CN111004229A

CN111004229A - Coumarin derivative for specifically identifying and distinguishing DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), and preparation method and application thereof

Info

Publication number: CN111004229A
Application number: CN201911308324.0A
Authority: CN
Inventors: 孙远强; 李朝辉; 曾华金; 王霞; 杨冉; 屈凌波; 孟红敏
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2019-12-18
Filing date: 2019-12-18
Publication date: 2020-04-14
Anticipated expiration: 2039-12-18
Also published as: CN111004229B

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a coumarin derivative for specifically identifying and distinguishing DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), and a preparation method and application thereof. The coumarin derivative is a probe 1, and the structural formula is

Or probe 2 has the structural formula

. The probe of the invention has simple synthesis, high yield, good specificity and high sensitivity, and can specifically identify and distinguish DNA and RNA. Of DNA and RNAThe development of the specific differential small molecule fluorescent probe provides a new idea and method.

Description

Coumarin derivative for specifically identifying and distinguishing DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), and preparation method and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a coumarin derivative for specifically identifying and distinguishing DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), and a preparation method and application thereof.

Background

DNA is a double helix structure and belongs to genetic material. RNA is generally single-stranded and is not used as genetic material. RNA is a single strand formed by transcription by taking a strand of DNA as a template and using a base complementary pairing principle, has the main function of realizing the expression of genetic information on protein, and is a bridge in the process of transforming the genetic information into phenotype. Unlike DNA, RNA is generally a single-stranded long molecule that does not form a double helix structure, but many RNAs also require base-pairing to form certain secondary or tertiary structures for biological function. RNA has essentially the same base pairing rules as DNA, although G-U can be paired in addition to the A-U, G-C pair. In the aspect of viruses, many viruses only use RNA as their sole genetic information carrier (unlike cellular organisms which generally use double-stranded DNA as a carrier). Among the RNAs, mRNA is a template for synthesizing a protein, the content is transcribed according to DNA in the nucleus, tRNA is a recognizer of a base sequence (i.e., a genetic codon) on mRNA and a transporter of amino acids, and rRNA is a component constituting a ribosome, which is a site of work for protein synthesis. DNA is a high molecular polymer, and a DNA solution is a high molecular solution, has high viscosity and can be dyed into green by methyl green. DNA absorbs ultraviolet rays (260 nm), and the content of DNA can be measured by utilizing this characteristic. When nucleic acids are denatured, the absorbance increases, called the hyperchromic effect. In contrast, other proteins can only specifically bind to a specific DNA sequence. Most of the research on such proteins has focused on various transcription factors that regulate transcription. Each of these proteins binds to a specific DNA sequence and activates or inhibits gene transcription of a sequence located in the vicinity of a promoter. Transcription factors act in two ways, the first of which binds, directly or via other intermediary proteins, the RNA polymerase responsible for transcription, which in turn binds the promoter and initiates transcription. The second is combined with enzymes that specifically modify tissue proteins on the promoter, which alters the difficulty of contacting the DNA template with the polymerase.

DNA and RNA are both present in eukaryotic cells, have similar basic compositions, and are mostly chain-shaped in structure, so that the difficulty of distinguishing the DNA from the RNA is still achieved. Many excellent fluorescent small molecule probes have been developed for the detection of DNA, but probes for RNA-specific detection are still lacking. The probes for detecting RNA reported so far generate large interference when DNA exists in the system, so that it is necessary to design fluorescent probes for detecting RNA and distinguishing from DNA.

In the research, coumarin is used as a basic structure as a fluorescent group, and on the basis, a group with positive charges is connected as a functional group for specifically targeting mitochondria; in addition, the probe 2 introduces thiophene heterocycle at the 4-position on the basis of the structure of the probe 1, and the main reason for changing the design is that the whole molecular structure of the probe 2 tends to a curved surface due to steric effect after the heterocycle is introduced, and the probe 2 can have better specific binding capacity with the RNA structure because the RNA structure is easy to fold and bend by itself. Therefore, it is very interesting to develop probes that specifically recognize and detect RNA by the structural design of the probe molecule.

