CN112110992B

CN112110992B - Method for cutting DNA based on flax cyclopeptide A

Info

Publication number: CN112110992B
Application number: CN202011054823.4A
Authority: CN
Inventors: 杨兆琪; 陈维霞; 钱月; 金坚
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-09-28
Filing date: 2020-09-28
Publication date: 2021-11-23
Anticipated expiration: 2040-09-28
Also published as: CN112110992A

Abstract

The invention discloses a method for cutting DNA based on flax cyclic peptide A, belonging to the technical field of biology. The invention utilizes flax cyclic peptide A to cut DNA, under the appropriate condition, the cutting efficiency can reach 100 percent, the cutting is free radical cutting, the irreversible damage can be caused to the DNA, the invention has certain guiding significance for the aspects of exploring the action of plant cyclic peptide and DNA, designing new DNA structure probes and the like, and the invention makes the flax cyclic peptide A possible to be used as a new anti-tumor medicament in the field of medicament research and development.

Description

Method for cutting DNA based on flax cyclopeptide A

Technical Field

The invention relates to a method for cutting DNA based on flax cyclopeptide A, in particular to a novel DNA cutting agent flax cyclopeptide A, belonging to the technical field of biology.

Background

Nucleic acids are important biological macromolecules and important constituent substances of organisms, carriers of genetic information and material bases of gene expression, and have very important roles in activities such as growth, development and reproduction of organisms. The structure of nucleic acid directly affects its function and is associated with carcinogenesis and anticancer. Therefore, how to effectively manipulate the nucleic acid molecules has potential application value not only in the aspect of treating diseases such as cancer, but also in the biotechnology book. Some important information in organisms can be obtained through the research on the nucleic acid cutting agent, the action mechanism of nuclease in the organisms is provided, and the nucleic acid cutting agent has great research value in the medical and biological directions, so the nucleic acid cutting agent is always one of the most active leading fields in biology.

Plant cyclic peptides (plant cyclopeptides) generally refer to a class of cyclic nitrogen-containing compounds formed primarily by amino acid peptide bonds in higher plants. Flax cyclic peptides are generally referred to as hydrophobic cyclic flax peptides extracted from flax seeds. Cyclic peptide a is a nonapeptide consisting of the primary sequence proline-phenylalanine-leucine-isoleucine-leucine-valine. The role of cyclic peptide a in flax is unknown and in current studies these peptides exhibit potent immunosuppressive activity and therefore can be used as template molecules in polypeptide drug design for structural modification or as carriers for active polypeptides. As the plant cyclic peptide has better biological activity and great potential for medicinal use and functional food, the plant cyclic peptide can also obtain greater economic value with the continuous and deep research.

At present, the research at home and abroad mainly focuses on the extraction and separation of plant cyclic peptides, homology and classification of structures and properties and sequences, structural identification, chemical synthesis, biosynthesis, bioactivity, application prospect and the like. However, no studies have been made on the interaction between the cyclic peptide and DNA.

Disclosure of Invention

Aiming at the problems in the prior art, the flax cyclic peptide A (CLA) with the function of cutting DNA is obtained by screening, and can play a good cutting effect under proper conditions. Provides a new idea for cutting DNA.

The present invention provides a method for cleaving DNA, which is characterized by cleaving DNA with a cyclic peptide.

In one embodiment of the present invention, the cyclic peptide has the structural formula:

in one embodiment of the present invention, the reaction time is 2 to 24 hours.

In one embodiment of the present invention, the reaction time is preferably 12 to 24 hours.

In one embodiment of the invention, the metal ions comprise Mg²⁺，Cu²⁺，Zn²⁺Or Ca²⁺。

In one embodiment of the invention, the metal ion is preferably Mg²⁺Or Ca²⁺。

In one embodiment of the present invention, the metal ion concentration is 1 to 100 mM.

In one embodiment of the present invention, the metal ion concentration is preferably 50 to 100 mM.

