CN115046973A - FRET-based DNA damage detection method and functional protein - Google Patents

Abstract

The invention discloses a FRET-based DNA damage detection method and a functional protein. Connecting yellow fluorescent protein eYFP and cyan fluorescent protein eFP to the N end and the C end of the protein respectively by using repressor Ddr O in deinococcus radiodurans to construct functional protein YDC with the fluorescent energy transfer characteristic; meanwhile, the protein PprI-D91A is obtained by mutating aspartic acid (D) at the position 91 of the protein PprI into alanine (A) by utilizing the deinococcus radiodurans damage response regulatory factor PprI protein capable of specifically cutting Ddr O, so that the enzyme digestion activity is reduced when single-stranded DNA does not exist; when single-stranded DNA generated by damage exists in the system, PprI-D91A can rapidly cut YDC, and under the action of 440 nm exciting light, the 480 nm peak of the light emitted by YDC is increased and the 530 nm peak is decreased; meanwhile, the amino acid sequences of YDC and PprI-D91A and a reaction system for detecting DNA damage are also provided. The invention has important guiding significance for developing novel molecular biology tools.

Description

FRET-based DNA damage detection method and functional protein

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a fluorescence detection method for detecting DNA damage and a functional protein.

Background

Current methods for detecting DNA damage can be divided into three major categories according to experimental principles: a detection method designed according to the change of the physicochemical property of DNA, a detection method designed according to the principle of molecular interaction, and a detection method designed according to the change of a substance after damage. The single cell gel electrophoresis method (SCGE, also called comet experiment) is a detection method designed according to the change of the physicochemical properties of DNA, can effectively determine the damage degree of single chains and double chains of DNA in cells, and mainly comprises three steps of packaging, cracking and electrophoresis, wherein the electrophoresis at high pH forms a comet-like structure. The DNA fragmentation fluorescence in situ hybridization (DBD-FISH) is based on the molecular hybridization principle, DNA is uncoiled into single-stranded DNA at a fragmentation part under an alkaline condition, and then the fragmentation part is displayed by hybridization of a fluorescent DNA probe. The method for detecting the change of substances comprises a gamma-H2 AX detection method, wherein gamma-H2 AX is a marker of DNA damage, and once DNA Double Strand Break (DSB) occurs, gamma-H2 AX is generated immediately and is in one-to-one correspondence with the number of the DSB, namely, one focus of gamma-H2 AX corresponds to one DSB site.

However, in the aforementioned DNA damage detection method, the first two processing steps are complicated, and the waiting time is long, which is not favorable for improving the experimental efficiency. The last one can achieve damage detection with good specificity, but depends on specific proteins, which is not favorable for popularization and application of the technology.

Recent studies have shown that deinococcus radiodurans (R.) (Deinococcus radioduransDR) took a different damage response repair pathway than the classical bacterial SOS system-the PprI-DdrO pathway, where two proteins, PprI and DdrO, played a key role, and the addition of ssDNA facilitated the enzymatic cleavage process therein.

Disclosure of Invention

In order to overcome the defects of the existing DNA damage detection technology, the invention aims to provide a FRET-based DNA damage detection method and a functional protein.

A FRET-based DNA damage detection method comprises the following steps:

1) preparing a reaction solution comprising: PprI-D91A, YDC, a sample to be detected and Buffer; reacting at 37 ℃ for 30 min;

2) after the reaction is finished, absorbing the reaction solution and adding the reaction solution into a black 386 pore plate, scanning the emitted light with the wavelength of 460 nm-600 nm by using a multifunctional microplate reader and light with the wavelength of 440 nm as exciting light, and obtaining the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nmR _530/480 ；

3) Subjecting the sample toR _530/480 In comparison with a blankR _530/480 Make a ratio to obtainrR _530/480 Obtaining the ssDNA content in the sample to be detected according to a standard curve equation, and accordingly evaluating the damage degree of the DNA in the sample to be detected;

the YDC contains three structural regions of eYFP, DdrO and eCFP; the amino acid sequence is shown as SEQ ID NO. 1;

the amino acid sequence of the PprI-D91A is shown in SEQ ID NO. 2.

