Nucleic acid fluorescent dye and preparation and application thereof

文档序号：183250 发布日期：2021-11-02 浏览：42次中文

阅读说明：本技术 一种核酸荧光染料及其制备和应用 (Nucleic acid fluorescent dye and preparation and application thereof ) 是由吴鹏王彦莹董真李显明杨琴于 2021-08-20 设计创作，主要内容包括：本发明涉及荧光染料领域,特别是涉及一种新型的核酸荧光染料。本发明提供一种荧光染料,所述荧光染料的结构式如式I所示,其中,A～(-)为氯离子、溴离子或碘离子。本发明对染料进行结构改造,提供另一种灵敏度高、稳定性好、原料易得、合成简单、序列偏好低、对PCR的抑制性小的化合物。(The invention relates to the field of fluorescent dyes, in particular to a novel nucleic acid fluorescent dye. The invention provides a fluorescent dye, which has a structural formula shown as a formula I, wherein A ‑ Is chloride ion, bromide ion or iodide ion. The invention carries out structural modification on the dye and provides another compound which has high sensitivity, good stability, easily obtained raw materials, simple synthesis, low sequence preference and small inhibition on PCR.)

1. A fluorescent dye, wherein the structural formula of the fluorescent dye is shown as formula I:

wherein A is^-Is chloride ion, bromide ion or iodide ion.

2. The method of preparing a fluorescent dye according to claim 1, wherein the method comprises: stirring and reacting the compound shown in the formula II and the monomer M in a solvent at room temperature for 6-24 hours to obtain a mixture, and then purifying the mixture to obtain the fluorescent dye shown in the formula I; wherein the monomer M is propylamine substituted by alkylamino;

3. the method of claim 2, wherein the equivalent ratio of the compound of formula II to monomer M is 1: 1-1: 5.

4. the method of claim 2 or 3, wherein the monomer M is selected from the group consisting of: n, N-dimethyl-1, 3-diaminopropane or 3-diethylaminopropylamine.

5. The method of claim 2 or 3, wherein the solvent is ethanol or dichloromethane.

6. A method for preparing a fluorescent dye according to any one of claims 2 to 5, wherein the stirring time is 6 to 12 hours.

7. The method for preparing a fluorescent dye according to any one of claims 2 to 6, wherein the compound represented by the formula II is prepared by the following method:

firstly, preparing the compound shown as the formula III

Then preparing the compound shown as the formula IV

Carrying out reflux reaction on a compound shown as a formula IV and phosphorus oxychloride in a solvent 1 at 65-85 ℃ to obtain a compound shown as a formula VWherein the solvent 1 is selected from at least one of acetonitrile, dichloroethane or dimethylformamide;

in a solvent 2, a compound shown as a formula V and a compound shown as a formula III are mixed in an equivalent ratio of 1: 1-1: 3 mixing at room temperature to obtain a compound shown as a formula II; the solvent 2 is ethanol or dichloromethane.

8. The method of claim 7, wherein the compound of formula III is prepared by the following method: and (3) carrying out methylation reaction on the 2-methylmercaptobenzothiazole by using a methylation reagent to obtain the compound shown in the formula III.

9. The method of claim 7, wherein the compound of formula iv is prepared by the following method: preparing a compound shown in a formula IV by reacting 2-hydroxy-4-methylquinoline with iodobenzene or phenylboronic acid under the action of a catalyst; wherein the catalyst is copper acetate or copper powder.

10. Use of a fluorescent dye in qPCR analysis or gel electrophoresis imaging, the fluorescent dye being as defined in claim 1 or prepared by the method of any one of claims 2 to 9.

Technical Field

The invention relates to the field of fluorescent dyes, in particular to a novel nucleic acid fluorescent dye.

Background

Fluorescent dye refers to a fluorescent substance that absorbs excitation light of a certain wavelength and emits light of a specific wavelength. The fluorescent dye is combined with a substance which does not emit fluorescence in a certain mode, and the chemical signal of the substance can be selectively converted into a fluorescent signal which is easy to be measured by an analysis instrument, so that the fluorescence detection of a specific target is realized. The detection method has the advantages of high sensitivity, good selectivity, capability of realizing visualization, real-time and nondestructive online monitoring in a living body and the like, and is widely applied to the field of biological analysis.