In one embodiment of the present invention, the pH in the reaction system is 6.0 to 7.0.

In one embodiment of the present invention, the concentration of the cyclic peptide is 5 to 37.5 mg/L.

In one embodiment of the present invention, the concentration of the cyclic peptide is preferably 25 to 37.5 mg/L.

In one embodiment of the present invention, the reaction system contains the target DNA and a BR buffer.

In one embodiment of the present invention, the final concentration of the target DNA is 0.01-0.02mg/mL, and the final concentration of the BR buffer is 100-120 mmol/L.

The invention provides a kit for cutting DNA, which contains cyclic peptide, BR buffer solution and metal ions.

The invention also protects the application of the method or the kit in DNA cutting.

In one embodiment of the invention, the use comprises free radical cleavage of DNA.

In one embodiment of the invention, the application comprises the design of a DNA structure probe, or the auxiliary cutting of cancer gene DNA.

The beneficial technical effects of the invention are as follows:

1. the invention takes plant cyclopeptide A and plasmid pUC19 DNA as main materials, the materials are mixed for reaction, and the reaction result of the cyclopeptide A on the DNA is detected by using a DNA agarose gel electrophoresis technology, so that the interaction between the cyclopeptide A and the DNA is explored, and a theoretical basis is provided for exploring physiological functions of polypeptide and polypeptide drugs. Meanwhile, reaction conditions such as reactant concentration, pH value, reaction time and the like are changed, a reaction result is determined by a gel electrophoresis method, the influence of the reaction conditions on the reaction result is examined, and the optimal condition of the interaction of the flax cyclic peptide A and the DNA is determined, so that the cutting efficiency can reach 100%.

2. The cyclopeptide A has nuclease activity, can cut DNA to a certain extent, is free radical cutting, can cause irreversible damage to the DNA, has certain guiding significance in aspects of exploring the action of plant cyclopeptide and the DNA, designing a new DNA structure probe and the like, and makes the cyclopeptide A possible to be used as a new anti-tumor medicament in the field of medicament research and development.

Drawings

FIG. 1 is a graph showing the results of optimal reaction time for cleavage of DNA by cyclic peptide A; (A) lanes 1-5 in (a) represent cleavage for 2, 4, 8, 12, 24h, respectively;

FIG. 2 is a schematic diagram showing the optimal result of cyclic peptide A cleaving DNA with auxiliary metal ions; (A) lane 1 in (1): no ions are added; lane 2: mg (magnesium)²⁺10 mM; lane 3: ca ²⁺10 mM; lane 4: cu ²⁺10 mM; lane 5: zn²⁺，10mM；

FIG. 3 is a graph showing the optimal concentrations of auxiliary metal ions for cleavage of DNA by cyclic peptide A; (A)) Lane 1 in (1): no ions are added; mg in lanes 2-6²⁺The concentrations are respectively: 100mM, 50mM, 10mM, 5mM, 1 mM;

FIG. 4 is a graph showing the optimal concentration of cyclopeptide A for cleaving DNA at pH 6; (A) lanes 1-6 in (b) are 5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L, 37.5mg/L, respectively, of cyclic peptide A;

FIG. 5 is a graph showing the optimal concentration of cyclopeptide A for cleaving DNA at pH 7; (A) lanes 1-6 in (b) are 5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L, 37.5mg/L, respectively, of cyclic peptide A;

FIG. 6 is a mixed system cyclic peptide A cleavage map; lanes 1-5 are control and experimental groups 1-4, respectively;

FIG. 7 is a ligation map of the cleaved DNA; lanes 1-4 are control and experimental groups 1-3, respectively.

Detailed Description

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the examples, the plasmid pUC19 was used as the target DNA, and the circular DNA was able to more intuitively show the nicks or damages on the DNA strand, so that the plasmid pUC19 was able to be used as a model DNA for verifying the cleavage effect, and the effect shown in the examples can be obtained by acting the cyclic peptide A on other target DNA.