The method comprises the following more specific steps:

1) preparing a reaction solution: PprI-D91A, final concentration 0.10. mu.M; YDC, final concentration 3.0 μ M; 10 mu L of sample to be detected; 2 mM MnCl ₂ 10 μL；65.7 μL Buffer；ddH ₂ Filling the reaction solution with O to 100 mu L, and reacting at 37 ℃ for 30 min;

the Buffer: 250 mM NaCl, 5% glycerol, 20 mM Tris-HCl pH 7.5;

2) after the reaction is finished, 70 mu L of reaction solution is absorbed and added into a black 386 pore plate, a multifunctional microplate reader is utilized, light with the wavelength of 440 nm is used as exciting light, emitted light with the wavelength of 460 nm-600 nm is scanned, and the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nm is obtainedR _530/480 ；

3) Subjecting the sample toR _530/480 In comparison with a blankR _530/480 Make a ratio to obtainrR _530/480 And obtaining the ssDNA amount contained in the sample to be detected according to the standard curve equation, and accordingly evaluating the damage degree of the DNA in the sample to be detected.

A functional protein YDC with fluorescence energy transfer characteristics, which contains three structural regions of eYFP, DdrO and eCFP; the amino acid sequence is shown as SEQ ID NO. 1.

A protease PprI-D91A has an amino acid sequence shown in SEQ ID NO. 2.

A FRET-based functional protein combination comprising PprI-D91A and YDC; the amino acid sequence of the PprI-D91A is shown in SEQ ID NO. 2;

the YDC contains three structural regions of eYFP, DdrO and eCFP; the amino acid sequence is shown as SEQ ID NO. 1;

PprI-D91A can accurately identify the specific sequence of the functional protein YDC and carry out enzyme digestion on the functional protein YDC, the fluorescence energy transfer efficiency of the YDC is reduced after the enzyme digestion reaction, and the addition of ssDNA promotes the enzyme digestion process.

The invention has the beneficial effects that:

the invention provides a simple, convenient and rapid fluorescence method for detecting DNA damage, and the content of ssDNA in a sample can be obtained by measuring fluorescence intensity only in 30 min by adding the sample into a premixed reaction solution, so that the damage condition of the DNA is reflected, and the fluorescence method has important significance for developing a novel molecular biology tool.

Drawings

FIG. 1 is a schematic diagram showing the constitution of the recombinant protein YDC.

FIG. 2 is a SDS-PAGE result of PprI, PprI-D91A and YDC after purification.

FIG. 3 is a standard curve obtained after a systematic reaction using a prepared ssDNA sample, with the horizontal axis representing the ssDNA concentration in the sample and the vertical axis representing the relative ratio of emitted fluorescence (rR _530/480 ）。

FIG. 4 is a diagram showing the results of electrophoresis of the E.coli genome after purification in a 1% agarose gel.

FIG. 5 is a graph showing the relative fluorescence values obtained by the detection system for the purified E.coli genome.

FIG. 6 is the purifiedegfpThe results of the electrophoresis of the genes on a 1% agarose gel are shown.

FIG. 7 is a drawing showing the purificationegfpAnd obtaining a fluorescence relative ratio value after the gene reacts through a detection system.

Detailed Description

The invention is further illustrated with reference to the following figures and specific examples.

A functional protein YDC with fluorescence energy transfer characteristic, wherein the amino acid sequence of the functional protein YDC is coded and is shown in SEQ ID NO. 1; the functional protein YDC contains three protein regions of eYFP, DdrO and eFP (figure 1), wherein the DdrO can be recognized and cut by the protein PprI. In FIG. 1, eYFP protein is at the N-terminus, Ddr protein is in the middle, and eCFP protein is at the C-terminus. The proteins are connected through GGGGSGGGS flexible peptide chains.

A protease PprI-D91A having a zinc finger-protease domain, a helix-turn-helix domain and a GAF domain, said protease holoenzyme and its independent structure zinc finger-protease domain having equivalent protease activity and having aspartic acid Asp at position 91 mutated to alanine Ala compared to wild type PprI from DR. The core protein sequence of the zinc finger-protease structural domain is shown in SEQ ID NO. 2.

The functional protein YDC and the protease PprI-D91A, PprI-D91A can accurately identify the functional protein YDC and carry out enzyme digestion on the functional protein YDC, the fluorescence energy transfer efficiency of the YDC is reduced after enzyme digestion, and the addition of ssDNA can promote the enzyme digestion process of the YDC.

The reaction buffer solution (100. mu.L system) in which the cleavage reaction proceeded contained 65.7. mu.L of a buffer solution (250 mM NaCl, 5% glycerol, 20 mM Tris-HCl pH 7.5) and 1 mM MnCl ₂ 0.10 mu M PprI-D91A protein, 3 mu M YDC protein, 10 mu L of detection sample (concentration range is 0.05 mu M-1 mu M), and the rest is ddH ₂ O make up to 100. mu.L.

