A nucleic acid gel dye based on an aggregation-induced emission material and uses thereof

Abstract

This invention discloses a nucleic acid gel dye based on aggregation-induced emission materials and its applications. The nucleic acid gel dye based on aggregation-induced emission materials includes AIE molecules with any structure of formula (1) and formula (2). The nucleic acid gel dye based on aggregation-induced emission materials of this invention is used for common gel imaging staining in nucleic acid electrophoresis. It is applicable to both agarose gel electrophoresis and polyacrylamide gel electrophoresis, and is particularly suitable for blue light imaging. The nucleic acid gel dye based on aggregation-induced emission materials of this invention combines good staining effect, high sensitivity, short staining time, and compatibility with all existing dye excitation channels, making it an ideal choice for use with laser scanners. It effectively solves the defects of existing nucleic acid dyes, such as poor staining effect and low sensitivity when used in polyacrylamide gel electrophoresis, and especially addresses the problem of the relatively limited excitation channels of existing dyes.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to a nucleic acid gel dye based on aggregation-induced emission materials and its applications. Background Technology

[0002] Nucleic acid electrophoresis is a relatively simple, rapid, and highly sensitive tool for studying the properties of nucleic acids. It is an indispensable component of techniques such as nucleic acid probes, nucleic acid amplification, and sequence analysis. Nucleic acid electrophoresis is generally divided into two types: agarose gel electrophoresis and polyacrylamide gel electrophoresis. Agarose is a chain-like polysaccharide prepared from agar. Many agarose molecules intertwine to form rope-like agarose bundles, creating a large-pore gel. Different concentrations of agarose can form gels with different molecular sieve pore sizes, used to separate nucleic acid fragments of different molecular weights. It can distinguish DNA fragments differing by 100 bp, making it suitable for samples with larger molecular weights, such as bacterial or viral genome sequences and large nucleic acid fragments. Polyacrylamide gel is a highly dense polymer with molecular sieving, concentration, and charge effects, making it most effective for separating small DNA fragments (5 bp-500 bp). The resolution of agarose gel is lower than that of polyacrylamide gel. For electrophoresis requiring particularly high resolution, especially for fragments differing by only a few bp, polyacrylamide gel electrophoresis should be chosen.

[0003] Commonly used nucleic acid electrophoresis dyes include EB, SYBR Green, SYBR Gold, GelRed, and GelGreen. Different nucleic acid dyes have different excitation wavelengths, and their excitation wavelength ranges are relatively small, resulting in limited compatibility with a single channel in gel imaging systems. Currently, gel imaging instruments commonly used for nucleic acid electrophoresis fall into two categories: ultraviolet gel imaging systems and blue light gel imaging systems (or blue light gel cutters). Prolonged ultraviolet irradiation poses certain hazards to nucleic acids (DNA) and to laboratory personnel; therefore, future nucleic acid dyes will increasingly lean towards safe and non-toxic blue light nucleic acid dyes.

[0004] Existing nucleic acid dyes, such as GelGreen, are safe, non-toxic, and highly sensitive; however, their staining effect in polyacrylamide gel electrophoresis is poor, and the staining time is long. This is because polyacrylamide gels are highly dense three-dimensional polymers. This dense polymer structure makes it difficult for fluorescent dyes, used as nucleic acid gel dyes, to penetrate when separating nucleic acid molecules of different sizes and concentrations, resulting in poor nucleic acid staining. Furthermore, existing dyes have many limitations in application; for example, for LAMP amplification products, many existing nucleic acid dyes tend to produce dragging patterns, making it difficult to present clearly visible trapezoidal bands.

[0005] In summary, existing technologies have two main problems:

[0006] The first problem is that the excitation wavelengths of existing dye molecules are relatively narrow, resulting in a limited number of excitation channels in instruments and restricting the use of dyes. Furthermore, some dyes do not excite well under blue light, or cannot be excited at all under blue light, and prolonged exposure to ultraviolet light poses certain risks to nucleic acids (DNA) and to laboratory personnel.