Example 1: optimal reaction time for cleavage of DNA by cyclic peptide A

The optimal reaction time for the cleavage of DNA by cyclic peptide A was determined by gel electrophoresis.

Selection of Mg²⁺As an auxiliary ion, BR buffer solution of pH7.0 was used as a reaction system, and a cyclic peptide A concentration of 25mg/L was used as a reaction concentration, and the optimum reaction time for cleavage of DNA by cyclic peptide A was selected by varying the reaction time.

(1) Preparing a sample: adding 15.6 mu L of BR buffer solution, 2 mu L of target compound solution and 2 mu L of metal ion solution into a 1.5mL EP tube in sequence, fully and uniformly mixing by a vortex mixer, adding 0.4 mu L of 0.5mg/mL pUC19 DNA plasmid after centrifugation, uniformly mixing after numbering and labeling, centrifuging by a centrifuge, sealing and placing in a 37 ℃ incubator for reaction for different times.

(2) Preparation of a 1% agarose gel: 1g of agarose was weighed into a 200mL beaker containing 100mL of a 1 XTAE solution, and the solution was dissolved completely by repeated heating and shaking in a microwave oven until the solution became transparent. After cooling below 60 ℃ at room temperature, 6. mu.L of EB solution (ethidium bromide) was added, mixed well and poured into a horizontal rubber frame and a comb was inserted. And after the agarose gel is completely cooled and solidified, pulling out the baffle and the comb.

(3) Sample adding: and taking out a completely reacted sample, sucking 4mL of 6 XLoading Buffer solution by using a pipette gun, adding into a centrifuge tube, fully oscillating and uniformly mixing by using a turbine mixer, and centrifuging. After mixing, 20. mu.L of the sample was carefully added to the spotting wells by pipetting with a pipette.

pUC19 DNA (0.01 mg/mL); cyclic peptide A (37.5Mg/L), BR buffer (120mmol/L, pH7.0), 37 ℃, 24h, Mg²⁺: 1 mM. Lanes 1-5: 2, 4, 8, 12 and 24 h.

(4) Electrophoresis: and (3) after the positive electrode and the negative electrode of the electrophoresis tank are correctly connected, turning on a switch of the electrophoresis apparatus, electrifying, regulating the voltage to 100V, regulating the current to 260mA, setting the electrophoresis gel running time to 1h, and allowing the pUC19 plasmid DNA to migrate in the agarose gel.

(5) And (3) photographing: the agarose gel was photographed using a gel imaging system.

As shown in FIG. 1, in samples No. 1-5, the cleavage rate of DNA by the mixture gradually increased with the increase of the reaction time, and finally reached about 50%, so that 24h was initially determined to be the optimum reaction time for cleaving DNA by cyclic peptide A.

Example 2: optimal auxiliary metal ions for cyclic peptide A to cut DNA

The effect of ionic species on the cleavage of DNA by cyclic peptide A was examined using gel electrophoresis.

Four divalent metal ions are selected as auxiliary ions, namely Mg²⁺，Cu²⁺，Zn²⁺And Ca²⁺。

(1) Preparing a sample: adding 15.6 mu L of BR buffer solution with the pH value of 7.0, 2 mu L of target compound solution and 2 mu L of metal ion solution into a 1.5mL EP tube, fully and uniformly mixing by using a vortex mixer, adding 0.4 mu L of 0.5mg/mL pUC19 DNA plasmid after centrifugation, uniformly mixing after numbering and labeling, centrifuging by using a centrifuge, sealing and placing in a 37 ℃ incubator for reaction for 24 hours.

pUC19 DNA: 0.01mg/mL, BR buffer (120mmol/L, pH7.0), 24h at 37 ℃, cyclic peptide A25 mg/L. Lane 1: a DNA control; lane 2: mg (magnesium)²⁺10 mM; lane 3: ca ²⁺10 mM; lane 4: cu ²⁺10 mM; lane 5: zn²⁺，10mM。

