A nucleic acid dye and its preparation method and application

Abstract

This application discloses a method for preparing and using a nucleic acid dye. The method comprises the following steps: preparing an intermediate compound I by reacting acridine orange and an alkynyl-containing compound; and mixing the intermediate compound I with a diazide compound to produce the nucleic acid dye. The nucleic acid dye prepared in this application has a short synthesis process and simple purification. The nucleic acid dye prepared in this application has good performance, as demonstrated by testing its optical properties, such as ultraviolet absorption and fluorescence, as well as by agarose gel electrophoresis.

Description

Technical Field

[0001] The present application relates to the technical field of nucleic acid dyes, and in particular to a preparation method and application of nucleic acid dyes. Background Art

[0002] Nucleic acids, including DNA and RNA, are an important class of biological macromolecules, and their study is crucial in the field of molecular biology. Because nucleic acids themselves are invisible and lack optical properties such as fluorescence emission, the development of nucleic acid dyes has garnered widespread attention. Nucleic acid dyes primarily detect nucleic acids by interacting with them, altering their optical properties before and after interaction. Commonly used fluorescent dyes, for example, detect nucleic acids through changes in fluorescence intensity. Traditional nucleic acid dyes, such as ethidium bromide (EB), have been gradually replaced by other dyes due to their strong genotoxicity and carcinogenicity.

[0003] Currently, dyes used in gel electrophoresis include the SYBR series, GoldView series, and GelGreen series. GelGreen, with its acridine orange chromophore and dimer structure, has low background fluorescence in aqueous environments, is safe, and has low toxicity, making it widely used. However, the synthetic route for GelGreen dyes is long, making purification difficult. Separation of monomers and dimers using common purification methods, such as column chromatography, is difficult. Therefore, a simpler method for preparing nucleic acid dyes is needed. Summary of the Invention

[0004] The present application provides a method for preparing a nucleic acid dye to solve the above problems.

[0005] The technical solutions adopted in the present invention are as follows:

[0006] A nucleic acid dye, the structural formula of the nucleic acid dye is shown in Formula 1:

[0007]

[0008] Wherein, R1 and R2 are one of alkyl, alkoxy, alkylamino, alkylacyl, alkylsulfonyl, substituted aryl, heterocycle, aromatic heterocycle, and substituted aromatic heterocycle; Y - is an anion, wherein the anion is a halide ion or a sulfonate ion (OTs - ).

[0009] Furthermore, the preparation method of the nucleic acid dye comprises the following steps:

[0010] (1) Acridine orange and an alkynyl-containing compound are added to a solvent at a molar ratio of 1:(2-10), and potassium carbonate is added to obtain a mixed solution 1. The mixed solution 1 is refluxed at 75-85° C. for 45-50 hours. The reflux liquid is collected, filtered, eluted with dichloromethane, and dried to obtain a crude product. The crude product is purified by a silica gel column to obtain an intermediate compound I. The intermediate compound I has a structural formula shown in Formula 2:

[0011]

[0012] (2) The intermediate compound I and the diazide compound are added to a tetrahydrofuran aqueous solution, and copper sulfate pentahydrate and sodium ascorbate are added to prepare a mixed solution 2. The mixed solution 2 is heated to 55-65° C. and allowed to react for 12-18 hours. The mixed solution 2 is then purified by a silica gel column and dried to obtain a nucleic acid dye.

[0013] Furthermore, in step (1), the structural formula of the alkynyl-containing compound is as shown in Formula 3:

[0014]

[0015] Furthermore, in step (2), the chemical structural formula of the diazide compound is:

[0016] N3-(CH2) n -N3 or N3-(CH2CH2O) n -N3, n=1~12.

[0017] Furthermore, in step (2), the mass ratio of the intermediate compound I to the diazide compound is 15-20:3-5.

[0018] Furthermore, the nucleic acid dye is used in gel imaging.

[0019] Furthermore, the nucleic acid dye is used in the preparation of nucleic acid gel imaging dye.

[0020] Furthermore, the nucleic acid dye is used in the preparation of gel imaging reagents.

