Electrochemical method for detecting DNA damage based on CRISPR-Cas12a system

Abstract

The invention discloses an electrochemical method for detecting DNA (deoxyribonucleic acid) damage based on a CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats-associated 12a) system. Normal DNA can activate the activity of the CRISPR-Cas12a compound, so that the DNA probe fixed on the surface of the electrode is cut. The cut DNA probe cannot be subjected to HCR reaction in the presence of hairpin DNA H1 and H2, so that a relatively small electrochemical signal of methylene blue is generated; the damaged DNA cannot completely activate the activity of the CRISPR-Cas12a compound, at the moment, the uncut DNA probe induces HCR and generates longer double-stranded DNA in the presence of hairpin DNA H1 and H2, and a large number of methylene blue molecules are combined with the double-stranded DNA, so that a stronger electrochemical signal is generated. The damage degree of the DNA corresponds to the light damage level of the DNA, the electrochemical signal of the methylene blue and the light damage level of the DNA are in a linear relation within the range of 0.021-0.42 kJ m <-2 >, and the lowest detection limit is 0.0088 kJ m <-2 >.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and specifically relates to an electrochemical method for detecting DNA damage based on a CRISPR-Cas12a system. Background Art

[0002] The integrity of DNA is essential for maintaining genetic stability and cell function. DNA damage is ubiquitous in organisms and may lead to gene mutations and even cancer and other diseases. Ultraviolet radiation can damage DNA in cells. When DNA absorbs ultraviolet radiation, cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are formed between adjacent pyrimidine bases. The generation of the above photoproducts can interfere with the normal functions of cells, such as DNA replication and transcription. Therefore, the detection of DNA damage induced by ultraviolet radiation has important biological significance.

[0003] Currently, there are many methods for detecting UV-induced DNA damage, such as chromatography-mass spectrometry, surface plasmon resonance, electrochemical impedance spectroscopy, and fluorescence. Among them, chromatography-mass spectrometry has been used to quantitatively analyze CPDs and 6-4PPs in genomic DNA. By inserting fluorescent probes into DNA, fluorescent detection of DNA photodamage levels can be achieved.

[0004] In recent years, the CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and Associated Proteins) system has become a powerful tool for gene editing and has been widely used in the field of biosensing. Among them, CRISPR-Cas12a, as an RNA-guided nuclease, can specifically recognize target DNA and activate trans-cleavage activity. At present, most studies usually focus on the recognition and quantification of specific DNA sequences by the CRISPR-Cas12a system. There are no reports on the use of the CRISPR-Cas12a system to detect DNA damage. Summary of the invention

[0005] The object of the present invention is to provide an electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system, using a DNA probe fixed on the electrode surface, after being cut by the CRISPR-Cas12a complex, hairpin DNA H1 and H2 are subsequently added to react by hybridization chain reaction (HCR), and the HCR product is combined with methylene blue; the strength of the methylene blue electrochemical signal corresponds to the difference in the degree of activation of the CRISPR-Cas12a complex; and then according to the principle that DNA with different degrees of damage corresponds to CRISPR-Cas12a complexes with different degrees of activation, different levels of DNA photodamage are obtained, and therefore it has good application value.

[0006] The present invention provides an electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system, comprising the following steps:

[0007] S1. Obtain the DNA nucleotide sequence to be tested and construct sample DNA; irradiate several sample DNAs under ultraviolet light for a preset time to obtain a group of sample DNAs with different degrees of damage; mix Cas12a and crRNA to generate a Cas12a / crRNA complex, and then add sample DNAs with different degrees of damage to them and incubate them to obtain a group of CRISPR-Cas12a complexes with different activation degrees;

[0008] S2. After polishing the gold electrode, a mixture of thiolated DNA probe and TCEP (tri(2-carboxyethyl)phosphine) was added to the electrode surface for incubation; the electrode surface was cleaned with water and ethanol respectively and dried, and 6-mercapto-1-hexanol was added for incubation; the electrode surface was cleaned with water and ethanol respectively and dried, and the CRISPR-Cas12a complexes with different activation degrees obtained in step S1 were added for incubation to obtain a set of pretreated electrodes;