Nucleic acids (such as DNA and RNA) are carriers of genetic information of living bodies, and are often used as target analytes because of their large amount of information carried and their relatively stable intracellular content. Currently, fluorescence detection is the mainstream method for achieving nucleic acid analysis. In addition to labeling fluorescent dyes by covalent coupling, non-covalently bound "luminescent probes" (i.e., nucleic acid fluorescent dyes) are the most advantageous means for qualitative and quantitative analysis of nucleic acids. The fluorescent dye can be non-covalently combined with nucleic acid molecules, and the detection of the target nucleic acid molecules is realized through the huge difference of the self fluorescence properties before and after the combination. Such fluorescent dyes are widely used in various states such as solution, gel electrophoresis, nucleic acid analysis in cells and in the environment, and the like. Currently, there are many commercially available nucleic acid analysis fluorescent dyes, and among them, SYBR series dyes sold by Invitrogen corporation in the united states have excellent performance, and occupy most of the nucleic acid dye markets internationally.

Among the SYBR series of dyes, SYBR Green I is most widely used because it has low background, low toxicity and ultra-high sensitivity, and has been widely used in DNA routine analysis, DNA gel staining and as a nucleic acid dye dedicated to real-time quantitative pcr (qpcr) routine detection. However, SYBR Green I still has the following disadvantages:

(1) first, the dye applied to qPCR should have excellent chemical stability during PCR (105 ℃ C. -55 ℃ C.) and storage (-20 ℃ C. -4 ℃ C.). This requires that the dye and its complex with the nucleic acid molecule should have a certain thermal stability. In addition, Tris is often used as a buffer in qPCR, and is alkaline at low temperatures and slightly acidic at high temperatures. Therefore, the dye is required to have better acid-base stability. However, SYBR Green I does not have good stability under the above conditions;

(2) SYBR Green I has concentration-dependent inhibition effect on the PCR process, and the PCR process can be inhibited by using high-concentration dye;

(3) in routine analysis of nucleic acids (including qPCR and gel electrophoresis imaging), the sequence of dye-bound DNA should be favored to be low or absent. However, SYBR Green I has been reported to have a more significant A-T sequence preference (see Zipper H, Brunner H, Bernhagen J, Vitzthim F (2004) Nucleic Acids Res 32:103 e);

(4) according to the instructions provided by Invitrogen company, SYBR Green I can maintain good stability in DMSO and has poor stability in aqueous solution;

(5) in addition, although SYBR Green I has excellent fluorescence properties, its synthesis is extremely difficult and the yield is extremely low (less than 2%) according to the reverse synthesis analysis. In addition, the price of the raw materials is high, so that the price of SYBR Green I is high (the official selling price of stock solution with the specification of 10000x and 1mL is as high as about 1 ten thousand yuan), and the cost is high in practical application.

Although another dye, SYBR Safe, is also sold by Invitrogen, is less expensive and has low mutagenicity and may be used as a replacement for SYBR Green I, the water solubility and sensitivity of this alternative dye is less than ideal.

Disclosure of Invention

Aiming at the defects that the high-efficiency nucleic acid fluorescent dye (such as SYBR Green I) in the prior art is unstable, influences the PCR process due to high concentration, is difficult to synthesize and the like, the invention carries out structural modification on the dye and provides another compound which has high sensitivity, good stability, easily obtained raw materials, simple synthesis, low sequence preference and small PCR inhibition.

The technical scheme of the invention is as follows:

the first technical problem to be solved by the present invention is to provide a fluorescent dye, wherein the structural formula of the fluorescent dye is shown as formula I:

wherein A is^-Is chloride, bromide or iodineIons.

The second technical problem to be solved by the present invention is to provide a preparation method of the above fluorescent dye, wherein the preparation method comprises: stirring and reacting the compound shown in the formula II and the monomer M in a solvent at room temperature for 6-24 hours to obtain a mixture, and then purifying the mixture to obtain the fluorescent dye shown in the formula I; wherein the monomer M is propylamine substituted by alkylamino.

Further, in the preparation method, the equivalent ratio of the compound shown in the formula II to the monomer M is 1: 1-1: 5.

Further, in the above production method, the monomer M is selected from: n, N-dimethyl-1, 3-diaminopropane or 3-diethylaminopropylamine.