Disclosure of Invention

The invention provides a coumarin derivative for specifically identifying and distinguishing DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) as well as a preparation method and application thereof, wherein coumarin is used as a basic structure as a fluorescent group, and on the basis, a group with positive charges is connected as a functional group of a specific targeting mitochondrion; in addition, the probe 2 introduces thiophene heterocycle at the 4-position on the basis of the structure of the probe 1, and the main reason for changing the design is that the whole molecular structure of the probe 2 tends to a curved surface due to steric effect after the heterocycle is introduced, and the probe 2 can have better specific binding capacity with the RNA structure because the RNA structure is easy to fold and bend by itself.

The technical scheme of the invention is realized as follows:

coumarin derivative for specifically identifying and distinguishing DNA and RNA, wherein the coumarin derivative is a probe 1, and the structural formula is shown in the specification

Or probe 2 has the structural formula

。

The preparation steps of the probe 1 are as follows: 7-Diethylaminocoumarin-3 aldehyde and 2, 3-dimethylbenzothiazole ammonium iodide were dissolved in EtOH. The reaction mixture was refluxed with stirring for 12 hours, then cooled to room temperature, and the precipitate was collected with a filter and then subjected to silica gel column Chromatography (CH)₂Cl₂MeOH, 50:1 v/v) to give a dark blue solid, probe 1.

The molar ratio of the 7-diethylamine coumarin-3 aldehyde to the 2, 3-dimethylbenzothiazole ammonium iodide is 1: (1-1.5).

The preparation steps of the probe 2 are as follows: 4- (2-thienyl) -7-diethylaminoazo-3-aldehyde and 2, 3-dimethylbenzothiazole ammonium iodide were dissolved in 10 mL of ethanol solution. The reaction mixture was refluxed with stirring for 12 hours, then cooled to room temperature, and the precipitate was collected with a filter and then subjected to silica gel column Chromatography (CH)₂Cl₂MeOH, 50:1 v/v) to give a dark blue solid, probe 2.

The molar ratio of the 4- (2-thienyl) -7-diethylamine azo-3-aldehyde to the 2, 3-dimethylbenzothiazole ammonium iodide is 1: (1-1.5).

The coumarin derivative is applied to the aspect of specifically identifying and distinguishing DNA and RNA, and comprises the following steps: dissolving coumarin derivatives in DMSO to prepare probe stock solution, adding into PBS buffer solution containing cells to be tested, incubating for a period of time, eluting for 2-3 times with PBS buffer solution, and performing fluorescence spectrum test at excitation wavelength of 550/560 nm.

The concentration of coumarin derivative in the probe stock solution is 2 mM, and the concentration of PBS buffer solution is pH7.4 and 10 mM.

The volume ratio of the probe stock solution to the PBS buffer solution was 1: 200.

The invention has the following beneficial effects:

1. the coumarin is used as a basic structure as a fluorescent group, has the advantages of good photostability, high biocompatibility and the like, and is connected with a group with positive charges as a functional group of a specific targeting mitochondrion; in addition, the thiophene heterocycle is introduced into the probe 2 at the 4-position on the basis of the structure of the probe 1, and the main reason for changing the design is that the whole molecular structure of the probe 2 tends to a curved surface due to steric effect after the heterocycle is introduced, and the probe 2 can have better specific binding capacity with the RNA because the RNA polymerization degree is lower and the RNA is easy to fold and bend by itself. When a cell feasibility experiment is carried out, the probe 1 can well target the cell nucleus, and the probe 2 can specifically target the cell nucleus nucleolus; corresponding in vitro spectral titration experiments and molecular simulation experiments are combined, and the two experiments can prove that the specific targeting discrimination of DNA and RNA can be realized. The probe in the invention has simple synthesis steps, is convenient to operate, can quickly target subcellular organelles-mitochondria in cells, simultaneously realizes the recognition of RNA in the mitochondria, has good specificity, and is convenient and feasible in design concept.

2. Coumarins are used as basic structures as fluorescent groups, and the fluorescent material has the advantages of good photostability, high quantum yield, easiness in modification and the like; in a confocal imaging experiment, the probe can well target subcellular organelles-mitochondria and cell nucleus in cells; the probe of the invention has simple synthesis, high yield, good specificity and high sensitivity, and can specifically identify and distinguish DNA and RNA. The method provides a new idea and method for the development of the small molecule fluorescent probe for the specificity distinction of DNA and RNA.

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 described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

Fig. 1 is a graph of fluorescence excitation vs. emission spectra for 2

μ M probes

1 and 2 in a PBS buffered (10 mM, pH = 7.4) system.

