNA glycosylase breaks the phosphodiester bond of the damaged single strand at the U-damaged DNA, forming a new 3' -OH terminus.

This example further uses gel electrophoresis to verify TDT-catalyzed template-free isothermal amplification after DNA glycosylase cleavage (fig. 3C). Only a 39bp band was observed for normal DNA (FIG. 3C, lane 1), indicating that ddATP blocked the 3' -OH end of the double-stranded DNA and no amplification occurred. In contrast, in the presence of U-damaged DNA, a distinct amplification band appeared, indicating the formation of a new 3' -OH terminus induced by DNA glycosylase and a poly-T structure catalyzed by TDT (FIG. 3C, lane 2). This result clearly shows that only damaged DNA can form poly-T structure under the catalysis of TdT.

Finally, bioluminescence measurements were performed in this example to monitor the cleavage product AMP produced by Lambda exonuclease digestion (FIG. 3D). In the control group, no bioluminescent signal was detected due to lack of AMP production, demonstrating DNA damage (fig. 3D). In contrast, enhanced bioluminescent signals were observed in the presence of U-damaged DNA (fig. 3D), suggesting that only U-damaged DNA can induce Lambda exonuclease digestion mediated release of AMP, producing a significant bioluminescent signal through conversion of AMP to ATP.

2. Different numbers and locations of DNA damage were studied.

This example designs a damaged DNA containing 1 uracil base, i.e., U-damaged DNA and two damaged DNAs containing 2 uracil bases, i.e., U-6 damaged DNA and U-17 damaged DNA, with the U-6 damaged DNA having a 6bp spacing between the two uracil bases (i.e., aggregated DNA damage) and the U-17 damaged DNA having a 17bp spacing between the two uracil bases (i.e., single base DNA damage). Notably, aggregated DNA lesions are typically tightly distributed within one or two helices of DNA by two or more lesions, whereas in single base DNA lesions, the two lesions are relatively distant. In the absence of the injury site, no bioluminescent signal was detected (fig. 4). In contrast, intense bioluminescent signals were observed in the presence of 1 damaged base of DNA (fig. 4), aggregated damaged bases (fig. 4) and single base damaged bases (fig. 4). Regardless of the position of the two damaged bases, the bioluminescent signal of dsDNA containing two damaged sites is much higher than that of dsDNA containing only one damaged site, and the bioluminescent signal of U-6 damaged DNA is the same as that of U-17 damaged DNA. These results indicate that the method can distinguish dsDNA containing different numbers of lesions, and that aggregate lesions that are difficult to detect with conventional methods can also be accurately detected.

Notably, most reported BER-based damaged base detection methods rely on templates, which can only detect single-base DNA damage and cannot detect aggregated DNA damage, because the position of the 2 bases of the aggregated damage is within 1 helix, and the broken double-stranded DNA may produce unstable single-stranded DNA after excision repair, which prevents the DNA polymerase from labeling the aggregated damage site in a template-dependent manner. TdT recognizes and captures the very short 3' terminal oligonucleotide (only about 4 nucleotides) that is generated during the process of DNA glycosylase removing damaged bases from aggregated damaged DNA, and the method of this example detects single base damage and aggregation damage due to the introduction of TdT. The obtained single-stranded DNA forms a poly-T structure in a template-free mode under the catalysis of TdT, and then is paired with a rA-containing signal probe to form a double strand. Addition of Lambda exonuclease can hydrolyze the signaling probe in the duplex, releasing abundant AMP to initiate pyrophosphoryl-ATP pyrophosphorylation of cyclic AMP to produce enhanced bioluminescent signal.

TABLE 1 DNA sequences used in this example

Note: underlined bold U indicates uracil deoxyribonucleotides, and underlined bold G indicates 8-oxoguanine (8-oxoG) deoxyribonucleotides. Underlined bold letters "X" indicate AP sites. rA in the signaling probe represents adenine nucleotide.