In the enzyme digestion reaction process, the reaction temperature is 37 ℃, and the reaction time is 30 min.

After the reaction is finished, absorbing the reaction solution, scanning the 460 nm-600 nm emission light of the reaction solution by using a multifunctional microplate reader with 440 nm light as excitation light and 5 nm as a step length to obtain the intensity ratio of the 530 nm and 480 nm emission light (intensity ratio of light emitted at 530 nm and 480 nm,R _530/480 ）。

according to the ratio of the emitted light intensitiesThe relative ratio of the emitted light intensities (relative intensity ratio of light emitted at 530 nm and 480 nm,rR _530/480 ) And calculating the ssDNA content in the sample by using a standard curve so as to evaluate the damage degree of the DNA.

Example 1: construction of expression plasmid of recombinant protein YDC and purification of protein expression

(1) pET-28a (+) was linearized using SEQ ID NO.3 and SEQ ID NO.4 and amplified from DR using SEQ ID NO.5 and SEQ ID NO.6ddrORecombining the fragments by recombinase Exanase II, converting the fragments into Trans 5 alpha, and screening and sequencing to obtain a target vector 1; linearization of vector 1 with SEQ ID No.10 and SEQ ID No.4, amplification with SEQ ID No.11 and SEQ ID No.8ecfpCarrying out recombination, transformation, screening and sequencing on the fragments to obtain a target vector 2; linearization of vector 2 with SEQ ID No.9 and SEQ ID No.3, amplification with SEQ ID No.7 and SEQ ID No.12eyfpAnd (3) recombining, transforming, screening and sequencing the fragments to obtain a target vector pET-28a (+) -YDC.

(2) Completing the construction of the vector, and transforming the vector into a BL21(DE3) expression strain; the strain was inoculated into 500 mL of LB medium and shake-cultured at 37 ℃ to OD ₆₀₀ About 0.6; adding IPTG with final concentration of 0.2 μ M, and performing shaking table induction at 30 deg.C for 10 hr; after the induction culture is finished, centrifuging by using a centrifugal machine to collect thalli precipitates (8000 rpm, 8 min); about 30 mL of PBS was added to resuspend the cells, and after re-centrifugation (8000 rpm, 8 min), the supernatant was removed, leaving the cell pellet.

(3) Adding about 30 mL of Buffer 1 to resuspend the thalli, and after pressure crushing (800 bar and 3 min), performing ultrasonic treatment for 40 min (2.5 s on, 7.5s off and 60% of power); centrifuging the treated liquid at 12000 rpm for 30 min, collecting supernatant, and filtering with 0.22 μm microporous membrane; firstly, filling 1 mL of nickel column with Buffer 1 by using an ATKA system for balancing, filling the filtered liquid with the nickel column, and balancing with the Buffer 1 after the filling is finished; adding 5% of Buffer 2 to elute the hybrid protein, and adding 30% of Buffer 2 to elute the target protein after balancing; replacing the Buffer solution of the target protein with Buffer 3 by using Buffer 4 and a 50 mL desalting column; balancing a Heparin column by using Buffer 3, adding a target protein solution, adding 30% of Buffer 1 after balancing to elute the target protein, and finally obtaining a relatively pure YDC sample. FIG. 2 is a SDS-PAGE result of PprI, PprI-D91A and YDC after purification, and it can be seen that the three proteins all reached a purity of 95% or more. Wherein PprI and PprI-D91A have a molecular weight of about 37 kDa and YDC has a molecular weight of about 72 kDa.

Buffer formulation, the same applies below Buffer 1: 20 mM Tris-HCl 7.5, 5% glycerol, 1M NaCl; buffer 2: 20 mM Tris-HCl 7.5, 5% glycerol, 1M NaCl, 500 mM imidazole; buffer 3: 20 mM Tris-HCl 7.5, 5% glycerol, 150 mM NaCl, 1 mM EDTA; buffer 4: 20 mM Tris-HCl 7.5, 5% glycerol, 150 mM NaCl; buffer 5: 20 mM Tris-HCl 7.5, 5% glycerol, 250 mM NaCl.

Example 2: construction of expression vector and protein expression purification of PprI and PprI-D91A

(1) pET-28a (+) was linearized using SEQ ID NO.3 and SEQ ID NO.4 and amplified from DR using SEQ ID NO.13 and SEQ ID NO.14pprIThe fragment is recombined by recombinase Exanase II, transformed to Trans 5 alpha, screened and sequenced to obtain a target vector pET-28a (+) -PprI. The vector pET-28a (+) -plus was prepared using SEQ ID NO.15 and SEQ ID NO.16pprIPerforming PCR (pfuEnzyme), directly transforming the obtained PCR product into a Trans 5 alpha strain, and obtaining a target vector pET-28a (+) -PprI-D91A after screening and sequencing.