[0007] The second problem is that polyacrylamide gels are highly dense three-dimensional polymers. This high-density polymer structure makes it difficult for many existing fluorescent dyes used as nucleic acid gel dyes to penetrate when separating nucleic acid molecules of different sizes and concentrations, resulting in poor nucleic acid staining effects and excessively long staining times. Therefore, developing a nucleic acid dye that is suitable for both agarose gel electrophoresis and high-density polyacrylamide gel electrophoresis and can be excited under blue light has great market potential. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a nucleic acid gel dye based on aggregation-induced emission materials and its applications. The nucleic acid gel dye based on aggregation-induced emission materials of this invention is applicable to both agarose gel electrophoresis and polyacrylamide gel electrophoresis, and is particularly suitable for blue light imaging.

[0009] The technical solution of the present invention is as follows:

[0010] A nucleic acid gel dye based on aggregation-induced emission material, including AIE molecules with any one of the structures of formula (1) and formula (2);

[0011]

[0012] In equation (1), Q1 and Q2 are each independently selected from one of the following structures:

[0013]

[0014] Q3 is selected from one of the following structures:

[0015]

[0016] Q4 - Each is an anion independently;

[0017] Q5, Q6, Q7, Q8, Q9, Q 10 Each is independently selected from one of -H, -NH2, -COOH, -CN, -CHO, -OH, -CH3;

[0018] n1 is an integer greater than or equal to 1;

[0019] Where * indicates the replacement position;

[0020] In equation (2), Y1, Y2, Y3, and Y4 are each independently selected from one of the following structures:

[0021]

[0022] Y5 - Each is an anion independently;

[0023] Y6, Y7, Y8, Y9, Y 10 Each is independently selected from one of -H, -COOH, -CN, -CHO, -OH, -CH3;

[0024] n2 is an integer greater than or equal to 1;

[0025] In this context, * indicates a replacement position.

[0026] Preferably, in equation (1), Q1 and Q2 have the same structure; in equation (2), Y1 and Y2 have the same structure, or Y2 and Y3 have the same structure.

[0027] Preferably, the nucleic acid gel dye based on aggregation-induced emission material comprises an AIE molecule with any of the following structures:

[0028]

[0029] Preferably, the nucleic acid gel dye based on aggregation-induced emission material comprises an AIE molecule with any of the following structures:

[0030]

[0031] More preferably, Q6, Q7, Q9, Y9, Y 10 Each is independently selected from one of -H, -COOH, -OH, and -CH3.

[0032] Preferably, the nucleic acid gel dye based on aggregation-induced emission material comprises an AIE molecule with any of the following structures:

[0033]

[0034] Preferably, n1 is an integer from 1 to 4 (1, 2, 3, 4); n2 is an integer from 1 to 4 (1, 2, 3, 4).

[0035] More preferably, n1 is 2 or 3; n2 is 2 or 3.

[0036] Preferably, the anion is selected from F - Cl- ,Br - I - NO2 - NO3 - PF6 - CF3SO3 - ClO4 - One of them;

[0037] Preferably, the nucleic acid gel dye based on aggregation-induced emission material further includes a solvent;

[0038] More preferably, the solvent includes at least one selected from dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC), water, and methanol.

[0039] Preferably, the nucleic acid gel dye based on aggregation-induced emission material comprises any one of the following chemical formulas I-IV:

[0040]

[0041] The above-mentioned applications of nucleic acid gel dyes based on aggregation-induced emission materials in agarose gel electrophoresis or polyacrylamide gel electrophoresis.

[0042] Preferably, the excitation channel for agarose gel electrophoresis or polyacrylamide gel electrophoresis is blue light.

[0043] This invention provides a method for applying the aforementioned nucleic acid gel dye in agarose gel electrophoresis, comprising the following steps:

[0044] Weigh agarose powder and place it in a container, add electrophoresis buffer and the nucleic acid gel dye described in this invention, heat until the agarose powder is fully dissolved, cool and let it solidify to form a gel; place the agarose gel in an electrophoresis tank, load the sample, turn on the current, and after electrophoresis is completed, place the agarose gel on a gel imaging system to obtain a gel chromatogram.

