CN114891780A - Method for improving flexibility of DNA - Google Patents

Abstract

The invention relates to a method for improving flexibility of DNA, which comprises the following steps: peroxynitrite is added to a solution system containing DNA to a concentration in the range of 50 μ M to 200 μ M. In the process of researching the DNA damage mechanism caused by peroxynitrite, the invention finds that the low-concentration peroxynitrite can improve the DNA flexibility.

Description

Method for improving flexibility of DNA

Technical Field

The invention relates to a method for improving flexibility of DNA.

Background

DNA plays a decision-making role as a carrier of genetic information in the life process in an organism, and the behavioral influence of DNA even determines various events occurring in the whole organism. Classical theoretical studies suggest that double stranded DNA is a partially flexible polymer. The flexibility of double-stranded DNA is important for prokaryotic gene control, eukaryotic nucleosomes, and the primary behavior of viruses.

Peroxynitrite is a toxic chemical and can diffuse and transfer between cells, and thus in vivo, peroxynitrite is associated with many diseases, including neurodegenerative diseases, cardiovascular diseases, inflammation, cancer, and aging. Under physiological conditions, the anionic portion of peroxynitrite is protonated (pKa ═ 6.8) to form peroxynitrite hydrochloric acid, and due to its strong oxidizing and nitrating properties, many biomolecules are destroyed by peroxynitrite-derived radicals. Peroxynitrite has significant adverse effects, particularly on DNA molecules, and can cause strand breaks by attacking the phosphate backbone and modifying bases of DNA, and cause deoxyribose oxidation, leading to mismatches and mutations.

Disclosure of Invention

The present invention aims at providing a method for improving flexibility of DNA to overcome the defects and shortcomings of the prior art.

The technical scheme adopted by the invention is as follows: a method of increasing the pliability of DNA comprising the steps of: peroxynitrite is added to a solution system containing DNA to a concentration in the range of 50 μ M to 200 μ M.

Preferably, peroxynitrite is added to the DNA-containing solution system to a concentration of 200. mu.M.

Preferably, the solution system containing DNA employs 1mM MgCl ₂ 10mM HEPES, pH 7.5.

Preferably, the DNA is linear DNA.

The invention has the following beneficial effects: in the process of researching the DNA damage mechanism caused by peroxynitrite, the invention finds that the low-concentration peroxynitrite can improve the DNA flexibility.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.

FIG. 1 shows the pBR322 plasmid in deposition buffer [1mM MgCl ] at various concentrations of peroxynitrite ₂ ，10mM HEPES(pH7.5)]AFM imaging in (1). No peroxynitrite (a). (B-H) wherein the concentration of peroxynitrite is 50. mu.M, 100. mu.M, 150. mu.M, 200. mu.M, 300. mu.M, 400. mu.M, or 1mM, respectively;

FIG. 2 shows linear DNA of 4000bp in deposition buffer [1mM MgCl ] at different concentrations of PN ₂ ,10mM HEPES(pH 7.5)]AFM imaging in (1). No peroxynitrite (a). (B-H) peroxynitrite concentrations of 50. mu.M, 100. mu.M, 150. mu.M, 200. mu.M, 300. mu.M, 400. mu.M, 500. mu.M, respectively;

FIG. 3 shows the deposition buffer [1mM MgCl ] containing 500mM ectoine at different concentrations of peroxynitrite (300. mu.M, 400. mu.M, 500. mu.M, 1mM) ₂ 、10mM HEPES(pH7.5)]AFM images of 4000bp linear DNA presented in (1).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

In the following examples, the materials and experimental procedures used are as follows:

first, experimental material

(1) The DNAs used in the experiment were three kinds, pBR322 DNA, 4000bp linear DNA, pBR322 DNA being a typical circular DNA, pBR322 DNA having 4361 base pairs in total and having a length of about 1.5. mu.m, and 4000bp DNA having a length of about 1.3. mu.m, both of which were originally 0.5. mu.g/. mu.L, and were stored in a refrigerator at-20 ℃ by freezing. pBR322 DNA, 4000bp linear DNA was purchased from Thermo Fisher Scientific.

(2) Preparing solution and NaCl and MgCl needed for treatment ₂ 、NiCl ₂ HEPES, ONOOAn (purity 99%) and ectoine (purity 99%) were purchased from Sigma-Aldrich. Peroxynitrite was purchased from Cayman CHEMICAL company in a buffer of 1mM MgCl ₂ 10mM HEPES, pH7.5, and 10mM NiCl as an imaging solution ₂ 10mM HEPES, pH 7.5. The ultrapure water used in the experiments was obtained from a Milli-Q system (Millipore, Billerica, MA, USA).