3. And detecting the selectivity.

8-oxo-7, 8-dihydroguanine (8-oxoG) is the most prominent oxidized base adduct produced by oxidation of the guanine (G) heterocycle by Reactive Oxygen Species (ROS), and Fpg, a bifunctional DNA glycosylase, removes 8-oxo groups, cleaves the DNA backbone, forming mononucleotide gaps. This example designs a DNA probe containing an 8-oxoG base at 21 bases on the 5' end (8-oxoG damaged DNA) as a substrate for Fpg. The selectivity of the method was analyzed with two DNA lesions (U-lesion DNA and 8-oxoG-lesion DNA) and two DNA glycosylases (UDG and FPG). As shown in FIG. 5, U-damaged DNA (FIG. 5, red bars) produced a bioluminescent signal when UDG and APE1 were used as DNA glycosylases, whereas 8-oxoG-damaged DNA (FIG. 5) produced no significant bioluminescent signal. When FPG and APE1 were used as DNA glycosylases, 8-oxoG damaged DNA (FIG. 5) produced a high bioluminescent signal, whereas U damaged DNA (FIG. 5) produced no significant bioluminescent signal. The result shows that the method has good selectivity on specific DNA damage, and different DNA damages can be specifically detected by combining different DNA glycosylases.

4. And (3) detection sensitivity.

To evaluate the sensitivity of the method, this example measured the bioluminescent signals generated by different concentrations of U-6 damaged DNA under optimal conditions. As shown in FIG. 6A, the bioluminescent signal increased with increasing U-6 damage DNA concentration and time and reached a maximum within 25 minutes. When the concentration of U-6 damaged DNA is from 1.0X 10^-9Increase to 1.0X 10^-7At mol/L, the bioluminescence signal also increased (FIG. 6B). In a logarithmic coordinate system, the concentration of bioluminescence signals and damaged DNA is 1.0 × 10^-9～1.0×10^-7Linear correlation in the mol/L range (FIG. 6C), and regression equation B of 31976.75log₁₀C+294865.51(R²0.9878), wherein C represents the concentration of damaged DNA (mol/L) and B represents the bioluminescence intensity. Calculated according to the principle of adding 3 times of standard deviation to the control group signal, the detection limit is 8.26 multiplied by 10^-10mol/L。

5. Improved method sensitivity analysis

To verify the sensitivity of the improved method, this example measured the bioluminescent signal generated by different concentrations of damaged DNA (U-6 damaged DNA) under optimal conditions. When the concentration of U-6 damaged DNA is from 1.0X 10^-13Increase to 1.0X 10^-8At mol/L, the bioluminescence increased and plateaus within 25min (FIGS. 7A and 7B). In logarithmic scale, the bioluminescence signal is at 1.0X 10^-13To 1.0X 10^-8The mol/L range is linearly related to the concentration of U-6 damaged DNA (FIG. 7C). The regression equation is B-19734.53 log₁₀C+276062.34(R²0.9900), wherein C represents the concentration of damaged DNA (mol/L) and B represents the bioluminescence intensity. Detection limit of 3.26 × 10^-14mol/L. The improved method has sensitivity which is improved by 3 orders of magnitude compared with the original method (figure 6C); labeling of non-natural nucleoside analogs (1X 10)^-10mol/L) and high performance liquid chromatography-tandem mass spectrometry (1X 10)^-10mol/L) is improved by 4 orders of magnitude; than aptamer-based fluorescence (3X 10)^-9mol/L) is 5 orders of magnitude higher. To investigate the feasibility of this improved method for the detection of uracil in the mixtures, U-6 damaged DNA was mixed with normal DNA in different proportions and the uracil content of the artificial mixtures was determined. The measured U-6 damaged DNA level (Y) is well linear with the input U-6 damaged DNA level (X) (FIG. 7D). The regression equation is Y-0.9831X +0.0402 (R)²0.9993). It is noteworthy that this improved method can even distinguish uracil as low as 0.001% in the mixture, superior to the highly specific co-amplification low temperature PCR reaction (COLD-PCR) method of spiking (0.01%), comparable to the single molecule counting method (0.001%).

