CN106905399B - A kind of light control method that double-stranded DNA B-Z conformation is mutually converted - Google Patents

Abstract

The invention provides a light-controlled method for the mutual conversion of double-stranded DNA B-Z conformations, belonging to the fields of molecular biology and nucleic acid chemistry. The method firstly synthesizes short-chain DNA with modified guanine by DNA standard solid-phase synthesis method, and then reversibly controls the mutual conversion of B-type DNA and Z-type DNA by light of 365nm and 254nm wavelengths. The method utilizes light to reversibly control the DNA conformational transformation, and has spatiotemporal controllability in application.

Description

A light-controlled method for the interconversion of double-stranded DNA B-Z conformation

technical field

The invention belongs to the fields of molecular biology and nucleic acid chemistry, and relates to a light-controlled reversible control method for the mutual conversion of B-type DNA and Z-type DNA.

Background technique

DNA has three main conformations: A-DNA, B-DNA and Z-DNA. B-DNA is the right-handed helix model proposed by Watson and Click, which is the conformation of most double-helix DNA under physiological conditions. A-DNA is also a right-handed helix, but the helix is wider and shorter, the major groove is narrower and deeper, and the minor groove is shallower and wider. A-DNA can be obtained by partial dehydration of B-DNA, which is usually formed under non-physiological conditions, while in vivo, A-DNA may be generated from DNA-RNA hybridization or DNA-enzyme complex. Different from the above two, Z-DNA is a left-handed helix, the phosphate groups are distributed in a Z-shape, and there is only one major groove and no minor groove. Alternating purine-pyrimidine sequences, especially d(GC) alternating sequences, tend to form Z-DNA, and if C is methylated, Z-DNA is more likely to be formed. Z-shaped DNA can be recognized by specific proteins, and this process has important implications for the regulation of transcription. B-DNA and Z-DNA have been directly observed in vivo. .

Various secondary structures of DNA can be transformed into each other, and for biological macromolecules, this transformation has important physiological functions. However, scientists still have no understanding of the structure and function of Z-DNA. At present, the methods for studying Z-DNA are to explore the functions of some Z-DNA through Z-DNA binding proteins. Therefore, the light developed by the present invention The control and reversible method of controlling the mutual conversion of B-type DNA and Z-type DNA can realize the manipulation and regulation of biological macromolecules, which is particularly important for the study of the molecular mechanism of related physiological effects.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a method for controlling the B-Z conformational conversion of double-stranded DNA based on modified guanine by light, which has spatiotemporal controllability and little damage to the living body.

The technical scheme of the present invention is:

1), utilize the DNA solid-phase synthesis method, synthesize the short-chain DNA containing a modified guanine such as general formula (I), then form double-stranded DNA after annealing with the complementary strand; wherein, R1 is phenyl, 2-naphthyl or one of 2-fluorenyl;

2) In a 0.2-4 mol/L NaCl solution, illuminate with a wavelength of 365 nm for 10 minutes, and then incubate at 37 degrees for 6 hours, the DNA conformation changes from B to Z; illuminate the DNA in the Z conformation with a wavelength of 254 nm for 10 minutes, then incubate at 37 degrees for 6 hours hours, reversible control of DNA conformation from Z to B can be achieved.

The principle diagram of the present invention is shown in FIG. 1 .

The advantages and beneficial effects of the present invention are:

1. In the present invention, the control of the B-Z reversible transformation of DNA conformation uses illumination, which has spatiotemporal controllability and little damage to the living body, which is conducive to in situ control.

2. The B-Z transformation of DNA conformation in the present invention is reversible, which makes this method very suitable for studying the molecular mechanism of physiological functions related to B-Z transformation.

Description of drawings

Figure 1. Working principle of photo-controlled reversible control of DNA configuration B-Z conversion

Figure 2 CD spectra of double-stranded DNA under different NaCl concentrations

Figure 3. Light-controlled reversible transformation of B-Z configuration

Detailed ways

The features and advantages of the present invention can be further understood from the following detailed description in conjunction with the accompanying drawings. The examples provided are merely illustrative of the methods of the present invention, and are not intended to limit the remainder of the present disclosure in any way.

Example 1

1. DNA synthesis containing modified guanine in the present invention

DNA containing modified guanine was synthesized by DNA solid-phase synthesis method (Angew.Chem.Int.Ed.2008,47,8839–8842), its sequence is CGCXCGCGCGC, X represents structurally modified guanine in Figure 1, its complementary DNA For the general sequence GGCGCGCGCCG. Annealed in 5 mM sodium phosphate buffer pH 7.0 to form double-stranded DNA.

2. The effect of NaCl concentration on the structural transition of B-DNA to Z-DNA

Under the condition of 10 μM DNA double-strand concentration at 10°C, NaCl was added to the solution to the final concentration of 1, 2, 3, 4, and 5 mol/L, respectively, and the CD spectra of the solution were measured, and the results are shown in the figure 2, it can be seen that the absorption value at 295 nm changes from positive to negative, indicating that the DNA conformation changes from B to Z as the NaCl concentration increases.

3. Different wavelengths of light control the reversible transformation of DNA B-Z configuration

In the double-stranded DNA solution, as shown in Figure 3, add NaCl to a final concentration of 3.5 mol/L, and measure the CD spectrum, which is the green line in Figure 3; then irradiate at 365 nm for 10 minutes, and then bath at 37 degrees for 6 hours to measure CD value, as shown by the red line in Figure 3. Then, the wavelength was 254 nm for 10 minutes, and then the temperature was bathed at 37 degrees for 6 hours, and the CD value was measured, as shown by the light blue line in Figure 3. Illumination at 365 nm for 10 minutes, followed by a temperature bath at 37 degrees for 6 hours, and the CD value was measured, as shown by the dark blue line in Figure 3. Then the wavelength of 254nm was illuminated for 10 minutes, and then the temperature was bathed at 37 degrees for 6 hours, and the CD value was measured, as shown by the pink line in Figure 3. The change of the absorption value at 295 nm of the CD spectrum with the illumination indicates that the B-Z transition of DNA conformation can be regulated reversibly by different wavelengths of illumination.

Claims (1)

1. a kind of method mutually converted based on the guanine of modification using light control double-stranded DNA B-Z conformation, feature are existed In including the following steps:

1) DNA solid-phase synthesis is utilized, the short chain DNA of modification guanine of the synthesis containing one such as (I) is then moved back with complementary strand Double-stranded DNA is formed after fire；

2) in the NaCl solution of 3.5 mol/Ls, with wavelength 365nm illumination 10 minutes, then 37 degree warm bath 6 hours, DNA structure As turning to Z by B；With the DNA10 minute of wavelength 254nm illumination Z conformation, then 37 degree warm bath 6 hours, may be implemented DNA conformation The reversible control of B is turned to by Z；