[0009] S3. Add a mixture of hairpin DNA H1 and H2 to a set of pretreated electrodes obtained in step S2 to perform HCR reaction. After the reaction is completed, wash the electrodes and blow dry them, add a methylene blue solution and incubate them to obtain a set of methylene blue treated electrodes;

[0010] S4. The group of methylene blue treated electrodes obtained in step S3 are subjected to differential pulse voltammetry, using a three-electrode system to test the electrochemical signal of methylene blue in phosphate buffer (10 mM PBS, pH 7.4), to obtain the relationship between the photodamage level of DNA and the electrochemical signal of methylene blue;

[0011] S5. According to the relationship between the photodamage level of the DNA obtained in step S4 and the electrochemical signal of methylene blue, the DNA to be tested is processed to obtain the photodamage level of the DNA to be tested.

[0012] Furthermore, in step S1, the sample DNA is irradiated with ultraviolet light for a preset time gradient, the time gradient range is 0 to 120 min, the ultraviolet wavelength range is 230 to 270 nm, and the ultraviolet irradiation intensity range is 0.3 to 0.4 W m -2 .

[0013] The process conditions for incubating Cas12a and crRNA to generate Cas12a / crRNA complex are as follows: the concentration ratio of Cas12a to crRNA is 1:(0.8-1.2), the incubation time is 20-40 min, and the incubation temperature is 35-40 ° C;

[0014] The process conditions for the mixed incubation of Cas12a / crRNA complex and damaged DNA are as follows: the concentration ratio of Cas12a / crRNA complex to damaged DNA is 1:(0.8-1.2), the incubation time is 20-40 min, and the incubation temperature is 35-40°C.

[0015] Further, in step S2, in the mixture of thiolated DNA probe and TCEP, the concentration of thiolated DNA probe is 0.1 to 1 μM, and the concentration of TCEP is 0.8 to 1.2 mM;

[0016] The process conditions for incubating the mixture of thiolated DNA probe and TCEP on the gold electrode are as follows: the volume of the mixture of thiolated DNA probe and TCEP is 8 to 15 μL, the incubation temperature is 20 to 25° C., and the incubation time is 8 to 12 h;

[0017] The process conditions for incubation by dropping 6-mercapto-1-hexanol are as follows: the concentration of 6-mercapto-1-hexanol is 0.5-1 mM, the volume is 30-50 μL, the incubation temperature is 20-25° C., and the incubation time is 0.5-1 h;

[0018] The process conditions for incubating by dropping CRISPR-Cas12a complexes with different activation degrees are as follows: the volume of CRISPR-Cas12a complexes with different activation degrees dropped is 8 to 15 μL, the incubation temperature is 35 to 40° C., and the incubation time is 100 to 150 min.

[0019] Furthermore, in step S3, the concentration of the raw material hairpin DNA H1 used in the mixture of hairpin DNA H1 and H2 is 1.5-2.5 μM, the concentration of hairpin DNA H2 is 1.5-2.5 μM, and the concentration ratio during mixing is 1:(0.8-1.2); the amount of the mixture of hairpin DNA H1 and H2 added dropwise is 8-15 μL, the HCR reaction temperature is 35-40°C, and the time is 100-150 min.

[0020] The concentration of the added methylene blue solution is 15-25 mM, the added amount is 20-30 μL, the incubation temperature is 20-25° C., and the incubation time is 20-40 min.

[0021] Furthermore, in step S4, a three-electrode system is used, wherein the working electrode is a methylene blue-treated electrode, the reference electrode is an Ag / AgCl reference electrode, and the counter electrode is a platinum wire electrode; and the electrolyte solution is 10 mM PBS at pH 7.4.