6. Detection of cellular genomic DNA damage

To demonstrate the feasibility of the improved method to detect DNA damage in authentic biological samples, this example measured uracil base levels in the genomes of tumor cells and normal cells. The tumor cells comprise a human lung adenocarcinoma cell line (A549 cells) and a human cervical cancer cell line (HeLa cells), and the normal cells comprise a human embryonic kidney cell line (HEK-293 cells) and a human embryonic lung fibroblast cell line (MRC-5 cells). This example extracts all cellular genomic DNA using a QIAamp DNA minikit (Qiagen, Germany) and measures the concentration of genomic DNA (ng/. mu.L) using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA). Higher levels of genomic uracil were measured in cancer cells HeLa cells (FIG. 8A) and A549 cells (FIG. 8A) compared to normal cells including HEK-293 cells (FIG. 8A) and MRC-5 cells (FIG. 8A), consistent with the literature-reported results of the LC-MS/MS method. This example further uses HeLa cell genomic DNA asModel, the relationship between bioluminescence signal and DNA quality was studied. As shown in FIG. 8B, the bioluminescence signal increased as the genomic DNA content increased from 0.05 to 200ng, with a regression equation of B-24657.92 log₁₀ N+58484.54(R²0.9951), where N represents genomic DNA content (ng) and B represents bioluminescence intensity. The detection limit was calculated to be 0.011 ng. The result shows that the improved method can be used for uracil whole genome analysis and has higher accuracy and reliability.

Example 1

(1) Preparation of double-stranded DNA substrates

mu.M oligonucleotides which hybridize to each other are added to an annealing buffer (50mM NaCl,10mM Tris-HCl, pH 8.0), incubated at 95 ℃ for 5 minutes and then slowly cooled to room temperature to form a double-stranded DNA (dsDNA) structure. The annealed dsDNA substrates were diluted to different concentrations with annealing buffer solution. All dsDNA substrates obtained were stored at 4 ℃ for later use in experiments.

(2) Isothermal signal amplification reaction

The isothermal signal amplification reaction process comprises four sequential steps. In the first step, dsDNA of different concentrations, 30. mu.M ddATP, 16U TdT, 0.25mM CoCl₂And 1 XTdT buffer (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, pH 7.9) in a total volume of 10. mu.L, and incubated at 37 ℃ for 2 hours followed by incubation at 75 ℃ for 20 minutes to inactivate the TdT. In the second step, 0.3U rSAP, 1U UDG, 0.1U APE1 and 1 XTdT buffer were mixed and added to the reaction system to make a total volume of 15. mu.L. And the mixed solution was incubated at 37 ℃ for 60 minutes and then reacted at 95 ℃ for 10 minutes. The third step, consisting of 1.5. mu.L of 100mM dTTP, 6U TdT, 0.25mM CoCl₂The reaction solution, 1 XTDT buffer and the product of the previous step, which had a total volume of 20. mu.L, was reacted at 37 ℃ for 60 minutes to generate a ploy T structure, and then heated at 95 ℃ for 20 minutes to terminate the reaction. In the last step,200nM signal probe 2, 2U Lambda exonuclease and 1 × Lambda exonuclease reaction buffer (6.7mM Glycine-KOH, 0.25mM MgCl. sub.₂5. mu.g/ml BSA, pH 9.4) were added to the solution to a total volume of 50. mu.L. Subsequently mixing the mixtureIncubate at 37 ℃ for 30 minutes to generate large amounts of AMP.