(3) HEPES buffer solution: the pH of the prepared 100mM Tris solution is adjusted to 7.5 by hydrochloric acid solution, and the prepared 100mM HEPES solution is diluted to 1mM HEPES buffer solution for experiment.

(4) Measuring absorbance at a wavelength of 302nm by using an ultraviolet spectrophotometer before using the ONOOONa, calculating the mother liquor concentration of the experimental ONOOONa, and mixing the prepared solutions with different concentrations by using 100mM HEPES buffer solution, pure water and ONOOONa raw solution according to different volume ratios, wherein all the prepared medicines are placed in a refrigerator for refrigeration for later use in order to ensure the normal service life of the medicines, and particularly, peroxynitrite needs to be frozen and stored in the ultra-low temperature refrigerator at the temperature of-80 ℃.

Second, liquid phase Atomic Force Microscope (AFM) testing process

Liquid phase atomic force microscope sample configuration flow:

(1) 20 μ L of the DNA mixture was deposited on the surface for 2 seconds.

(2) Then 1ml of deposition buffer [1mM MgCl ] was pumped in with a syringe ₂ ，10mM HEPES(pH7.5)]The surface was continuously flushed horizontally with a flow rate of 2 min/ml. During the flushing process, 500. mu.l of liquid was stored in the liquid reservoir at all times, preventing the sample from dehydrating or coming into contact with air.

(3) The liquid cell was tilted 10 °, and then 8ml of deposition buffer [1mM MgCl ] was pumped in with a syringe ₂ ，10mM HEPES(pH7.5)]The surface was continuously flushed horizontally with a flow rate of 2 min/ml.

(4) Finally, 2mL of imaging buffer [10mM NiCl ] was used ₂ ，10mM HEPES(pH7.5)]The samples were rinsed in the same way.

(5) And placing the sample on an atomic force microscope stage for scanning.

Liquid phase AFM operation

1. Firstly, an Atomic Force Microscope (AFM) is adjusted to laser in the air without a sample, so that the laser is shot on a needle point, and a red point is positioned in the center of a laser panel.

2. The well treated liquid cell or BioCell was placed on an Atomic Force Microscope (AFM) stage, the frequency at the highest peak was selected using AC mode, needle drop was suspended when the probe was about to touch the liquid surface, 50 μ L of pipette was dropped onto the probe holder, liquid was allowed to run down the probe holder and wet the probe, at which time the laser panel SUM value suddenly decreased. Then, the needle is manually inserted by a stepping motor in 50 steps, so that the probe is completely immersed in the liquid, and then the laser is adjusted.

3. Opening the CCD, observing the position of the laser on the probe, firstly finely adjusting the position of the laser to enable the laser to be on the probe tip, then adjusting the reflector to enable the SUM value to be maximum, and finally adjusting the laser panel to enable the red spot to be in the center of the panel.

4. The cantilever beam is stabilized in the liquid for 5 minutes, then the laser panel is finely adjusted, and then a proper frequency (taking SNL-10A as an example, the resonance frequency of the laser panel in the liquid is about 16 KHz) and a lower setpoint value (about 0.4) are selected, and a baseline adjustable mode is selected for needling (the setpoint value is automatically reduced when the selection is difficult to needle, and the needling force is increased). After the needle is withdrawn and the needle is moved back once again, the peak value is adjusted, the imaging force is reduced, the higher setpoint value is used, about 0.7, and the scanning speed is 1Hz, so that the imaging can be carried out in the liquid phase. And adjusting specific parameters such as a setpoint value, a gain value, a scanning rate, a Target Amplitude and the like in real time according to the scanning condition.

Thirdly, the principle of quantitative analysis of DNA flexibility change is as follows:

polymers with intrinsic elasticity exhibit rigid behavior on the small length scale, while on the large length scale they resemble flexible chains, which are called semi-flexible polymers, such as DNA, collagen, actin filaments, microtubules, etc. all are semi-flexible. Understanding the effect of molecular rigidity on polymer behavior is of great importance to the study of macromolecular systems. The worm chain model (WLC) is applied to many important biopolymers (e.g. DNA) that can well describe the flexibility and rigidity of biopolymer chains.

In fact, WLC is a continuous limitation of discrete free-wheeling chains. For a discrete chain of length L (L ═ nl, and length L per segment, and n segments), the end distance R is obtained by integrating the tangent unit vector u(s) at the arc length position s

WLC expresses bending deformation caused by thermal fluctuation by a classical elastic energy function (where k is _B T Boltzmann constant, T being absolute constant)

The unit tangent vectors are separated by a curvilinear (arc length) distance d, with an included angle θ, and an elastic bending constant P is related to them by the following relation

The elastic bending constant P, referred to as the persistence length, i.e., the length of memory retention of the initial orientation of the chain, quantifies the stiffness of the long polymer.