[0022] Further, step S5 includes the following steps:

[0023] A. Prepare Cas12a / crRNA complex, then add the DNA to be tested for incubation to obtain an activated CRISPR-Cas12a complex;

[0024] The process conditions for preparing the Cas12a / crRNA complex are the same as the process conditions for mixing Cas12a and crRNA in step S1 to generate the Cas12a / crRNA complex;

[0025] The incubation conditions are the same as those for the mixed incubation of the Cas12a / crRNA complex and damaged DNA in step S1;

[0026] B. polishing the gold electrode, adding a mixture of thiolated DNA probe and TCEP (tri(2-carboxyethyl)phosphine), incubating overnight, washing the electrode surface with water and ethanol respectively and drying it, then adding 6-mercapto-1-hexanol for incubation; then washing the electrode surface with water and ethanol respectively and drying it, adding the activated CRISPR-Cas12a complex for incubation to obtain a complex-pretreated electrode;

[0027] The process conditions of this step are the same as those of the corresponding step in step S2;

[0028] C. DNAH1 and H2 are added dropwise to the electrode pretreated with the complex to carry out HCR reaction. After the reaction is completed, the electrode is cleaned and blown dry, and a methylene blue solution is added dropwise and incubated to obtain a methylene blue-treated electrode;

[0029] The process conditions of this step are the same as those of the corresponding step in step S3;

[0030] D. According to the relationship between the photodamage level of DNA and the electrochemical signal of methylene blue, the electrochemical signal of methylene blue is tested in PBS by differential pulse voltammetry using a three-electrode system to obtain the photodamage level of the DNA to be tested; the process conditions of this step are the same as those of the corresponding step in step S4.

[0031] Principle of the present invention:

[0032] Ultraviolet radiation can induce adjacent pyrimidine bases in DNA to form cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). When the sample DNA is not damaged, the CRISPR-Cas12a system can be activated normally; at this time, the thiol-fixed DNA probe on the electrode surface is cut, resulting in the inability to trigger the HCR reaction in the presence of hairpin DNA H1 and H2, and a small electrochemical signal is generated after the introduction of methylene blue. After the sample DNA is damaged by ultraviolet irradiation, the CRISPR-Cas12a system cannot be activated and cannot normally cut the DNA probe fixed on the surface. At this time, the DNA probe triggers the HCR reaction in the presence of H1 and H2, first opening the stem-loop structure of hairpin H1, and then opening the stem-loop structure of hairpin H2, at which time a large amount of double-stranded DNA is generated on the electrode surface; the generated double-stranded DNA interacts with the methylene blue molecules through electrostatic action or insertion mechanism, generating a significant electrochemical signal. The degree of DNA damage can be measured by the level of photodamage. As the degree of damage changes, the electrochemical signal of methylene blue changes. The difference in the electrochemical signal of methylene blue corresponds to different levels of DNA photodamage.

[0033] Beneficial effects of the present invention:

[0034] (1) The present invention uses the CRISPR-Cas12a system for the first time to detect DNA damage, and different degrees of damage correspond to different levels of photodamage;

[0035] (2) The HCR reaction initiated on the electrode surface in the method of the present invention is highly dependent on the integrity of the DNA probe, which reduces background noise and improves analytical performance;

[0036] (3) The method of electrochemical detection of DNA photodamage level based on the CRISPR-Cas12a system of the present invention has the advantages of high sensitivity and wide linear range, and is expected to be used for the detection of DNA damage in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the electrochemical detection of DNA damage based on the CRISPR-Cas12a system in the embodiment;

[0038] Figure 2 The gel electrophoresis diagram in the embodiment, wherein lane 1: DNA probe without thiol group, lane 2: H1, lane 3: H2, lane 4: DNA probe + hairpin H1, lane 5: hairpin H1 + H2, lane 6: DNA probe + hairpin H1 + H2, lane 7: CRISPR-Cas12a complex + DNA probe + H1 + H2, lane 8: damaged T 21 +Cas12a / crRNA+DNA probe+H1+H2;