(3) The improved method comprises the following steps

In the first step, dsDNA of different concentrations, 30. mu.M ddATP, 16U TdT, 0.25mM CoCl₂And 1 XTdT buffer in a total volume of 10. mu.L were mixed and incubated at 37 ℃ for 2 hours followed by incubation at 75 ℃ for 20 minutes to inactivate the TdT. In the second step, 0.3U rSAP, 1U UDG, 0.1U APE1 and 1 XTdT buffer were mixed and added to the reaction system to make thetotal volume 15. mu.L, and the mixed solution was incubated at 37 ℃ for 60min and then reacted at 95 ℃ for 10 min. Third, 1.5. mu.L of 100mM dTTP, 6U TdT, 0.25mM CoCl₂1 XTdT buffer and 20. mu.L of the reaction solution of the reaction product of the previous step were reacted at 37 ℃ for 60 minutes to generate a ploy T structure, and then reacted at 95 ℃ for 20 minutes to terminate the reaction. Next, 0.5. mu.L of 10. mu.M AP probe, 2U of APE1 and 1 XTdT buffer were added to the reaction system so that the total volume was 25. mu.L, and reacted at 37 ℃ for 40 minutes, resulting in a large amount of 3' -OH ends. To generate more poly T structure, 6U of TdT, 1. mu.L of 100mM dTTP and 1 XTDT buffer were added again to a volume of 30. mu.L, reacted at 37 ℃ for 60 minutes, and then reacted at 95 ℃ for 20 minutes to terminate the reaction. In the last step,200nM signal probe 2, 2U Lambda exonuclease and 1 × Lambda exonuclease reaction buffer were added to the solution to a total volume of 50 μ L. The mixture was then incubated at 37 ℃ for 30 minutes to generate large amounts of AMP.

(4) Bioluminescence measurement and data analysis

The reaction solution containing AMP was transferred to an ATP detection system and mixed, and then a bioluminescent signal was monitored in real time at room temperature using a Glomax bio/chemiluminescence detector. The ATP detection System consisted of 4.0. mu.L AMP-ATP conversion buffer (1.0. mu.L 1U/. mu.L AK, 1.0. mu.L 1U/. mu.L PK, 1.0. mu.L 10mM dCTP and 1.0. mu.L 4.8mM PEP) and 6.0. mu.L ATP detection buffer (0.5mM D-luciferin, 1.25. mu.g/mL firefly luciferase, 10mg/mL BSA, 500mM Tricine buffer (pH 7.8),100mM MgSO 2₄2mM EDTA, 100mM DTT). The parameter settings of the instrument are respectively: integration time was 1s and the number of runs was 25. And selectThe maximum of the 25 measurements was used as bioluminescent signal for subsequent data analysis.

(5) Gel electrophoresis measurement

The TDT-mediated DNA amplification product was stained by SYBR Gold and electrophoresed in 1 XTBE buffer (44.5mM Tris-boric acid, 1mM EDTA, pH 8.2) for 45 minutes on 12% native polyacrylamide gel at a constant pressure of 110V at room temperature. Finally the gel was visualized using a Bio-Rad ChemiDoc MP imaging system.

(6) Fluorescence measurement

After the TdT-mediated DNA amplification product was stained with SYBR Gold, the fluorescence signal of the reaction product was measured using an F-7000 fluorescence spectrophotometer from Hitachi, Japan, using a xenon lamp as an excitation light source. The excitation wavelength is 488nm, and the spectrum recording range is 500-700 nm. Both excitation and emission slits were 5.0 nm. The fluorescence intensity at 540nm (maximum emission wavelength of SYBR Gold) was used for subsequent data analysis.

(7) Measurement of melt chain temperature

100nM damaged dsDNA and 100nM normal dsDNA were incubated with UDG and APE1 for 1h at 37 ℃. After the reaction, the sample was stained with SYBR Green I, and the fluorescence value was measured every 30 seconds at a temperature of 55 ℃ to 95 ℃ using a Bio-Rad real-time fluorescence detection system. The temperature of the melting chain is the corresponding temperature value when-dF/dT reaches the maximum value. (wherein F is fluorescence intensity and T is temperature).

(8) Cell culture and extraction of genomic DNA

Human cervical cancer cell lines (HeLa cells), human lung adenocarcinoma cell lines (A549 cells), human embryonic kidney cell lines (HEK-293 cells), and normal human lung cell lines (MRC-5 cells) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin mixture. All cells contained 5% CO at 37 deg.C₂Culturing in a humid incubator. During exponential growth phase, cells were harvested and intracellular DNA was extracted using the QIAamp DNA Mini kit according to the instructions and the concentration of extracted DNA (ng/, L) was determined with a ultramicro uv spectrophotometer.