FIG. 2 is a fluorescent titration curve of 2

μ M probes

1 and 2 reacted with DNA and RNA in a PBS buffered (10 mM, pH = 7.4) system.

FIG. 3 is a molecular simulation experiment of probes 1 (a) and 2 (b) with DNA in a PBS buffered (10 mM, pH = 7.4) system with corresponding binding constants of-10.83 kcal/mol (a) and-5.82 kcal/mol (b).

FIG. 4 is a molecular simulation experiment of probes 1 (a) and 2 (b) with RNA in a PBS buffered (10 mM, pH = 7.4) system with corresponding binding constants of-7.30 kcal/mol (a) and-7.55 kcal/mol (b).

FIG. 5 shows the relative cell viability of the serum-containing DMEM medium after 24h incubation of the cells with two probes at different concentrations.

FIG. 6 is an imaging experiment at 550nm excitation when 1 μ M probes 1 (left) and 2 (right) were incubated with fixed cells in colorless DMEM medium.

FIG. 7 is a co-localization experiment of RNA in targeted mitochondria when 1 μ M probes were incubated with cells in colorless DMEM medium, where the green channel is the commercial dye and the red channel is the emission wavelength collection channel of the fluorescent probe molecule itself.

Fig. 8 is a nuclear magnetic hydrogen spectrum of the probe 1.

Fig. 9 is a nuclear magnetic hydrogen spectrum of the probe 2.

FIG. 10 shows nuclear magnetic carbon spectrum of probe 1.

FIG. 11 is a nuclear magnetic carbon spectrum of probe 2.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

The preparation principle of the coumarin derivative probe 1 of the embodiment is as follows:

the preparation method comprises the following specific steps: 7-Diethylaminocoumarin-3 aldehyde (76 mg, 0.31 mmol) and 2, 3-dimethylbenzothiazole ammonium iodide (98 mg, 0.34 mmol) were dissolved in 10 mL of ethanol solution. The reaction mixture was refluxed with stirring for 12 hours, then cooled to room temperature, and the precipitate was collected with a filter and then subjected to silica gel column Chromatography (CH)₂Cl₂MeOH, 50:1 v/v) to give a dark blue solid (135 mg, 84% yield) as probe 1, of the formula:

(Probe 1), the nuclear magnetic hydrogen spectrum of probe 1 is shown in FIG. 8, and the nuclear magnetic carbon spectrum is shown in FIG. 10.

¹H NMR (600 MHz, DMSO-d6) δ 8.61 (s,1H), 8.38 (d, J =7.4 Hz, 1H),8.22 (d, J =8.5 Hz, 1H) ,8.05 (q, J₁=15.4 Hz, J₂= 12.6 Hz ,2H), 7.84 (t, J =7.56 Hz, 1H), 7.75 (t, J =7.74 Hz, 1H), 7.58 (d, J =9.06 Hz, 1H), 6.87 (dd,J₁= J₂=2.2 Hz, 1H), 6.67 (d, J =2.04 Hz, 1H), 4.23 (s,3H) ,3.54 (q, J =7.08Hz, 4H), 1.17 (t, J =7.02 Hz, 6H)；¹³C NMR (150 MHz, DMSO-d6 ): d=172.15,160.01, 157.66, 153.77, 148.85, 144.80, 142.48, 132.26, 129.74, 128.51,127.93, 124.60, 116.98, 112.47, 111.83, 111.37, 109.38,96.96,55.38,45.16,36.39, 12.91 ppm。

Example 2

the preparation method comprises the following specific steps: 7-Diethylaminocoumarin-3 aldehyde (76 mg, 0.31 mmol) and 2, 3-dimethylbenzothiazole ammonium iodide (89.4 mg, 0.31 mmol) were dissolved in 10 mL of ethanol solution. The reaction mixture was refluxed with stirring for 12 hours, then cooled to room temperature, and the precipitate was collected with a filter and then subjected to silica gel column Chromatography (CH)₂Cl₂MeOH, 50:1 v/v) to give a dark blue solid, probe 1.