Further, in the above preparation method, the solvent is ethanol or dichloromethane.

Further, in the reaction, the stirring time is 6 to 12 hours.

Further, the compound shown in the formula II is prepared by adopting the following method:

firstly, preparing the compound shown as the formula III

Then preparing the compound shown as the formula IV

mixing a compound shown as a formula V and a compound shown as a formula III in a solvent 2 at an equivalent ratio of 1: 1-1: 3 at room temperature to obtain a compound shown as a formula II; the solvent 2 is ethanol or dichloromethane.

Further, the compound represented by the formula III is prepared by the following method: and (3) carrying out methylation reaction on the 2-methylmercaptobenzothiazole (with or without substituent groups) by using a methylation reagent (such as methyl iodide) to obtain the compound shown in the formula III.

Further, the compound shown in the formula IV is prepared by adopting the following method: preparing a compound shown in a formula IV by reacting 2-hydroxy-4-methylquinoline with iodobenzene or phenylboronic acid under the action of a catalyst; wherein the catalyst is copper acetate, potassium carbonate and copper powder.

The third technical problem to be solved by the present invention is to provide the use of the above fluorescent dye, which can be used in qPCR analysis or gel electrophoresis imaging.

The invention discovers for the first time that when the compound shown in the formula II and monomers such as N, N-dimethyl-1, 3-diaminopropane and the like are used as raw materials, a novel fluorescent dye (shown in the formula I) can be obtained only by stirring and reacting for 6-24 hours at room temperature, and the obtained fluorescent dye has higher response sensitivity of nucleic acid molecules compared with SYBR Green I, has higher thermal stability after being combined with nucleic acid, and can also improve the melting temperature of the nucleic acid molecules.

In addition, the benzothiazole and quinoline rings in the fluorescent dye are connected through a methine bond, and the single bond can rotate by itself, so that the dye molecule has strong intramolecular torsional charge transfer (TICT) in a free state. And moreover, due to the steric hindrance of the phenyl substituent and the alkylamino substituent on the quinoline ring, the compactness of dye molecule aggregation and accumulation is reduced, and the dye monomer almost has no fluorescence. When the fluorescent dye is combined with nucleic acid molecules, a methine bond connecting benzothiazole and quinoline rings is limited by the structure of the nucleic acid molecules and fixed, so that a conjugated structure is formed conveniently, and an enhanced fluorescent signal can be obtained.

The invention has the beneficial effects that:

1) the invention provides a design of a novel nucleic acid fluorescent dye, the dye is prepared from easily available raw materials, has multiple purchasing channels and low cost, and is simple in synthesis condition, easy to synthesize (the reaction can be completed in a short time at room temperature), and an intermediate product does not need to be additionally purified.

2) The fluorescent dye has excellent fluorescence performance and good stability, can be directly used in common PCR buffer solution (even pure water), and does not need to add a protective reagent; secondary structures that specifically recognize nucleic acid molecules; the dye has good thermal stability after reacting with nucleic acid molecules, and can effectively improve the melting temperature of the nucleic acid molecules.

3) The fluorescent dye obtained by the invention has low sequence preference on nucleic acid molecules and high sensitivity. The high-sensitivity detection of the nucleic acid molecules in solution, gel, cells and environment can be realized by detecting the fluorescent signal of the dye and nucleic acid molecule mixed system.

Description of the drawings:

FIG. 1 shows an absorption spectrum and a fluorescence spectrum of compound c in example 1 of the present invention.

FIG. 2 is a graph showing the relationship between the fluorescence intensity of the compound c (- ■ -), SYBR Green I (- ● -), and SYBR Green I analog (-. tangle-solidup. -) combined with double-stranded DNA of different concentrations and the corresponding double-stranded DNA concentration.

FIG. 3A is a graph of fluorescence intensity versus temperature for a double stranded DNA sample dsDNA-2 bound by compound c of the invention (solid line) and SYBR Green I (dashed line), and FIG. 3B is a melting curve for the corresponding dsDNA-2.

FIG. 4A is a graph of Harpin-1 fluorescence intensity versus temperature for a double stranded DNA sample combined with compound c of the present invention (solid line) and SYBR Green I (dashed line), and FIG. 4B is a melting curve for the corresponding Harpin-1.