[0021] The beneficial effects of the embodiments of the present application compared with the prior art are:

[0022] The preparation method of the nucleic acid dye of the present application is simple, the prepared nucleic acid dye has a good application effect in gel imaging, and the nucleic acid dye prepared in the present application is applied in the detection of DNA with low concentration and has the advantage of high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Synthesis route of alkynyl compound 2 in Example 1

[0024] Figure 2 Synthesis route of intermediate compound 3 in Example 1

[0025] Figure 3 Synthesis route of diazide compound 4 in Example 1

[0026] Figure 4 Synthesis route of nucleic acid dye I in Example 1

[0027] Figure 5 The nucleic acid dye I prepared in Example 1 1 H NMR spectrum

[0028] Figure 6 Synthesis route of diazide compound 5 in Example 2

[0029] Figure 7 Synthesis route of nucleic acid dye II in Example 2

[0030] Figure 8 Example 2 Nucleic acid dye I prepared 1 H NMR spectrum

[0031] Figure 9 Nucleic acid dye I prepared in Example 1 of the present invention and its UV absorption spectrum of interaction with nucleic acid.

[0032] Figure 10 Nucleic acid dye I prepared in Example 1 of the present invention and its fluorescence emission spectrum of interaction with nucleic acid.

[0033] Figure 11 Nucleic acid dye II prepared in Example 2 of the present invention and its ultraviolet absorption spectrum of interaction with nucleic acid.

[0034] Figure 12 Nucleic acid dye II prepared in Example 2 of the present invention and its fluorescence emission spectrum of interaction with nucleic acid.

[0035] Figure 13 Image of an agarose gel using GelGreen dye.

[0036] Figure 14 Agarose gel image of nucleic acid dye I prepared in Example 1 of the present invention.

[0037] Figure 15 Agarose gel image of nucleic acid dye II prepared in Example 2 of the present invention. DETAILED DESCRIPTION

[0038] It should be understood that the present invention is not limited to the specific compositions, methods or protocols described herein. In addition, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is defined solely by the claims. The experimental methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources.

[0039] Example 1

[0040] Preparation and characterization of dimer nucleic acid dye I.

[0041] (1) Preparation of alkynyl-containing compound 2

[0042] The specific steps are as follows: 694 mg of bromohexanoic acid and 181 mg of propargylamine were mixed in 50 mL of dichloromethane, 455 mL of triethylamine and 630 mg of EDC were added, and the mixture was stirred at room temperature overnight. After quenching with 100 mL of water, the mixture was extracted with 100 mL of dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and the filtrates were combined and the solvent was removed to obtain a crude product. The crude product was purified on a silica gel column (silica gel:crude product mass ratio of 5:1) using petroleum ether:ethyl acetate = 2:1 as eluent. The product was collected and dried to dryness to obtain 570 mg of the intermediate product as a light yellow solid. This intermediate product was dissolved in 50 mL of acetone, 442 mg of sodium iodide was added, and the reaction was carried out at 75°C for 12 hours. After cooling to room temperature, the product was filtered and dried to dryness to obtain 480 mg of the product as a light yellow solid.

[0043] (2) Preparation of intermediate compound 3

[0044] The specific steps are as follows: 280 mg of acridine orange and 2 g of compound 2 were mixed in 10 mL of acetonitrile, to which 800 mg of potassium carbonate was added. The mixture was heated under reflux for 48 hours. After cooling to room temperature, it was filtered and washed with dichloromethane. The filtrates were combined and the solvent was removed to obtain a crude product. The crude product was purified using a silica gel column (silica gel:crude product ratio ~5:1) with a dichloromethane:methanol ratio of 20:1 as eluent. The product was collected and dried to obtain 501 mg of the product as a red solid.

[0045] (3) Preparation of diazide compound 4

[0046] The specific steps are as follows: 488 mg of 1,2-bis(2-chloroethoxy)ethane was dissolved in 5 mL of DMF, 373 mg of sodium azide and 860 mg of sodium iodide were added, and the mixture was reacted at 80°C overnight. After cooling to room temperature, 50 mL of saturated brine and 50 mL of ethyl acetate were added for extraction. The organic phase was dried over anhydrous sodium sulfate and then spin-dried to yield 610 mg of the product as a yellow oil.

[0047] (4) Preparation of Nucleic Acid Dye I

[0048] The specific steps were as follows: 90 mg of compound 3 and 16 mg of compound 4 were mixed in 10 mL of tetrahydrofuran and 5 mL of water, to which 41 mg of copper sulfate pentahydrate and 65 mg of sodium ascorbate were added. The mixture was heated to 60°C and reacted overnight. After cooling to room temperature, the solvent was evaporated and the product was purified using a silica gel column (silica gel:crude product ratio ~5:1) with a dichloromethane:methanol ratio of 15:1 as eluent. The product was collected and dried in a spin-drying machine to yield 83 mg of the product as a red solid.