As shown in FIG. 2, in the BR buffer system at pH7.0, the cleavage rate of sample No. 1 by cyclic peptide A alone was only about 33.6% without adding metal ions, and it was found that the effect of cyclic peptide A in cleaving DNA was not significant without the aid of metal ions. From the results of samples No. 2-5, the addition of metal auxiliary ions has a better promotion result on the cleavage of DNA by cyclic peptide A. Ca²⁺、Cu²⁺And Zn²⁺The effect of the three ions on the DNA cleavage by the cyclic peptide A is improved to a certain extent, but all the ionsMg of less than the same concentration²⁺In Mg²⁺Under the assistance of ions, the cleavage rate of the cyclic peptide A on the DNA is increased to 70.7 percent, which is the highest cleavage rate of four metal ions, so that Mg can be considered as²⁺The best auxiliary metal ion for cutting DNA by the cyclic peptide A.

Example 3: optimal concentration of auxiliary metal ions for cleavage of DNA by cyclic peptide A

As can be seen from example 2, Mg²⁺The optimum auxiliary metal ion for cleaving DNA for cyclic peptide A was investigated, thereby finding the optimum ion concentration.

Selection of Mg²⁺Are 100mM, 50mM, 10mM, 5mM and 1mM, respectively.

(1) Preparing a sample: adding 15.6 mu L of BR buffer solution with the pH value of 7.0, 2 mu L of target compound solution and 2 mu L of metal ion solution into a 1.5mL EP tube, fully and uniformly mixing by a vortex mixer, adding 0.4 mu L of 0.5mg/mL pUC19 DNA plasmid after centrifugation, uniformly mixing after numbering and labeling, centrifuging by a centrifuge, sealing and placing in a 37 ℃ incubator for reaction for 24 hours.

pUC19 DNA: 0.01mg/mL, BR buffer (120mmol/L, pH7.0), 37 ℃, 24h, cyclic peptide A25 mg/L. Lane 1: a DNA control; lanes 2-6, Mg²⁺：100mM，50mM，10mM，5mM，1mM。

The cutting results are shown in FIG. 3, where Mg when added can be seen at the optimum ion concentration²⁺When the ion concentration reaches 100mM, the cutting effect is optimal, the cutting rate is 100%, and the DNA is completely cut into bulk without circular nick DNA and linear DNA. Therefore, it was preliminarily concluded that the concentration of Mg was 100mM²⁺Is the optimum ion concentration for the cyclic peptide A to cleave the DNA.

Example 4: cleavage of DNA with Cyclic peptide A in an Environment of pH6

The concentration of cyclic peptide A with the best DNA cleavage effect when the pH value is 6 is detected and selected by using a gel electrophoresis method.

Selection of Mg²⁺As an auxiliary ion, BR buffer solution with pH value of 6.0 was used as the reaction system. The concentrations of cyclic peptide A were 5, 10, 15, 20, 25, 37.5mg/L, respectively.

(1) Preparing a sample: adding 15.6 mu L of BR buffer solution, 2 mu L of target compound solution and 2 mu L of metal ion solution into a 1.5mL EP tube in sequence, fully and uniformly mixing the solution through a vortex mixer, adding 0.4 mu L of 0.5mg/mL pUC19 DNA plasmid after centrifugation, uniformly mixing the mixture after numbering and labeling, centrifuging the mixture by using a centrifuge, sealing the mixture and placing the mixture in a 37 ℃ incubator for reaction for 24 hours.

pUC19 DNA: 0.01Mg/m L, BR buffer (120mmol/L, pH6.0), 37 deg.C, 24h, Mg²⁺: 1 mM; lanes 1-6: mg/L: 5, 10, 15, 20, 25, 37.5;