Example 3

the preparation method comprises the following specific steps: 7-Diethylaminocoumarin-3 aldehyde (76 mg, 0.3 mmol) and 2, 3-dimethylbenzothiazole ammonium iodide (129.7 mg, 0.45 mmol) were dissolved in 10 mL of ethanol solution. The reaction mixture was refluxed with stirring for 12 hours, then cooled to room temperature, and the precipitate was collected with a filter and then subjected to silica gel column Chromatography (CH)₂Cl₂MeOH, 50:1 v/v) to give a dark blue solid, probe 1.

Example 4

The preparation principle of the coumarin derivative probe 2 in the embodiment is as follows:

the preparation method comprises the following specific steps:

4- (2-thienyl) -7-diethylaminoazo-3-aldehyde (100 mg, 0.3 mmol) and ammonium 2, 3-dimethylbenzothiazole iodide (98 mg, 0.34 mmol) were dissolved in 10 mL of ethanol. The reaction mixture was refluxed with stirring for 12 hours, then cooled to room temperature, and the precipitate was collected with a filter and then subjected to silica gel column Chromatography (CH)₂Cl₂MeOH, 50:1 v/v) to give a dark blue solid (130 mg, 71% yield) as probe 2, with a nuclear magnetic hydrogen spectrum as shown in FIG. 9 and a nuclear magnetic carbon spectrum as shown in FIG. 11.

¹H NMR (600 MHz, DMSO-d6) δ 8.31 (d, J =6.0 Hz ,1H), 8.38 (q, J₁=3.8Hz, J₂=2.1 Hz, 2H), 8.22 (dd, J =2.22,2.16 Hz, 1H) ,7.81 (t, J=7.08 Hz, 1H),7.71 (t, J=7.56 Hz, 1H), 7.55 (d, J =15.12 Hz, 1H), 7.44 (t, J =4.08 Hz, 2H),7.16 (d, J =9.9 Hz, 1H), 6.67 (dd, J =2.4 Hz, 1H), 6.75 (d, J =2.04 Hz ,1H) ,4.18 (s, 3H), 3.53 (q, J₁=6.96 Hz, J₂=4.0 Hz, 4H) , 1,16 (t, J =7.08 Hz,6H)；

¹³C NMR (150MHz,DMSO-d6)δ172.43,159.35,156.62,153.59,152.98,142.61,142.31,132.56,

131.74,129.80,128.67,128.53,127.80,124.70,117.02,113.33,111.99,111.59,109.89,97.12,45.17,36.37,12.95 ppm。

Example 5

the preparation method comprises the following specific steps:

4- (2-thienyl) -7-diethylaminoazo-3-aldehyde (100 mg, 0.3 mmol) and ammonium 2, 3-dimethylbenzothiazole iodide (86.5 mg, 0.3 mmol) were dissolved in 10 mL of ethanol. The reaction mixture was refluxed with stirring for 12 hours, then cooled to room temperature, and the precipitate was collected with a filter and then subjected to silica gel column Chromatography (CH)₂Cl₂MeOH, 50:1 v/v) to give a dark blue solid, probe 2.

Example 6

the preparation method comprises the following specific steps:

4- (2-thienyl) -7-diethylamineazo-3-al (100 mg, 0.3 mmol) and ammonium 2, 3-dimethylbenzothiazole iodide (129.7 mg, 0.45 mmol) were dissolved in 10 mL of ethanol. The reaction mixture was stirred at reflux for 12 hours, then cooled to room temperature and usedThe precipitate was collected by a filter and then subjected to silica gel column Chromatography (CH)₂Cl₂MeOH, 50:1 v/v) to give a dark blue solid, probe 2.

Examples of the effects of the invention

1. Fluorescence spectra of probe 1 and probe 2 in PBS buffered (10 mM, pH = 7.4) system

Preparing a PBS (10 mM) buffer solution at pH = 7.4; probe 1 and probe 2 were weighed, dissolved in DMSO, and a 2 mM probe stock solution was prepared accurately. After 2 mL of PBS buffer solution is added into the cuvette, 2 muL of probe storage solution with the concentration of 2 mM is added, and fluorescence excitation and emission spectrum test are carried out. As shown in FIG. 1, the excitation wavelength of the probe 1 prepared in example 1 was 560 nm, and the excitation wavelength of the probe 2 prepared in example 2 was 550nm, both of which had an emission wavelength of 650 nm.