[0049] (5) The nucleic acid dye prepared in this embodiment was subjected to NMR testing. 1 H NMR spectrum Figure 1 As shown; the specific NMR data of the dye are: 1 H NMR (400MHz, DMSO-d6) δ8.73 (s, 1H), 8.32 (t, J = 5.7Hz, 1H), 7.93–7.83 (m, 3H), 7.26 (dd,J=9.3,2.0Hz,2H),6.62–6.54(m,2H),4.64(t,J=8.1Hz,2H),4.47(t,J=5.2Hz, 2H),4.28(d,J=5.6Hz,2H),3.74(t,J=5.2Hz,2H),3.48(s,2H),3.26(s,12H),2.16( t, J=7.2Hz, 2H), 1.82 (t, J=7.8Hz, 2H), 1.66 (q, J=7.4Hz, 2H), 1.56 (d, J=8.8Hz, 2H).

[0050] Example 2

[0051] Preparation and Characterization of Dimeric Nucleic Acid Dye II

[0052] (1) Preparation of diazide compound 5

[0053] The specific steps are as follows: 460 mg of diethylene glycol bis(chloroethyl)ester was dissolved in 5 ml of DMF, 360 mg of sodium azide and 800 mg of sodium iodide were added, and the mixture was reacted at 80°C overnight. After cooling to room temperature, 50 ml of saturated sodium chloride solution and 50 ml of ethyl acetate were added for extraction. The organic phase was dried over anhydrous sodium sulfate and then spin-dried to obtain 470 mg of the product as a yellow oil.

[0054] (2) Preparation of Nucleic Acid Dye II

[0055] The specific steps were as follows: 180 mg of compound 3 and 40 mg of compound 5 were mixed in 30 mL of tetrahydrofuran and 15 mL of water, to which 83 mg of copper sulfate pentahydrate and 130 mg of sodium ascorbate were added. The mixture was heated to 60°C and reacted for 12 hours. After cooling to room temperature, the solvent was evaporated and the product was purified using a silica gel column (silica gel:crude product ratio ~5:1) with a dichloromethane:methanol ratio of 15:1 as eluent. The product was collected and dried to obtain 170 mg of the product as a red solid.

[0056] (3) The nucleic acid dye prepared in this embodiment was subjected to NMR testing. 1 H NMR spectrum Figure 2 As shown; the specific NMR data of the dye are: 1 H NMR (400MHz, DMSO-d6) δ8.73 (s, 1H), 8.32 (t, J = 5.8Hz, 1H), 7.95–7.82 (m, 3H), 7.2 6(d,J=9.3Hz,2H),6.58(s,2H),4.65(t,J=8.0Hz,2H),4.47(t,J=5.2Hz,2H),4.28 (d,J=5.6Hz,2H),3.77(t,J=5.2Hz,2H),3.48(t,J=4.6Hz,2H),3.26(s,12H),2.16 (t,J=7.2Hz,2H),1.82(d,J=8.7Hz,2H),1.66(q,J=7.6Hz,2H),1.60–1.50(m,2H).

[0057] The preparation method of compound 3 is the same as that of Example 1.

[0058] Example 3

[0059] UV absorption detection of nucleic acid dyes I and II

[0060] UV absorption spectra of nucleic acid dyes I and II in organic solvents, aqueous phases, and DNA-bound aqueous phases were measured using a UV-visible spectrophotometer. Nucleic acid dyes I and II were dissolved in DMSO to a 10 mM stock solution. 0.5 mL of each stock solution of nucleic acid dyes I and II was added to 1 mL of methanol, 50 mM Tris-HCl (pH 7.4), and 0.1 mM dsDNA, and UV absorbance was measured from 200 nm to 600 nm.