[0039] Figure 3 A is the damage T of different ultraviolet doses in the embodiment 21 The electrochemical signal corresponding to Figure 3 B is the linear relationship curve between electrochemical signal and UV dose. DETAILED DESCRIPTION

[0040] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0041] The embodiment of the present invention adopts DNAT 21 As the DNA sample to be tested, an electrochemical method for evaluating DNA damage was constructed based on the enzymatic cleavage reaction of the CRISPR-Cas12a complex on the DNA probe. Figure 1 As shown in the figure. First, the thiolated DNA probe is fixed on the surface of the gold electrode. 21 When damaged, the CRISPR-Cas12a complex cannot be activated normally, resulting in the inability to cut the DNA probe. The intact DNA probe undergoes an HCR reaction in the presence of hairpin DNA H1 and H2 to generate a longer double-stranded DNA. Subsequently, the methylene blue molecule is embedded in the double-stranded DNA, generating a stronger electrochemical signal; normal or undamaged DNA can activate the enzymatic cleavage activity of the CRISPR-Cas12a complex, resulting in the cutting of the DNA probe fixed on the surface, and the HCR reaction cannot be triggered in the presence of hairpin DNA H1 and H2, resulting in a smaller amount of methylene blue binding to the electrode surface, thereby generating a smaller electrochemical signal.

[0042] Among them, DNA T 21 The nucleotide sequences of , crRNA, thiolated DNA probe, and hairpin DNA H1 and H2 are shown in the following table.

[0043] Table 1 Nucleotide sequences

[0044]

[0045] Example 1 Validation method for detecting DNA damage based on CRISPR-Cas12a system

[0046] Take 10 groups of 0.1 mL, 0.5 μM T 21 The intensity of exposure at 254nm is 0.35Wm -2 The UV light was used for 0min, 1min, 5min, 7.5min, 10min, 15min, 20min, 30min, 60min, and 120min to obtain 10 groups of UV-damaged T 21 ;

[0047] 150 nM crRNA and 150 nM Cas12a were mixed and incubated, the concentration ratio of Cas12a to crRNA was 1:1, the incubation time was 30 min, and the incubation temperature was 37 ° C to obtain a Cas12a / crRNA complex;

[0048] 10 groups of UV-damaged T 21 Cas12a / crRNA complex was added to the culture medium, the concentration ratio of Cas12a / crRNA complex to damaged DNA was 1:1, the incubation time was 30 min, and the incubation temperature was 37°C, to obtain 10 groups of CRISPR-Cas12a complexes with different activation degrees;

[0049] The gold electrode was polished on a polishing cloth with 0.3μm and 0.05μm alumina slurry and ultrasonically cleaned in water and ethanol. A 0.5μM thiolated DNA probe was mixed with a 1mM TCEP and incubated in the dark for 1h, and then 10μL of the thiolated DNA probe and TCEP mixture was added to the polished gold electrode and incubated at room temperature overnight. The gold electrode was rinsed with PBS, and 10μL of 1.0mM 6-mercapto-1-hexanol was added and incubated for 0.5h. 10 groups of CRISPR-Cas12a complexes with different activation degrees were added to the electrode surface and incubated at 37°C for 120min. Then a mixed solution of hairpin DNA H1 and H2 was added to the electrode and reacted at 37°C for 2h. The above electrode was immersed in a 20mM methylene blue solution and incubated at room temperature for 30min. Finally, differential pulse voltammetry was performed in 10 mM PBS in the range of -0.5 V to 0 V to obtain the relationship between the degree of DNA damage and the electrochemical signal of methylene blue.

[0050] The damage of T by different UV doses was detected. 21 The corresponding electrochemical signal is Figure 3 As shown in A, the linear relationship curve between electrochemical signal and UV dose is as follows Figure 3 B. In this example, UV dose can be used to indicate the degree of DNA damage. Figure 3 As shown in A and 3B, the oxidation current of methylene blue increases with the increase of UV dose, and the current is in the range of 0.021-0.42 kJ m -2 Within this range, the oxidation current is linearly related to the UV dose, and the linear equation is i(μA)=7.27UV dose(kJ m -2 )+1.64(R 2 =0.9959). Based on S / N=3, the minimum detection limit of the method is 0.0088 kJ m -2 .