2. Molecular simulation experiment of interaction between probe 1 and probe 2 with DNA and RNA

To further support the above fluorescence test results, we performed molecular simulation experiments relating two probes to DNA and RNA, respectively (molecular docking experimental conditions: the crystal structures of DNA and RNA are taken from the protein database RCS ProteinDataBank, DNA is coded as 453D (calf thymus DNA), RNA is coded as 1FFZ (23 s RNA is from Haloarcula marimortui), the probe structures are drawn by the Chem3D Pro14.0 software, the optimal conformations of the probe compounds are calculated using the lowest energy. the probe molecules and DNA and RNA are simulated using AutoDock 4.0. the molecular docking is performed in a cubic lattice whose center position is the center of the DNA molecule, coordinates are 15.115, 20.786 and 8.718, the length, width and height of the cubic lattice are 60, 60 and 80, respectively, the coordinates of the center position of RNA are 67.775, 110.6 and 92.858, the length, width and height of the cubic lattice are 60, the lattice spacing is 0.6, and the number of ligands generated is 100, the remaining parameters are performed by default). As can be seen from the results of the correlation simulation in FIG. 3, for DNA, the binding energies of probes 1 (a) and 2 (b) corresponding thereto are-10.83 kcal/mol (a) and-5.82 kcal/mol (b), respectively; and for RNA, see FIG. 4, which shows the corresponding binding energies of-7.30 kcal/mol (a) and-7.55 kcal/mol (b), respectively, and the results after the above simulation are consistent with the in vitro fluorescence titration results, indicating that it is feasible to design two small molecule fluorescent probes for specifically distinguishing DNA from RNA. As can be known from relevant literature, the action types of different types of substances and DNA or RNA mainly comprise covalent binding and non-covalent binding, the covalent binding is mainly a specific action mode of a complex and the action of the complex, the non-covalent binding is divided into three action modes of insertion, groove and electrostatic binding, wherein the groove is a specific action mode of small molecules and DNA or RNA, so that the conclusion that the fluorescent signal response of the designed molecules to the target is caused by the fact that the compound is inserted into the groove of the target molecules to cause the change of the planar structure configuration of the compound, and further cause the change of the intensity of the fluorescent signal can be deduced. It can be seen from the simulation that the DNA molecule is a double-helix molecule with a certain rigid planar structure, the RNA molecule is relatively curled in structure, and as for the specific structure of the designed probe, the thiophene heterocycle is introduced at the 4-position of the probe 2 on the basis of the structure of the probe 1, and the main reason for the design change is that the whole molecular structure of the probe 2 tends to a curved surface due to steric hindrance effect after the heterocycle is introduced, and the RNA is a single-strand structure and is easy to fold and bend by itself, so the probe 2 can have a good specific binding ability with the RNA molecule. Therefore, by combining the intrinsic properties of the target detection substance's own structure and the three-dimensional configuration of the fluorescent probe molecule at the lowest energy, it can be fully demonstrated which probe 2 has a higher affinity for RNA than probe 1.

3. Viability of cells after co-incubation of probe 1 and probe 2 with cells.

Firstly, preparing HepG-2 cell suspension, then adding cell suspension with the volume of 100 muL and the density of 2000/well into a 96-well plate, placing the cell suspension in an incubator to be cultured to an adherent state, adding probe 1 and probe 2 solutions (1, 2,4,6, 8, 10 muM) with different concentrations, incubating the cell suspension with the cell for 24h, then determining cytotoxicity by an MTT method, replacing the culture medium in each well with a fresh culture medium, adding 10 muL of 5 mg/mL MTT reagent into each well, after incubating for 4h at 37 ℃, sucking out the culture medium, adding purple crystal dye dissolved by 100 muL of LDMSO solution into each well, detecting the absorbance value of each well at 470 nm by using a microplate reader (TECAN SPAGELLAN), and finally calculating the cell survival rate by the formula = [ (As-)/(Ac-Ab) ] x 100% (As: Ab: control well; blank well: Ab Further evaluation of the toxicity of the probe was carried out. As can be seen from FIG. 5, even if the concentration of the two probe molecules reaches 10 μ M, the cell survival rate can still reach more than 80%, and the toxicity of the molecules on cells is proved to be small, so that the molecules can be applied to the research of cell biological imaging.