(2) Completing the construction of the vector, and transforming the vector into a BL21(DE3) expression strain; the strain was inoculated into 500 mL of LB medium and shake-cultured at 37 ℃ to OD ₆₀₀ About 0.6; adding IPTG with final concentration of 0.2 μ M, and performing shaking table induction at 30 deg.C for 10 hr; after the induction culture is finished, centrifuging by using a centrifugal machine to collect thalli precipitates (8000 rpm, 8 min); about 30 mL of PBS was added to resuspend the cells, and after re-centrifugation (8000 rpm, 8 min), the supernatant was removed, leaving the cell pellet.

Adding about 30 mL of Buffer 1 to resuspend the thalli, and carrying out ultrasonic treatment for 40 min (2.5 s on, 7.5s off and 60% of power); centrifuging the treated liquid at 12000 rpm for 30 min, collecting supernatant, and filtering with 0.22 μm microporous filter membrane; firstly, filling 1 mL of nickel column into Buffer 1 by using an ATKA system for balancing, filling the filtered liquid into the nickel column, and balancing by using the Buffer 1 after the filling is finished; adding 5% of Buffer 2 to elute the hybrid protein, and adding 30% of Buffer 2 to elute the target protein after balancing;

the Buffer 4 of the target protein was replaced with Buffer 4 using Buffer 4 and a 50 mL desalting column to obtain samples of PprI and PprI-D91A (FIG. 2).

Example 3: drawing of standard curve

(1) After the target protein was purified by the methods of examples 1 and 2, aqueous ssDNA solutions were prepared at different concentrations, and reaction solutions were prepared according to the following formulations: PprI-D91A (final concentration about 0.10. mu.M), YDC (final concentration about 3.0. mu.M), ssDNA (10. mu.L), MnCl ₂ （2 mM，10 μL）、Buffer 5（65.7 μL）、ddH ₂ The reaction solution was filled with O to 100. mu.L, and reacted at 37 ℃ for 30 min.

(2) After the reaction is finished, 70 mu L of reaction solution is sucked and added into a black 386 pore plate, a multifunctional microplate reader is utilized, light with the wavelength of 440 nm is used as exciting light, emitted light with the wavelength of 460 nm-600 nm is scanned, and the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nm is obtained (the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nm is obtained by the method: (the ratio is the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nm) (R _530/480 ). Blank control (sample without DNA)R _530/480 Taking the ratio as a standard value, and dividing the ratio by the standard value to obtain a relative ratio (rR _530/480 ). ssDNA concentration is plotted on the abscissa and the relative ratio is calculatedrR _530/480 A standard curve is plotted for the ordinate (fig. 3). As can be seen from the standard curve, the optimal detection range of the detection method is 0.05 μ M to 1 μ M (10 μ L of sample).

Example 4: genomic DNA purification sample degradation analysis

(1) Taking overnight cultured escherichia coli, extracting genome DNA, dividing the obtained DNA sample into four parts, and carrying out the following treatment: i: storing at 4 ℃; II: incubating at 37 ℃ for 5 days; III: incubating at 37 ℃ for 10 days; IV: incubate at 70 ℃ for 10 days. After completion of the incubation, the degradation of the genomic DNA was checked by electrophoresis on a 1% agarose gel (FIG. 4), in which the genome (IV) at 70 ℃ treatment had been completely degraded.

(2) mu.L of the obtained DNA sample was added to the reaction system of example 3 in place of ssDNA while keeping the same5 mu.L of double distilled water is added to make up the reaction volume, and the reaction is carried out for 30 min at 37 ℃. After the reaction is finished, 70 mu L of reaction solution is sucked and added into a black 386 pore plate, a multifunctional microplate reader is utilized, light with the wavelength of 440 nm is used as exciting light, emitted light with the wavelength of 460 nm-600 nm is scanned, and the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nm is obtained (the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nm is obtained by the method: (the ratio is the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nm) (R _530/480 ) Comparing the blank to obtain a relative ratio (rR _530/480 See fig. 5, consistent with the results of fig. 4). The degree of DNA damage was evaluated by extrapolating back the amount of ssDNA contained in the sample through the standard curve equation in example 3. The DNA damage assessment results were consistent with the agarose gel electrophoresis results.