The probability distribution of the angular distribution function is obtained by Boltzmann statistical method

The odd moments are all 0 and the even moments are all 0

Then, segmented by the distance d, the cosine average of the angle theta between the polymer segments is

Mean square end distance of polymer

The above formula is a calculation formula of the lasting length of the semi-flexible polymer, when the semi-flexible polymer is DNA, the elastic bending constant P is the lasting length of the DNA, and L is set during calculation _P By mean square terminal distance of DNA<R ² >And the contour length L to obtain the persistence length of DNA.

Example 1:

to examine the effect of peroxynitrite on plasmid DNA, liquid phase AFM images of plasmid DNA on the surface of mica were examined by adding peroxynitrite to a buffer containing pBR322 plasmid DNA to a final concentration of 0. mu.M, 50. mu.M, 100. mu.M, 150. mu.M, 200. mu.M, 300. mu.M, 400. mu.M, 1mM, respectively. As shown in FIG. 1(A), this shows a typical circular conformation of DNA in the absence of peroxynitrite. From FIGS. 1(B) to (H), the corresponding peroxynitrite concentration in the solution was gradually increased from 50. mu.M to 1mM, and it was found that the DNA conformation exhibited a dose-dependent change.

In FIG. 1(A), it was found that plasmid DNA takes a natural loose circular shape and little kinks occur when no peroxynitrite solution is applied. When 50. mu.M peroxynitrite solution was added, it was found from FIG. 1(B) that more than half of the plasmid DNA had kinked and the plasmid DNA became softer. As shown in FIGS. 1(C) and (D), when the concentration of peroxynitrite solution reached 100. mu.M to 150. mu.M, the proportion of plasmid DNA with kinks gradually increased, and in addition, the number of kinks on the individual plasmid DNA also gradually increased, indicating that the degree of DNA distortion also further increased, peroxynitrite caused the DNA to become softer. In FIG. 1(E), almost all plasmid DNA was kinked and the proportion of kinked DNA reached a maximum when the peroxynitrite solution concentration reached 200. mu.M. When the concentration of peroxynitrite reaches 300. mu.M, a part of plasmid DNA in FIG. 1(F) has obvious double-strand breaks, the circular structure is destroyed, and the DNA is in linear segments and is also intertwined with each other. As shown in FIG. 1(G), when the peroxynitrite concentration reached 400. mu.M, all plasmid DNAs were kinked and intertwined, which is similar to a slight DNA aggregation. Increasing the peroxynitrite solution concentration to 1mM in fig. 1(H), the pBR322 plasmid DNA almost completely became linear DNA, and a significant decrease in the amount of DNA deposited on the mica plates was observed under AFM.

Example 2:

to examine the effect of peroxynitrite on linear DNA, the liquid phase AFM images of plasmid DNA on the surface of mica were examined by adding peroxynitrite to a buffer containing 4000bp linear DNA to a final concentration of 0. mu.M, 50. mu.M, 100. mu.M, 150. mu.M, 200. mu.M, 300. mu.M, 400. mu.M, 1mM, respectively.

Linear DNA images of the mica surface obtained by AFM with increasing peroxynitrite concentration are shown in figure 2. FIG. 2(A) shows that in the absence of peroxynitrite, naturally extended DNA can be seen on the surface of fresh mica. When the peroxynitrite concentration was from 50. mu.M to 200. mu.M, as shown in FIGS. 2(B) to (E), DNA double strand breaks were gradually made flexible, but DNA double strand breaks were hardly observed at these several concentrations, and only the DNA bending increased with the increase of peroxynitrite, consistent with the results exhibited by plasmid DNA. In FIG. 2(F), as the peroxynitrite concentration increased to 300. mu.M, portions of DNA were aggregated together and the number of fragments of DNA double strand breaks caused by DNA double strand breaks increased. When the peroxynitrite concentration reached 400. mu.M in FIG. 2(G), almost all of the DNAs were entangled with each other, and a very small amount of DNA fragments caused by double strand breaks were found. In FIG. 2(H), when the concentration of peroxynitrite reached 500. mu.M, the amount of DNA on mica was very small and the DNA fragments were all after double strand break.

In peroxynitrite with different concentrations, the configuration change trends of plasmid DNA and linear DNA with 4000bp are basically the same, but the shape change degree of the plasmid DNA is obviously higher than that of the linear DNA within the peroxynitrite concentration range of 50-200 mu M. To better illustrate the increase in flexibility of linear DNA, a quantitative method was chosen. In order to quantitatively study the influence of peroxynitrite (50-200 μ M) on the flexibility of linear DNA, the mean square end distance of linear DNA fragments in peroxynitrite (50-200 μ M) solution was determined, and the durable length of the linear DNA fragments at different peroxynitrite concentrations was calculated by using a WLC model. The root mean square end distances of the 4000bp linear DNA fragments are shown in Table 1. It can be seen that the root mean square terminal distance of the DNA decreases monotonically with increasing peroxynitrite concentration. For example, the average square root distance from end to end of 4000bp linear DNA is 863nm when there is no peroxynitrite in solution, and it drops sharply to 720nm when there is 100. mu.M peroxynitrite in solution. This indicates that very low concentrations of peroxynitrite have an effect on changes in the mean square end distance of the DNA, and thus on the persistence length of the DNA.