4. Confocal imaging after co-incubation of probes with cells

And washing the adherent cells with PBS three times, diluting the probe solution to the required concentration by using a colorless DMEM culture medium, adding the diluted probe solution into a dish containing the cells, incubating for a certain time, eluting with PBS three times again, and carrying out confocal imaging. From the literature, there are a large number of DNAs and RNAs in cells to carry the relevant genetic material, but the distribution of the two in the cells is different: DNA is predominantly present in the nucleus and RNA is predominantly present in the cytoplasm and nucleoli of the nucleus. When a cell is used for carrying out a relevant verification experiment, firstly, the cell is fixed by 4% paraformaldehyde for 30 min, then solutions (with final concentration of 1 [ mu ] M) of two probe molecules and the fixed cell are respectively incubated together, then confocal imaging is carried out under the conditions that the excitation wavelength is 550nm and the emission and collection wave band is 570 nm-700 nm, the result is shown in figure 6 (the length represented by the size of a scale is 30 [ mu ] M), and it can be seen from the left figure that the light-emitting position of the probe 1 is mainly enriched in the cell nucleus and the fluorescence in the cytoplasm is weak after the probe 1 and the cell are incubated together; from the right panel, it can be seen that the primary luminescence sites of probe 2 after incubation with cells are nucleolus and cytoplasm of the nucleus. Therefore, the above experimental results prove that specific region and recognition of DNA and RNA can be realized through the slight difference of the structures between the two coumarin derivatives.

When carrying out the intracorporeal RNA of mitochondria and co-locating the experiment, add commercial mitochondrial dye MitoTracker Deep Red FM (100 nM) and cell and incubate 15 min together earlier, later add probe solution and continue to incubate 20 min, elute the cubic with PBS after finishing, add colorless DMEM culture medium at last. When confocal imaging is carried out, different excitations are respectively used, and different emission wave bands are collected so as to avoid mutual interference between the two. The results of the experiment are shown in figure 7, and it can be seen that there is no interference of the excitation and emission of the commercial dye with the probe itself, and the imaging results when the two are incubated together show that the probe molecule is able to target well to RNA within the mitochondria.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A coumarin derivative which specifically recognizes and distinguishes DNA from RNA, characterized in that: the coumarin derivative is a probe 1, and the structural formula is

Or probe 2 has the structural formula

。

2. The method for preparing coumarin derivatives capable of specifically recognizing and distinguishing DNA from RNA as claimed in claim 1, wherein said probe 1 is prepared by the steps of: dissolving 7-diethylamine coumarin-3 aldehyde and 2, 3-dimethyl benzothiazole ammonium iodide in EtOH for reaction, refluxing and stirring the reaction mixture for 12 hours, then cooling to room temperature, collecting precipitates by using a filter, and then purifying by using a silica gel column chromatography to obtain a dark blue solid, namely the probe 1.

3. The method of claim 2, wherein: the molar ratio of the 7-diethylamine coumarin-3 aldehyde to the 2, 3-dimethylbenzothiazole ammonium iodide is 1: (1-1.5).

4. The method for preparing coumarin derivatives capable of specifically recognizing and differentiating DNA and RNA according to claim 1, wherein the probe 2 is prepared by the steps of: dissolving 4- (2-thienyl) -7-diethylamine azo-3-aldehyde and 2, 3-dimethyl benzothiazole ammonium iodide in an ethanol solution for reaction, refluxing and stirring the reaction mixture for 12 hours, then cooling to room temperature, collecting precipitates by using a filter, and then purifying by using a silica gel column chromatography to obtain a dark blue solid, namely the probe 2.

5. The method of claim 4, wherein: the molar ratio of the 4- (2-thienyl) -7-diethylamine azo-3-aldehyde to the 2, 3-dimethylbenzothiazole ammonium iodide is 1: (1-1.5).

6. The use of a coumarin derivative as claimed in claim 1 for the specific recognition and discrimination of DNA and RNA by the steps of: dissolving coumarin derivatives in DMSO to prepare probe stock solution, adding into PBS buffer solution containing cells to be tested, incubating for a period of time, eluting for 2-3 times with PBS buffer solution, and performing fluorescence spectrum test at excitation wavelength of 550/560 nm.

7. Use according to claim 6, characterized in that: the concentration of coumarin derivative in the probe stock solution is 2 mM, and the concentration of PBS buffer solution is pH7.4 and 10 mM.

8. Use according to claim 7, characterized in that: the volume ratio of the probe stock solution to the PBS buffer solution was 1: 200.