[0061] The results are as follows Figure 9-10As shown in the figure, in methanol solution, the nucleic acid dyes I and II with a final concentration of 5mM have the strongest ultraviolet absorption peaks at 502-504nm, and a shoulder peak at 472nm; in Tris-HCl solution, the maximum absorption peaks of nucleic acid dyes I and II are around 472nm, and there is a shoulder peak at 502-504nm; after adding dsDNA with a final concentration of 0.1mM to Tris-HCl, the peak shapes of nucleic acid dyes I and II change, the main peak at 472nm decreases, and the shoulder peak at 502-504nm increases, indicating that nucleic acid dyes I and II can bind to dsDNA and cause their ultraviolet absorption spectra to change.

[0062] Example 4

[0063] Fluorescence detection of nucleic acid dyes I and II

[0064] Fluorescence emission spectra of nucleic acid dyes I and II, as well as changes in fluorescence upon addition of DNA / RNA, were measured using a fluorescence spectrometer. 10 mM stock solutions of nucleic acid dyes I and II were diluted to 1 mM with DMSO. 1 mL of nucleic acid dye I or II was added to 200 mL of methanol, 50 mM Tris-HCl (pH 7.4), and Tris-HCl containing 0.1 mM dsDNA or 400 ng RNA, respectively. Fluorescence values ​​were measured using the fluorescence spectrometer parameters set to Ex = 472 nm, V = 500 V, and Ex Slit / Em Slit = 5 nm.

[0065] The results are as follows Figure 11-12 As shown, since nucleic acid dyes I and II are dispersed and active in the organic phase, they exhibit maximum fluorescence emission at 528 nm after excitation at 472 nm. However, in aqueous Tris solution, the nucleic acid dyes hydrophobically aggregate into an inactive form, exhibiting almost no fluorescence. Adding RNA to the Tris solution significantly increases fluorescence at 528 nm, and adding 0.1 μM dsDNA further increases fluorescence at 528 nm. These results demonstrate that nucleic acid dyes I and II exhibit virtually no fluorescence in the native state, but their fluorescence is significantly enhanced upon binding to DNA / RNA, demonstrating their excellent performance as nucleic acid dyes.

[0066] Example 5

[0067] Applications of Nucleic Acid Dyes: Agarose Gel Electrophoresis

[0068] Practical application of nucleic acid dyes I and II using agarose gel electrophoresis. 10mM nucleic acid dyes I and II were diluted 10,000-fold with 1*TAE and used to prepare 1% agarose gel. 1ng, 2ng, 5ng, 10ng, 20ng, 50ng, and 100ng of DNA marker were spotted on a 1% agarose gel and electrophoresed at 130V for 30 minutes. GelGreen was used as a reference control. The results are shown below. Figure 13-15As shown, the imaging shows that nucleic acid dyes I and II can detect as low as 20 ng of DNA with clear bands.

[0069] In summary, the nucleic acid dyes prepared in this application have shorter synthesis steps and simpler purification than existing products. The dyes have good performance as determined by testing their optical properties such as UV absorption and fluorescence, as well as by agarose gel electrophoresis.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A nucleic acid dye, characterized in that The structural formula of the nucleic acid dye is shown in any of the following:

2. The method for preparing a nucleic acid dye according to claim 1, wherein The following steps are involved: (1) Acridine orange and an alkynyl-containing compound in a mass ratio of 0.15 to 0.2:1 are added to a solvent, and potassium carbonate is added to obtain a mixed solution 1. The mixed solution 1 is refluxed at 75-85° C. for 45-50 hours. The reflux liquid is collected, filtered, eluted with dichloromethane, and dried to obtain a crude product. The crude product is purified by a silica gel column to obtain an intermediate compound I. The structural formula of the intermediate compound I is shown below: (2) The intermediate compound I and the diazide compound are added to a tetrahydrofuran aqueous solution, and copper sulfate pentahydrate and sodium ascorbate are added to prepare a mixed solution 2. The mixed solution 2 is heated to 55-65°C and allowed to react for 12-18 hours. The mixed solution 2 is then purified by a silica gel column and dried to obtain a nucleic acid dye.

3. The method for preparing a nucleic acid dye according to claim 2, wherein: In step (1), the structural formula of the alkynyl-containing compound is shown in Formula 3: 。 4. The method for preparing a nucleic acid dye according to claim 2, wherein: In step (2), the mass ratio of the intermediate compound I to the diazide compound is 15-20:3-5.

5. Use of the nucleic acid dye according to claim 1 in preparing a nucleic acid gel imaging dye.

6. Use of the nucleic acid dye according to claim 1 in preparing a gel imaging reagent.

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