TABLE 14000 bp Linear DNA root mean square terminal distance at different concentrations of peroxynitrite

Table.4-1root-mean-square end-to-end distance of 4000bp linear DNA at different concentrations of PN

According to WLC, the persistence length of a linear DNA can be estimated from its mean square end distance, the relationship between them being as follows (L) _p )：

Wherein<R ² >Is the mean square end distance of the polymer, L is the original contour length of the polymer, L _p Is a lasting length of polymerAnd (4) degree. The mean square end distance of DNA under different concentrations of peroxynitrite (A), (B), (C), (D) and (D)<R ² >) Respectively inserted into the expression to obtain corresponding DNA persistence length L _p See table 2. For example, in a sample with a peroxynitrite concentration of 200. mu.M, the initial persistence length (L) of the naked DNA fragment _p ) From 47nm down to 4 nm.

TABLE 24000 bp Linear DNA persistence at different peroxynitrite concentrations

Table 2Persistence length of 5000bp linear DNA at different concentrations of PN

Analysis of the data by liquid phase AFM, DNA became more flexible as peroxynitrite concentration increased. Low concentrations of peroxynitrite (50. mu.M to 200. mu.M) cause little double strand breaks. When the peroxynitrite concentration reached 300. mu.M, the DNA began to show significant double strand breaks and slight aggregation. If the peroxynitrite concentration is increased further to 400. mu.M, the DNA will become entangled with each other under the action of peroxynitrite and the number of DNA molecules observed on the mica will decrease. With increasing peroxynitrite concentrations, further degradation of the DNA will result in shorter fragments, with a lesser amount of DNA on the mica.

Example 3:

to investigate whether ectoine protected DNA from peroxynitrite, 4000bp linear DNA was mixed in deposition buffer [1mM MgCl ] containing ectoine (500mM) ₂ ，10mM HEPES(pH7.5)]Exposure to peroxynitrite (300. mu.M, 400. mu.M, 500. mu.M, 1 mM). As shown in FIG. 3(A), no aggregation or double strand break occurred in the DNA as compared with FIG. 1 (F). As the concentration of peroxynitrite was increased to 400. mu.M, some DNA was slightly aggregated, but almost no DNA fragments due to double strand breaks were observed. Comparing 3(C) and 2(D), it was found that the length of DNA remained normal, while the amount of DNA observed on the mica plate remained at a very high level under the protection of ectoine. Finally, directly in FIG. 3(D)A high concentration of peroxynitrite up to 1mM still gave a longer line of intact DNA, but due to the peroxynitrite concentration being too high, some of the DNA had coagulated and fragmented DNA fragments around the coagulated DNA appeared to be undergoing fragmentation prior to immobilization on mica plates. This also indicates that the protective effect of ectoine at 500mM seems to have reached a limit in response to 1mM peroxynitrite.

Therefore, ectoine can protect DNA from peroxynitrite-mediated strand breaks, while DNA aggregation is an intermediate state of peroxynitrite-mediated DNA damage conversion.

In conclusion, in the low concentration of peroxynitrite, the DNA takes a more bent conformation from a relaxed state, and gradually forms a mostly compact supercoiled structure from a local supercoiled structure with the increase of the concentration, and the linear DNA also curls from a natural state. Secondly, by measuring the root-mean-square terminal distance and the persistence length, the rule that the DNA damage caused by peroxynitrite is accompanied by the reduction of the root-mean-square terminal distance and the persistence length of the DNA from 0 to 200 mu M is obtained. And the generation of single stranded DNA was first discovered during the damage process using liquid phase atomic force microscopy.

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.

Claims (4)

1. A method for increasing the flexibility of DNA, comprising the steps of: peroxynitrite is added to a solution system containing DNA to a concentration in the range of 50 μ M to 200 μ M.

2. The method of claim 1, wherein the DNA pliability is increased by: peroxynitrite was added to the DNA-containing solution system to a concentration of 200. mu.M.

3. The method for improving flexibility of DNA according to claim 1, wherein: the solution system containing DNA employs 1mM MgCl ₂ 10mM HEPES, pH 7.5.

4. The method of claim 1, wherein the DNA pliability is increased by: the DNA is linear DNA.

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