As shown in FIG. 4, it can be seen from samples No. 1 and No. 2 that the concentration of cyclic peptide A is too low or even lower than the amount of DNA in the reaction system, and thus the cleavage effect is very poor in these two groups. When the concentration of the cyclic peptide A reaches 15mg/L or more, the cutting rate of the DNA can be greatly improved to more than 70%, so that the obvious cutting effect can be realized only when the concentration of the cyclic peptide A is higher than that of the DNA. Furthermore, it can be seen from the figure that when the concentration of cyclic peptide A reached 37.5mg/L, the cleavage rate of DNA had reached substantially 100%, and the presence of supercoiled DNA had not been observed in the image, so that the optimum reaction concentration of cyclic peptide A was 37.5mg/L at pH 6.0.

Example 5: cleavage of DNA with Cyclic peptide A in an Environment of pH7

The concentration of cyclic peptide A with the best DNA cleavage effect when the pH value is 7 is selected is detected by using a gel electrophoresis method.

Selection of Mg²⁺As an auxiliary ion, BR buffer solution with pH value of 7.0 was used as a reaction system. The concentrations of the cyclic peptides A were 5, 10, 15, 20, 25, and 37.5mg/L, respectively, and the optimum concentration of the cyclic peptide A for DNA cleavage in the BR buffer system at pH7.0 was found, and the optimum pH for the cyclic peptide A reaction was selected as compared with the reaction system at pH 6.0.

(1) Preparing a sample: adding 15.6 mu L of BR buffer solution, 2 mu L of target compound solution and 2 mu L of metal ion solution into a 1.5mL EP tube in sequence, fully and uniformly mixing by a vortex mixer, adding 0.4 mu L of 0.5mg/mL pUC19 DNA plasmid after centrifugation, uniformly mixing after numbering and labeling, centrifuging by a centrifuge, sealing and placing in a 37 ℃ incubator for reaction for 24 hours.

pUC19 DNA: 0.01Mg/m L, BR buffer (120mmol/L, pH7.0), 37 deg.C, 24h, Mg²⁺: 1mM lanes 1-6: mg/L: 5, 10, 15, 20, 25, 37.5;

As shown in FIG. 5, the cleavage results were substantially the same as those in the reaction system at pH6.0, but the concentration of cyclic peptide A was relatively low in the early stage, so that the cleavage effect on DNA was relatively poor, and the cleavage efficiency was maintained at about 20% and was lower than that in the case of the same concentration at pH 6.0. When the concentration of cyclic peptide A reached 20mg/L, the cleavage effect began to increase, substantially maintaining above 40%, while when the maximum concentration was reached it was found that the DNA had been substantially completely cleaved and substantially no supercoiled DNA was observed. Although the cleavage rate has not reached 100%, it has been considered that a concentration of 37.5mg/L is the optimum reaction concentration for achieving the maximum reaction concentration under the current realization conditions. Furthermore, as compared with the experiment of pH6.0, the cleavage effect of the cyclic peptide A on DNA is basically consistent when the reaction concentration of the cyclic peptide A reaches 37.5 mg/L. However, pH7.0 is relatively close to the physiological environment of human body, and therefore, reaction concentrations of pH7.0 and 37.5mg/L were used as optimum reaction conditions.

Example 6: verification of DNA cleavage mechanism

(1) Cutting experiment

And adding a free radical scavenger into the reaction system to observe the change of the cutting result so as to judge the mechanism of cutting the DNA by the cyclic peptide. Wherein the pH value of the BR buffer solution is 7.0, the DNA concentration is 50 mu g/mL, the DMSO and the n-butanol are both analytically pure, and the KI solution concentration is 1 g/mL. After the reagents are added in sequence according to the following table, the mixture is shaken, centrifuged and placed in a constant temperature water bath box to react for 24 hours at the constant temperature of 37 ℃.