[0045] Preferably, the electrophoresis buffer is selected from TAE solution or TBE solution; the nucleic acid gel dye is a solution with a concentration of 8-15 mM.

[0046] More preferably, the volume ratio of the electrophoresis buffer to the nucleic acid gel dye is 10000:1.

[0047] This invention provides a method for applying the aforementioned nucleic acid gel dye in polyacrylamide gel electrophoresis, comprising the following steps:

[0048] The nucleic acid gel dye described in this invention is added to a sodium chloride solution to prepare a staining solution. The polyacrylamide gel after electrophoresis is immersed in the staining solution. After staining, the polyacrylamide gel is placed on a gel imaging system to obtain a gel chromatogram.

[0049] Preferably, the sodium chloride solution is a 0.1M NaCl aqueous solution; the nucleic acid gel dye is an 8-15mM solution.

[0050] More preferably, the volume ratio of the sodium chloride solution to the nucleic acid gel dye is 10000:3.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The nucleic acid gel dye based on aggregation-induced emission materials of this invention, compared with existing products, not only maintains high sensitivity in agarose gels but also maintains the same high sensitivity in polyacrylamide gel electrophoresis, and significantly shortens the staining time, producing clear and bright bands within 5 minutes. In particular, it broadens the excitation wavelength of the dye molecules, thus exhibiting excellent compatibility, allowing it to adapt to the excitation channels of all existing dyes, making it an ideal choice for use with laser scanners, especially for blue light imaging. Gel electrophoresis results obtained under the same conditions show that the compound of this invention, as a nucleic acid dye, has extremely high resolution for nucleic acids, even clearly distinguishing ladder-shaped products such as LAMP, thus ensuring both the sensitivity and accuracy of electrophoresis tests. Attached Figure Description

[0053] Figure 1 Images of fluorescent dyes formula I, II, III, IV, and Gelred on agarose gel.

[0054] Figure 2 Images of fluorescent dyes formulas V and VI on agarose gel.

[0055] Figure 3 Images of fluorescent dyes formulas VII and VIII on agarose gel.

[0056] Figure 4 Images of fluorescent dye Formula II and SYBR Gold on agarose gel.

[0057] Figure 5 Images of fluorescent dye Formula II and SYBR Gold on polyacrylamide gel.

[0058] Figure 6 This is an image of fluorescent dye type II on a polyacrylamide gel. Detailed Implementation

[0059] The technical solutions in the embodiments of the present invention will be clearly and specifically described below with reference to the accompanying drawings, but the implementation methods and protection scope of the present invention are not limited to the following embodiments.

[0060] The structures of the fluorescent molecules used in the examples, namely chemical formulas I, II, III, and IV, are as follows:

[0061]

[0062]

[0063] The structures of the comparative fluorescent molecules used in the examples, namely chemical formulas V, VI, VII, and VIII, are as follows:

[0064]

[0065]

[0066] Example 1:

[0067] Application of the dye of this invention

[0068] (I) Formulation of the dye of the present invention

[0069] Weigh out the fluorescent molecule solid of Formula I, add biological-grade anhydrous DMSO, and dissolve thoroughly to obtain a nucleic acid gel dye with a concentration of 10 mM. Using the same method, prepare nucleic acid gel dyes with a concentration of 10 mM for Formulas II, III, IV, V, VI, VII, and VIII respectively.

[0070] (II) The specific experimental method for applying the dye of the present invention in agarose gel electrophoresis is as follows:

[0071] 1. Weigh 0.3g of agarose powder into an Erlenmeyer flask;

[0072] 2. Add 30 mL of 1×TAE (TAE electrophoresis buffer was purchased from Beijing Kechuang Xinda Technology Co., Ltd., 50×TAE electrophoresis buffer was diluted 50 times to obtain 1×TAE solution) to the conical flask, and add 3 μL of the nucleic acid gel dye prepared in step (I).

[0073] 3. Heat in a microwave oven until boiling, until the agarose is fully dissolved, and the final solution volume is 30 mL;

[0074] 4. Simultaneously, measure an equal volume of agarose solution in another conical flask, add 3 μL of the control group's 10000×Biotium Gelred (41005) dye solution, and boil until fully dissolved. Pour the solutions into the prepared gelation apparatus.