(9) Principle of bioluminescence method for detecting DNA Damage (FIG. 1)

The scheme comprises four parts: (1) the TdT-mediated ddATP labeling reaction is used for blocking the 3 '-hydroxyl (3' -OH) end of DNA, (2) DNA glycosylase is used for catalyzing and removing DNA damage, (3) TdT-mediated template-free amplification is used for forming a poly-T structure, and (4) AMP is generated and bioluminescence reaction is carried out. The whole reaction system consists of ddATP, TdT, rSAP, DNA glycosylase, a signal probe and Lambda exonuclease. ddATP is first labeled on the DNA with the aid of TdT polymerase to block the initial 3'-OH terminus to prevent the effect of the initial 3' -OH terminus on subsequent reactions. Shrimp alkaline phosphatase (rSAP) was added to remove excess ddATP, avoiding excess ddATP affecting subsequent reactions. Then, DNA damage in the genome is removed by the action of DNA glycosylase and APE1, and a new 3' -OH site is generated. Multiple dTTPs were then incorporated into the newly generated 3' -OH sites by template-free amplification of the TdT to form a poly-T sequence. This example designs a signaling probe modified at the 5 'end with a phosphate group (5' -dRP) and containing multiple adenine ribonucleotides in the middle. The signal probe can be completely hybridized with the generated poly T sequence to form a double-stranded structure of the poly-T/signal probe. The signal probe is gradually degraded from 5' -dRP to release a large amount of AMP under the catalysis of Lambda exonuclease. This is because Lambda exonuclease can specifically digest a single strand in a double-stranded structure from the 5' -phosphorylated (5' -dRP) end, but has extremely low catalytic efficiency even for a sequence without the 5' -dRP end. The resulting AMP is converted to ATP by the enzymatic reaction of Adenylate Kinase (AK) and Pyruvate Kinase (PK) in the presence of PEP and dCTP, and subsequently ATP is converted to AMP with the aid of luciferase and luciferin and produces a strong bioluminescent signal. Notably, AMP can be converted to ATP through a new cycle, which can generate an amplified bioluminescent signal. If there is no damage in the genomic DNA, no new 3' -OH sites are generated under the catalysis of DNA glycosylase, and no obvious bioluminescent signal is generated.

(10) Improved DNA Damage detection method (FIG. 2)

This example further incorporates the APE 1-induced cyclic cleavage signal amplification technique to improve detection sensitivity. This example designed a DNA sequence containing a poly-A sequence and two alkali-free nucleotidesAP Probe for a base site (AP site) whose 3' terminal is NH-substituted₂Modified to prevent non-specific amplification (FIG. 2). ddATP was introduced via TDT catalysis, blocking all U-damaged DNA with free 3' -OH ends (FIG. 2), and then rSAP was added, stopping the blocking reaction by hydrolysis of the remaining ddATP. Subsequently, UDG and APE1 were added to remove uracil bases to generate new 3' -OH. The cleaved U-damaged DNA (FIG. 2) can be used as a primer to initiate TDT catalyzed template-free isothermal amplification, which results in the generation of a poly-T structure. The AP probe (FIG. 2) can hybridize to a poly-T structure to form an AP probe/poly-T double-stranded DNA (FIG. 2), followed by the APE1 enzyme initiating the cyclic cleavage of the double-stranded DNA, releasing a large amount of 3' -OH primer. TDT catalyzes repeated addition of dTTPs to the 3' -OH end of the newly generated primer to form a long poly-T sequence (FIG. 2). The resulting poly-T can hybridize to a poly-A signaling probe to form a poly-T/signaling probe duplex (FIG. 2). The signaling probe in the duplex can be specifically degraded by Lambda exonuclease from its 5' -phosphorylated end to release a large amount of AMPs, and the remaining poly-T sequence can hybridize with a new signaling probe to form a new poly-T/signaling probe duplex, initiating cyclic cleavage of the signaling probe and release of more and more AMPs. Released AMPs can be converted to ATPs by a combination of enzymatic reactions of Adenylate Kinase (AK) and Pyruvate Kinase (PK) in the presence of phosphoenolpyruvate monosodium salt hydrate (PEP) and dCTP, and the conversion of ATPs to AMPs with the aid of firefly luciferase and luciferin produces a strong bioluminescent signal. Compared with the previous method, the improved method can generate additional 3' -OH due to the introduction of the AP probe, and the newly generated primer can generate more poly-T sequences under the catalysis of TDT, form more poly-T/signal probe double-stranded structures and generate more AMP molecules to generate enhanced bioluminescent signals.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Qingdao university of science and technology