Example 5:egfpanalysis of gene fragment degradation at different temperatures

(1) Obtained by PCR reactionegfpThe gene fragment was collected, purified, and divided into six portions, which were incubated under conditions of I (4 ℃ C.), II (30 ℃ C.), III (40 ℃ C.), IV (50 ℃ C.), V (60 ℃ C.) and VI (70 ℃ C.) for 6 hours, respectively. After completion of the incubation, the degradation of the genomic DNA was examined by electrophoresis on a 1% agarose gel (FIG. 6), and the results of the electrophoresis slightly showed the degradation of the DNA.

(2) mu.L of the obtained DNA sample was added to the reaction system of example 3 instead of ssDNA, and 5. mu.L of double distilled water was added to complete the reaction volume, followed by reaction at 37 ℃ for 30 min. After the reaction is finished, 70 mu L of reaction solution is sucked and added into a black 386 pore plate, a multifunctional microplate reader is utilized, light with the wavelength of 440 nm is used as exciting light, emitted light with the wavelength of 460 nm-600 nm is scanned, and the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nm is obtained (the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nm is obtained by the method: (the ratio is the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nm) (R _530/480 ). Comparing the blank to obtain a relative ratio (rR _530/480 FIG. 7), the degree of DNA damage was evaluated by extrapolating back the amount of ssDNA contained in the sample using the standard curve equation of example 3. From the electrophoresis result (FIG. 6) and the fluorescence analysis result (FIG. 7), the two results are consistent, but the fluorescence analysis result (FIG. 7) can more directly characterize the damage condition of the DNA.

The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

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Claims (5)

1. A FRET-based DNA damage detection method is characterized by comprising the following steps:

1) preparing a reaction solution comprising: PprI-D91A, YDC, a sample to be detected and Buffer; reacting at 37 ℃ for 30 min;

2) after the reaction is finished, absorbing the reaction solution and adding the reaction solution into a black 386 pore plate, scanning the emitted light with the wavelength of 460 nm-600 nm by using a multifunctional microplate reader and light with the wavelength of 440 nm as exciting light, and obtaining the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nmR _530/480 ；

3) Subjecting the sample toR _530/480 In comparison with a blankR _530/480 Make a ratio to obtainrR _530/480 Obtaining the ssDNA content in the sample to be detected according to a standard curve equation, and accordingly evaluating the damage degree of the DNA in the sample to be detected;

the YDC contains three structural regions of eYFP, DdrO and eCFP; the amino acid sequence is shown as SEQ ID NO. 1;

the amino acid sequence of the PprI-D91A is shown in SEQ ID NO. 2.

2. The method according to claim 1, characterized by the steps of:

1) preparing a reaction solution: PprI-D91A, final concentration 0.10. mu.M; YDC, final concentration 3.0 μ M; 10 mu L of sample to be detected; 2 mM MnCl ₂ 10 μL；65.7 μL Buffer；ddH ₂ Filling the reaction solution with O to 100 mu L, and reacting at 37 ℃ for 30 min;

the Buffer: 250 mM NaCl, 5% glycerol, 20 mM Tris-HCl pH 7.5;

2) after the reaction is finished, 70 mu L of reaction solution is absorbed and added into a black 386 pore plate, a multifunctional microplate reader is utilized, light with the wavelength of 440 nm is used as exciting light, emitted light with the wavelength of 460 nm-600 nm is scanned, and the ratio of the emitted light with the wavelength of 530 nm to the emitted light with the wavelength of 480 nm is obtainedR _530/480 ；

3) Subjecting the sample toR _530/480 In comparison with blanksR _530/480 Make a ratio to obtainrR _530/480 And obtaining the ssDNA amount contained in the sample to be detected according to a standard curve equation, and accordingly evaluating the damage degree of the DNA in the sample to be detected.

3. The functional protein YDC with fluorescence energy transfer characteristics is characterized by comprising three structural regions of eYFP, Ddr O and eCFP; the amino acid sequence is shown as SEQ ID NO. 1.

4. A protease PprI-D91A is characterized in that the amino acid sequence is shown in SEQ ID NO. 2.

5. A FRET-based functional protein combination comprising PprI-D91A and YDC; the amino acid sequence of the PprI-D91A is shown in SEQ ID NO. 2;

the YDC contains three structural regions of eYFP, DdrO and eCFP; the amino acid sequence is shown as SEQ ID NO. 1;

PprI-D91A can accurately identify the specific sequence of the functional protein YDC and carry out enzyme digestion on the functional protein YDC, the fluorescence energy transfer efficiency of the YDC is reduced after the enzyme digestion reaction, and the addition of ssDNA promotes the enzyme digestion process.

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