[0075] 5. Allow to gel at room temperature for 1 hour (or allow to stand at room temperature for 20 minutes until the gel stops moving, then place it in a 4°C refrigerator for 15 minutes to allow it to solidify).

[0076] 6. Place the solidified agarose gel into an electrophoresis tank filled with electrophoresis buffer (1×TAE), and sequentially load 5 μL of nucleic acid marker (existing conventional substance) into the control group gel (Gelred) and the experimental group gel (dyed in this invention) as usual.

[0077] 7. After loading the sample, turn on the electrophoresis tank switch, set the voltage to 120V and the time to 30min, and start the electrophoresis process;

[0078] 8. After electrophoresis, remove the agarose gel and place it on different channels of the instrument (Gelred, 488nm) and the blue light gel cutter. Take pictures and save the images to the corresponding folders. The results are as follows: Figure 1 , Figure 2 and Figure 3 In each figure, the order of samples from left to right is: (1) 1Kb plus DNA; (2) DL 15000bp; (3) DL 5000bp; (4) DL 2000bb; (5) DL 100bp.

[0079] result:

[0080] from Figure 1 It can be seen that:

[0081] ① Compared with the control group (right column, Gelred), the dyes of this invention (left 4 columns, Formula I, Formula II, Formula III, Formula IV) have a wider range of applicable channels and greater compatibility, provided that the bands are clear;

[0082] ② Compared with the control group, the dye of the present invention can clearly separate large fragment bands without the phenomenon of diffuse tailing;

[0083] ③ This invention demonstrates that the dye can effectively solve the problem of single channel in existing products, is highly compatible, and can replace gel dyes such as Gelred without changing any imaging system. It is suitable for gel imaging and detection systems that previously used SYBR Green or SYBR Gold as nucleic acid dyes, making it an ideal choice for use with laser scanners.

[0084] from Figure 2 and 3 It can be seen that:

[0085] This invention compares the performance of fluorescent dyes with chemical formulas V and VI in gel imaging, noting that while fluorescent dyes with chemical formulas VII and VIII can produce brighter bands under blue light, their background color is darker and the color distribution of the gel block is uneven, affecting the observation and analysis of electrophoresis results.

[0086] 9. Using the same gel imaging method as described above, the dye (Formula II) of this invention was compared with SYBR Gold to obtain a gel spectrum (blue light excitation channel, 488nm excitation channel), see [link to relevant documentation]. Figure 4 The bands represent LAMP amplification products. It can be seen that, compared to SYBR Gold, the dye of this invention has higher resolution.

[0087] (III) The specific experimental method for applying the dye of the present invention in polyacrylamide gel electrophoresis is as follows:

[0088] 1. After assembling the gel preparation plate of the gel device, add water and let it stand for 20 minutes. Check the sealing of the device. If the sealing is good, pour out the water and prepare two 15wt% non-denatured PAGE gels.

[0089] 2. In a 50mL test tube, add 5.86mL of H2O, 10mL of 30wt% Acrylamid, and 4mL of 5×TBE (TBE electrophoresis buffer was purchased from Shanghai Zeye Biotechnology Co., Ltd., and 10×TBE electrophoresis buffer was diluted 2 times to obtain 5×TBE solution). Finally, add 0.14mL of 10wt% AP and 0.014mL of TEMED and mix well (H2O, 30wt% Acrylamid, and 5×TBE should be prepared in advance, while 10wt% AP and TEMED should be added before gel casting).

[0090] 3. Add the mixed non-modified glue to completely fill the glue-making plate with liquid, and insert the comb (note the front and back sides, with the smooth side of the comb closer to the thicker side of the glue-making plate inside).

[0091] 4. Allow the gel to solidify at room temperature for 1 hour to allow it to fully solidify.

[0092] 5. Load 5 μL of nucleic acid marker;

[0093] 6. The electrophoresis buffer is 1×TBE (TBE electrophoresis buffer was purchased from Shanghai Zeye Biotechnology Co., Ltd., 10×TBE electrophoresis buffer was diluted 10 times to obtain 1×TBE solution), the voltage is set to 100V, the electrophoresis time is 60min, and the electrophoresis is started.