<120> a label-free homogeneous phase bioluminescence DNA damage detection method

<130> 2010

<160> 10

<170> PatentIn version 3.3

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gaacaccatc aacctcgacc accgcatccg ttctcacgg 39

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Claims (10)

Translated fromChinese

1.一种无标记、均相生物发光的DNA损伤检测方法，其特征在于，所述检测方法如下：封闭待测基因组中所有的3'-OH末端，通过DNA糖基化酶识别并且切割待测DNA受损部分暴露新的3'-OH末端，在新的3'-OH末端构建富含腺嘌呤和胸腺嘧啶的杂交链，加入核酸外切酶对所述杂交链中富含腺嘌呤的单链进行剪切产生腺嘌呤核糖核苷酸分子，催化荧光素实现生物发光，通过检测光信号强度实现受损DNA的检测。1. a label-free, homogeneous bioluminescence DNA damage detection method, is characterized in that, described detection method is as follows: block all 3 '-OH ends in the genome to be tested, identify and cut by DNA glycosylase The damaged part of DNA is detected to expose the new 3'-OH end, and a hybrid chain rich in adenine and thymine is constructed at the new 3'-OH end, and exonuclease is added to the adenine-rich chain in the hybrid chain. The single strand is cleaved to generate adenine ribonucleotide molecules, which catalyze fluorescein to achieve bioluminescence, and detect damaged DNA by detecting the intensity of the light signal.2.如权利要求1所述无标记、均相生物发光的DNA损伤检测方法，其特征在于，所述封闭3'-OH的方式包括但不限于向待测样品中加入双脱氧的腺苷三磷酸对3'-OH末端进行封闭；2. The label-free, homogeneous bioluminescence DNA damage detection method of claim 1, wherein the method of closing 3'-OH includes, but is not limited to, adding dideoxy adenosine triglyceride to the sample to be tested Phosphoric acid blocks the 3'-OH end;优选的，通过末端转移酶催化双脱氧的腺苷三磷酸对3'-OH末端进行标记；Preferably, the 3'-OH end is labeled with dideoxy adenosine triphosphate catalyzed by terminal transferase;进一步的，所述封闭反应还包括向反应后的体系中加入虾碱性磷酸酶用于中和过量的ddATP。Further, the blocking reaction further includes adding shrimp alkaline phosphatase to the reacted system to neutralize excess ddATP.3.如权利要求1所述无标记、均相生物发光的DNA损伤检测方法，其特征在于，所述富含腺嘌呤和胸腺嘧啶的杂交链的构建方式如下：通过末端转移酶催化胸腺嘧啶的三磷酸脱氧核苷酸加入受损DNA的3'-OH末端，形成富含胸腺嘧啶的DNA单链，并对应的加入富含腺嘌呤的探针进行杂交构建富含腺嘌呤和胸腺嘧啶的杂交链；3. the DNA damage detection method of label-free, homogeneous bioluminescence as claimed in claim 1, is characterized in that, the construction mode of the hybrid chain that is rich in adenine and thymine is as follows: Deoxynucleotide triphosphate is added to the 3'-OH end of the damaged DNA to form a thymine-rich DNA single strand, and a corresponding adenine-rich probe is added for hybridization to construct adenine- and thymine-rich hybridization chain;优选的，所述富含腺嘌呤的探针5'端具有磷酸基团修饰。Preferably, the 5' end of the adenine-rich probe is modified with a phosphate group.4.如权利要求1所述无标记、均相生物发光的DNA损伤检测方法，其特征在于，所述核酸外切酶为Lambda核酸外切酶。4. the DNA damage detection method of label-free, homogeneous bioluminescence as claimed in claim 1, is characterized in that, described exonuclease is Lambda exonuclease.5.