TABLE 1 amount of reagent added

As shown in FIG. 6, the effect of using the cyclic peptide mixture under 2 normal reaction conditions to cut DNA can be clearly seen, and the DNA is not only opened but also completely cut into a diffuse band. The group with DMSO added was compared with group 2, and it was found that although most of DNA was still cleaved, the cleavage effect was significantly decreased. The group added with n-butanol showed no significant difference in DNA cleavage effect from the control, and the amount of linear DNA was smaller than that of the control. By the group of potassium iodide, the DNA was not even cut at all, and only a small amount of the circular DNA was cut with nicks. The three groups of results show that the addition of the free radical scavenger has obvious inhibition effect on the DNA cutting effect of the cyclic peptide, even the result of basically no cutting is generated, and the phenomenon can basically judge that the plant cyclic peptide cutting machine is supposed to be a free radical cutting mechanism. The reason that the cutting effect is different and weaker than that of the control group after the addition of the free radical scavenger is probably that the effect of the scavenger in the third group is different, the cutting effect is weaker than that of the control group, namely that the effect of removing the free radicals by potassium iodide and n-butyl alcohol is better, the original free radicals in the system are removed together, and the DNA is basically not broken.

(2) Connection experiment

The sample of the ligation reaction needs to be added with 2 mu L of DNA ligase buffer solution and 7.9 mu L of water in sequence, after shaking, centrifuging and mixing evenly, 0.5 mu L of T4 DNA ligase is added, and the mixture is placed at the constant temperature of 37 ℃ for 2 h.

TABLE 2 amount of reagent added

As shown in FIG. 7, the DNA control No. 1 and the set of the normal reactions without the addition of DNA T4 ligase No. 2 show that the DNA had been completely cleaved. The sample No. 3 is a group purified after normal reaction, and it can be seen that the final result is basically consistent with the sample No. 2 and has no difference, and thus, the purification hardly has any influence on the final result of the experiment. The sample No. 4 is a group which is added with DNA T4 ligase after purification and reacts for 2h, but the reaction result is almost the same as that of the sample No. 2 and the sample No. 3 from the final result, namely the reaction result is not different from that of the sample No. 3, namely the reaction result is not influenced by the addition of the DNA T4 ligase, namely the broken DNA chain cannot be reconnected after the addition of the DNA T4 ligase, and the free radical cutting mechanism is met, namely the free radical cutting mechanism is adopted for the plant cyclic peptide to cut the DNA.

Example 7: preparation of DNA cleavage kit

Mixing BR buffer solution (120mmol/L, pH7.0), cyclic peptide A (37.5Mg/L), and metal ions (such as Mg)²⁺，Cu²⁺，Zn²⁺And Ca²⁺Preferably Mg²⁺Or Ca²⁺The concentration of the metal ion is preferably 50 to 100 mM.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for cleaving DNA, which comprises cleaving DNA with a cyclic peptide, wherein the method is not intended for the diagnosis and treatment of a disease; the structural formula of the cyclopeptide is as follows:

2. the method of claim 1, wherein the reaction time is 2 to 24 hours.

3. The method of claim 1, wherein metal ions are also added to the reaction environment; the metal ions comprise Mg²⁺，Cu²⁺，Zn²⁺Or Ca²⁺。

4. The method according to claim 1, wherein the metal ion concentration is 1 to 100 mM.

5. The method of claim 1, wherein the concentration of cyclic peptide is 15-37.5 mg/L.

6. A kit for cutting DNA is characterized in that the kit contains cyclic peptide, BR buffer solution and metal ions; the structural formula of the cyclopeptide is as follows:

7. the kit of claim 6, wherein the metal ion comprises Mg²⁺，Cu²⁺，Zn²⁺Or Ca²⁺。

8. Use of the method of any one of claims 1 to 5, or the kit of claim 6 or 7, for cleaving DNA; such applications are not aimed at the diagnosis and treatment of diseases.

9. Use according to claim 8, wherein said use comprises free radical cleavage of DNA.

10. The use of claim 8, wherein said use comprises the design of a DNA structure probe.