[0094] 7. Prepare the dyeing solution: Take two containers and add 100 mL of 0.1 M NaCl solution (10 mL of 1 M NaCl mixed with 90 mL of deionized water) to each container. Add 30 μL of the control dye SYBR Gold (10000×) and the dye prepared in step (I) (Formula II) to the 100 mL solution respectively, and mix them thoroughly.

[0095] 8. After electrophoresis, carefully remove the gel from the glass plate and stain it in the staining solution for 30 min (SYBR Gold) and 5 min (Formula II), respectively.

[0096] 9. After staining, place the gel on a gel imaging system (488nm excitation channel), take pictures, and save the images to the corresponding folders;

[0097] 10. Results are shown below. Figure 5 The sample concentrations in the lanes from left to right are: (1) 25 ng; (2) 10 ng; (3) 5 ng; (4) 2.5 ng; (5) 1 ng; (6) 500 pg; (7) 250 pg; (8) 100 pg.

[0098] result:

[0099] Compared with the control group, the dye of the present invention can stain PAGE gel with a short staining time and high sensitivity, with a sensitivity as low as 100 pg while maintaining clear bands.

[0100] 11. After performing polyacrylamide gel electrophoresis on the LAMP amplification products as described above, stain with the dye of this invention (Formula II) for 5 min as described above. Using the same blue light and gel imaging device (488nm excitation channel) method (repeated three times), obtain the PAGE gel chromatography, see [link to PAGE gel chromatography]. Figure 6 .

[0101] As can be seen, the dye of this invention only needs to be soaked in polyacrylamide gel for 5 minutes, which greatly shortens the experimental time and is an ideal substitute for silver dyeing.

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A nucleic acid gel dye based on aggregation-induced emission materials, characterized in that, Including any aggregation-induced emission molecule with the structure shown in the following chemical formula; Chemical Formula I Chemical Formula II Chemical Formula III Chemical formula IV.

2. The nucleic acid gel dye based on aggregation-induced emission material according to claim 1, characterized in that, The aggregation-induced emission molecule has the structure shown in chemical formula I or chemical formula II.

3. The nucleic acid gel dye based on aggregation-induced emission material according to claim 1, characterized in that, The aggregation-induced emission molecule has the structure shown in chemical formula III or chemical formula IV.

4. The nucleic acid gel dye based on aggregation-induced emission material according to any one of claims 1-3, characterized in that, It also includes a solvent; the solvent includes at least one of dimethyl sulfoxide, dimethylformamide, dimethylacetamide, water, and methanol.

5. The application of the nucleic acid gel dye based on aggregation-induced emission material as described in any one of claims 1-4 in agarose gel electrophoresis.

6. The application of the nucleic acid gel dye based on aggregation-induced emission material as described in any one of claims 1-4 in polyacrylamide gel electrophoresis.

7. The application according to claim 5 or 6, characterized in that, The excitation channel for agarose gel electrophoresis or polyacrylamide gel electrophoresis is blue light.

8. The application according to claim 5, characterized in that, The procedure includes the following steps: weighing agarose powder and placing it in a container, adding electrophoresis buffer and the nucleic acid gel dye according to any one of claims 1-4, heating until the agarose powder is fully dissolved, cooling it to solidify it into a gel; placing the agarose gel in an electrophoresis tank, loading the sample, applying electricity, and after electrophoresis is completed, placing the agarose gel on a gel imaging system to obtain a gel chromatogram.

9. The application according to claim 8, characterized in that, The electrophoresis buffer is selected from TAE solution or TBE solution; the nucleic acid gel dye is a solution with a concentration of 8-15 mM.

10. The application according to claim 6, characterized in that, Includes the following steps: Add the nucleic acid gel dye according to any one of claims 1-4 to a sodium chloride solution to prepare a staining solution. Immerse the polyacrylamide gel after electrophoresis in the staining solution. After staining, place the polyacrylamide gel on a gel imaging system to obtain a gel chromatogram.

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