如权利要求1-4任一项所述无标记、均相生物发光的DNA损伤检测方法，其特征在于，所述检测方法如下：通过TdT催化ddATP封闭基因组DNA中所有的3'-OH，加入rSAP终止反应；DNA糖基化酶切割受损的碱基产生新的3'-OH末端；随后，TdT催化dTTPs重复加入受损DNA的3'-OH末端，产生丰富的poly-T结构单链，加入富A的信号探针杂交形成杂交链；所述杂交链在Lambda核酸外切酶水解下产生大量的AMP分子，通过萤火虫荧光素酶和荧光素的协助实现AMP-ATP-AMP的转化，产生生物发光。5. The label-free, homogeneous bioluminescence DNA damage detection method according to any one of claims 1-4, wherein the detection method is as follows: by TdT catalyzing ddATP to seal all 3'-OH in genomic DNA , adding rSAP to terminate the reaction; DNA glycosylase cleaves damaged bases to generate new 3'-OH ends; then, TdT catalyzes the repeated addition of dTTPs to the 3'-OH ends of damaged DNA, resulting in abundant poly-T structures Single chain, adding A-rich signal probe hybridization to form a hybrid chain; the hybrid chain is hydrolyzed by Lambda exonuclease to generate a large amount of AMP molecules, and the assistance of firefly luciferase and luciferin is realized. transformed to produce bioluminescence.6.如权利要求1所述无标记、均相生物发光的DNA损伤检测方法，其特征在于，所述检测方法通过核酸内切酶诱导的循环裂解对检测信号进行放大，所述检测方法如下：封闭待测基因组中的3'-OH末端，加入糖基化酶剪切受损碱基并产生新的3'-OH，在新的3'-OH末端扩展富含T碱基的单链，并与含有poly-A序列和AP位点的AP探针杂交形成双链，APE1酶识别AP位点并催化所述杂交双链循环裂解，释放出新的3'-OH序列，新释放的引物序列3'-OH末端重复扩增成为富含腺嘌呤及胸腺嘧啶的杂交双链，以实现再一次的信号放大。6. The DNA damage detection method of label-free and homogeneous bioluminescence as claimed in claim 1, wherein the detection method is amplified by the cyclic cleavage induced by endonuclease, and the detection method is as follows: Block the 3'-OH end of the genome to be tested, add glycosylase to cleave the damaged base and generate a new 3'-OH, and expand the T-rich single strand at the end of the new 3'-OH, And hybridize with the AP probe containing the poly-A sequence and AP site to form a double strand, APE1 enzyme recognizes the AP site and catalyzes the cyclic cleavage of the hybrid double strand, releasing a new 3'-OH sequence, the newly released primer The 3'-OH terminal of the sequence is repeatedly amplified to become a hybrid double-stranded rich in adenine and thymine to achieve signal amplification again.7.如权利要求6所述无标记、均相生物发光的DNA损伤检测方法，其特征在于，所述AP探针中的AP位点为至少一个无碱基的位点；7. The label-free, homogeneous bioluminescence DNA damage detection method of claim 6, wherein the AP site in the AP probe is at least one abasic site;优选的，所述AP探针含有poly-A序列和两个无碱基位点的AP探针，其3'端被NH₂修饰以防止非特异性扩增。Preferably, the AP probe contains a poly-A sequence and an AP probe with two abasic sites, the 3' end of which is modified by NH₂ to prevent non-specific amplification.8.如权利要求7所述无标记、均相生物发光的DNA损伤检测方法，其特征在于，所述APE1诱导的循环裂解信号放大的检测方法步骤如下：通过TdT催化将ddATP引入，封闭所有游离3'-OH末端；随后，添加糖基化酶识别并剪切受损碱基以产生新的3'-OH；被切割的受损DNA启动TdT催化的扩增，产生poly-T单链；AP探针与poly-T单链杂交以形成AP探针/poly-T双链DNA，随后APE1酶启动双链DNA的循环裂解，释放出大量3'-OH的引物；TdT催化dTTPs重复加到新产生的引物的3'-OH末端，形成长链poly-T序列；产生的poly-T与poly-A信号探针杂交，形成poly-T/信号探针双链；双链中的信号探针被Lambda核酸外切酶从其5'-磷酸化末端特异性降解以释放大量AMP，剩余的poly-T序列可以与新的信号探针杂交形成新的poly-T/信号探针双链，引发信号探针的循环切割和越来越多AMP的释放。