[0051] Example 2 Gel electrophoresis experiment to verify the occurrence of HCR reaction

[0052] Set up 8 experimental groups, which are 8 lanes of gel electrophoresis;

[0053] The first experimental group was DNA probe;

[0054] The second experimental group was hairpin DNA H1;

[0055] The third experimental group was hairpin DNA H2;

[0056] The fourth experimental group was DNA probe + hairpin DNA H1;

[0057] The fifth experimental group was hairpin DNA H1 + hairpin DNA H2;

[0058] The sixth experimental group consisted of DNA probe + hairpin DNA H1 + hairpin DNA H2;

[0059] The seventh experimental group consisted of CRISPR-Cas12a complex + DNA probe + hairpin DNA H1 + hairpin DNA H2;

[0060] The eighth experimental group was injured T 21 +Cas12a / crRNA+DNA probe+hairpin DNA H1+hairpin DNA H2;

[0061] Gel electrophoresis results Figure 2 As shown, the single bands in lanes 1, 2, and 3 correspond to DNA probe, H1, and H2, respectively. A new band with a slower migration rate appears in lane 4, indicating that the DNA probe can successfully open the card H1. The brighter bands in lane 5 correspond to H1 and H2, and the fuzzy bands with slower migration rates indicate the weak occurrence of HCR reaction. The continuous bright bands with slow migration rates in lane 6 correspond to the occurrence of HCR reaction. Lane 7 is similar to lane 5, with normal T 21 The enzyme activity of the CRISPR-Cas12a complex was successfully activated, thereby cutting the DNA probe and causing a weak HCR reaction. Lane 8 is similar to lane 6, damaging T 21 The inability to completely bind to the Cas12a-crRNA complex results in the DNA probe being almost unable to be cut, thereby inducing the HCR reaction.

Claims

1. An electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system, characterized in that: The following steps are involved: S1. Obtain the DNA nucleotide sequence to be tested and construct sample DNA; irradiate several sample DNAs under ultraviolet light for a preset time to obtain a group of sample DNAs with different degrees of damage; mix Cas12a and crRNA to generate a Cas12a / crRNA complex, and then add sample DNAs with different degrees of damage to them and incubate them to obtain a group of CRISPR-Cas12a complexes with different activation degrees; S2. After polishing the gold electrode, a mixture of thiolated DNA probe and TCEP (tri(2-carboxyethyl)phosphine) was added to the electrode surface for incubation; the electrode surface was cleaned with water and ethanol respectively and dried, and 6-mercapto-1-hexanol was added for incubation; the electrode surface was cleaned with water and ethanol respectively and dried, and the CRISPR-Cas12a complexes with different activation degrees obtained in step S1 were added for incubation to obtain a set of pretreated electrodes; S3. Add a mixture of hairpin DNA H1 and H2 to a set of pretreated electrodes obtained in step S2 to perform HCR reaction. After the reaction is completed, wash the electrodes and blow dry them, add a methylene blue solution and incubate them to obtain a set of methylene blue treated electrodes; S4. The set of methylene blue treated electrodes obtained in step S3 are subjected to differential pulse voltammetry, using a three-electrode system to test the electrochemical signal of methylene blue in PBS to obtain the relationship between the photodamage level of DNA and the electrochemical signal of methylene blue; S5. According to the relationship between the photodamage level of the DNA obtained in step S4 and the electrochemical signal of methylene blue, the DNA to be tested is processed to obtain the photodamage level of the DNA to be tested.

2. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that In step S1, the sample DNA is irradiated under ultraviolet light for a preset time gradient, the time gradient range is 0 to 120 min, the ultraviolet wavelength range is 230 to 270 nm, and the ultraviolet irradiation intensity range is 0.3 to 0.4 Wm -2 .

3. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that In step S1, the process conditions for mixing Cas12a and crRNA to generate a Cas12a / crRNA complex are as follows: the concentration ratio of Cas12a to crRNA is 1:(0.8-1.2), the incubation time is 20-40 min, and the incubation temperature is 35-40 ° C; The process conditions for the mixed incubation of Cas12a / crRNA complex and damaged DNA are as follows: the concentration ratio of Cas12a / crRNA complex to damaged DNA is 1:(0.8-1.2), the incubation time is 20-40 min, and the incubation temperature is 35-40°C.

4. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that In step S2, in the mixture of thiolated DNA probe and TCEP, the concentration of thiolated DNA probe is 0.1-1 μM, and the concentration of TCEP is 0.8-1.2 mM; the process conditions for incubating the mixture of thiolated DNA probe and TCEP on the gold electrode are as follows: the volume of the mixture of thiolated DNA probe and TCEP is 8-15 μL, the incubation temperature is 20-25° C., and the incubation time is 8-12 h.

5. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that In step S2, the process conditions for adding 6-mercapto-1-hexanol for incubation are: the concentration of 6-mercapto-1-hexanol is 0.5-1 mM, the volume is 30-50 μL, the incubation temperature is 20-25° C., and the incubation time is 0.5-1 h.

6. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that In step S2, the process conditions for adding CRISPR-Cas12a complexes with different activation degrees for incubation are as follows: the volume of CRISPR-Cas12a complexes with different activation degrees added is 8 to 15 μL, the incubation temperature is 35 to 40° C., and the incubation time is 100 to 150 min.

7. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that In step S3, the concentration of the raw material hairpin DNA H1 used in the mixture of hairpin DNA H1 and H2 is 1.5-2.5 μM, the concentration of hairpin DNA H2 is 1.5-2.5 μM, and the concentration ratio during mixing is 1:(0.8-1.2); the amount of the mixture of hairpin DNA H1 and H2 added dropwise is 8-15 μL, the HCR reaction temperature is 35-40°C, and the time is 100-150 min.

8. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that In step S3, the concentration of the methylene blue solution added dropwise is 15-25 mM, the amount added is 20-30 μL, the incubation temperature is 20-25° C., and the incubation time is 20-40 min.

9. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to claim 1, characterized in that In step S4, a three-electrode system is used, wherein the working electrode is a methylene blue treated electrode, the reference electrode is an Ag / AgCl reference electrode, the counter electrode is a platinum wire electrode; and the electrolyte solution is 10 mM PBS with a pH of 7.

4.

10. The electrochemical method for detecting DNA damage based on the CRISPR-Cas12a system according to any one of claims 1 to 9, characterized in that: Step S5 includes the following steps: A. Prepare Cas12a / crRNA complex, then add the DNA to be tested for incubation to obtain an activated CRISPR-Cas12a complex; B. polishing the gold electrode, adding a mixture of thiolated DNA probe and TCEP (tri(2-carboxyethyl)phosphine), incubating overnight, washing the electrode surface with water and ethanol respectively and drying it, then adding 6-mercapto-1-hexanol for incubation; then washing the electrode surface with water and ethanol respectively and drying it, then adding the activated CRISPR-Cas12a complex obtained in step S11 for incubation to obtain a pretreated electrode; C. Adding hairpin DNA H1 and H2 to the pretreated electrode obtained in step S12 to perform HCR reaction. After the reaction is completed, the electrode is cleaned and blown dry, and a methylene blue solution is added and incubated to obtain a methylene blue treated electrode; D. Based on the relationship between the photodamage level of DNA obtained in step S4 and the electrochemical signal of methylene blue, the methylene blue treated electrode obtained in step S13 is subjected to differential pulse voltammetry, and the electrochemical signal of methylene blue is tested in PBS using a three-electrode system to obtain the photodamage level of the DNA to be tested.

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