8. the DNA damage detection method of label-free, homogeneous bioluminescence as claimed in claim 7, is characterized in that, the detection method step of described APE1-induced cyclic cleavage signal amplification is as follows: ddATP is introduced by TdT catalysis, and all free 3'-OH end; then, glycosylase is added to recognize and cleave damaged bases to generate new 3'-OH; the cleaved damaged DNA initiates TdT-catalyzed amplification to generate poly-T single strands; The AP probe hybridizes with poly-T single-stranded to form AP probe/poly-T double-stranded DNA, and then APE1 enzyme initiates the cyclic cleavage of double-stranded DNA, releasing a large number of 3'-OH primers; TdT catalyzes the repeated addition of dTTPs to The 3'-OH end of the newly generated primer forms a long-chain poly-T sequence; the generated poly-T hybridizes with the poly-A signal probe to form a poly-T/signal probe duplex; the signal probe in the duplex The needle is specifically degraded from its 5'-phosphorylated end by Lambda exonuclease to release large amounts of AMP, and the remaining poly-T sequence can hybridize with the new signal probe to form a new poly-T/signal probe duplex, Cyclic cleavage of the signaling probe and release of more and more AMPs are initiated.9.一种用于DNA损伤检测的试剂盒，其特征在于，所述试剂盒中至少包括三磷酸腺嘌呤双脱氧核苷酸、胸腺嘧啶的三磷酸脱氧核苷酸、荧光素、末端转移酶、虾碱性磷酸酶、DNA糖基化酶、Lambda核酸外切酶、荧光素酶、腺苷酸激酶和丙酮酸激酶；9. A kit for DNA damage detection, characterized in that the kit comprises at least adenine triphosphate dideoxynucleotide, thymine triphosphate deoxynucleotide, fluorescein, terminal transferase , shrimp alkaline phosphatase, DNA glycosylase, Lambda exonuclease, luciferase, adenylate kinase and pyruvate kinase;优选的，所述试剂盒中，还包括核酸内切酶及AP探针。Preferably, the kit further includes endonuclease and AP probe.10.权利要求1-8任一项所述无标记、均相生物发光的DNA损伤检测方法或权利要求9所述DNA损伤检测试剂盒在疾病诊断、抗肿瘤活性成分筛选领域的应用；10. Application of the label-free, homogeneous bioluminescence DNA damage detection method according to any one of claims 1 to 8 or the application of the DNA damage detection kit according to claim 9 in the fields of disease diagnosis and anti-tumor active component screening;优选的，所述疾病诊断领域的应用包括用于肿瘤、神经退行性疾病或其他DNA损伤相关疾病；进一步的，所述其他DNA损伤相关疾病为着色性干皮病、华沙破损综合征或亨廷顿病。Preferably, the application in the field of disease diagnosis includes use in tumors, neurodegenerative diseases or other DNA damage-related diseases; further, the other DNA damage-related diseases are xeroderma pigmentosum, Warsaw breakage syndrome or Huntington's disease .

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