FIG. 5A is a graph of dsDNA-3 fluorescence intensity versus temperature for a double stranded DNA sample with compound c of the invention (solid line) combined with SYBR Green I (dashed line), and FIG. 5B is a melting curve for the corresponding dsDNA-3.

FIG. 6 is a bar graph of the fluorescence response of the compound c of the present invention and SYBR Green I to double-stranded DNA with different d (A-T) contents.

FIG. 7 shows the qPCR fluorescent amplification curves of the compound c of the present invention at the concentrations of 0.5, 1, 2 and 4. mu. mol/L on the left, and SYBR Green I at the concentrations of 0.5(0.55X), 1(1.1X), 2(2.2X) and 4(4.4X) mu. mol/L on the right.

FIG. 8 shows the qPCR amplification curves of 1. mu. mol/L compound c of the present invention at primer concentrations of 50, 100, 200, and 300nmol/L, and FIG. 8 shows the qPCR amplification curves of 1. mu. mol/L (1.1X) SYBR Green I under the same conditions.

FIG. 9 is a graph showing the melting curves of the amplification products of 1. mu. mol/L compound c of the present invention at primer concentrations of 50, 100, 200 and 300nmol/L, and FIG. 8 is a graph showing the melting curves of the amplification products of 1. mu. mol/L (1.1X) SYBR Green I under the same conditions.

FIG. 10 is a graph comparing the effect of standard molecular weight DNA on gel imaging in the prestained mode, with 1. mu. mol/L (1.1X) SYBR Green I on the left and 1. mu. mol/L Compound c of the present invention on the right.

FIG. 11 is a graph comparing the effect of standard molecular weight DNA on gel imaging in post-staining mode, with 1. mu. mol/L (1.1X) SYBR Green I on the left and 1. mu. mol/L Compound c of the present invention on the right.

FIG. 12 is a graph showing a comparison of gel imaging effects of 1. mu. mol/L (1.1X) SYBR Green I and 1. mu. mol/L compound c of the present invention on PCR amplified products in the pre-staining mode on the left, and a graph showing a comparison of gel imaging effects in the post-staining mode on the right.

FIG. 13 is a graph showing the relationship between the fluorescence intensity detected at 55 ℃ and the number of thermal cycles performed by Compound c of the present invention.

FIG. 14A is a graph showing the relationship between the absorbance and the time change in pure water for the compound c (- ● -) and SYBR Green I (- ■ -) of the present invention, and FIG. 14B is a graph showing the relationship between the absorbance and the time change in Tris-HCl buffer solution for the compound c (- ● -) and SYBR Green I (- ■ -).

FIG. 15 synthetic route, cost comparison of compound c with SYBR Green I.

Detailed Description

The fluorescent dye can be prepared by adopting the following preparation method, and the preparation method comprises the following steps:

1) 2-methylmercaptobenzothiazole (with or without substituents) is first methylated with a methylating agent (such as methyl iodide)Reacting to obtain a compound a

2) 2-hydroxy-4-methylquinoline and phenylboronic acid are catalyzed by copper acetate to obtain a compound b

3) Will be provided withMixing with phosphorus oxychloride to obtain the compoundFollowed by reacting it with a compoundMixing to obtain the compoundIt reacts with N, N-dimethyl-1, 3-diaminopropane to give the final dye structure

The specific reaction formula is as follows:

the following further describes specific embodiments of the present invention in conjunction with examples, which are not intended to limit the invention thereto.

Example 1: preparation of the nucleic acid fluorescent dye of the present invention

The preparation method comprises the following steps:

1) the starting material, 2-methylmercaptobenzothiazole (1.5g, 8.3mmol, 1eq), was dissolved in 30mL of dry ethanol, and iodomethane (1.4g, 10mmol, 1.2eq) was added and reacted at 60 ℃ for 5 hours. After the reaction is finished, removing the solvent, washing for multiple times by using petroleum ether, and collecting an off-white solid product a by using a suction filtration method:

2) the raw material 2-hydroxy-4-methylquinoline (3.0g, 18.8mmol, 1eq), phenylboronic acid (2.3g, 18.8mmol, 1eq) and copper acetate (3.75g, 18.8mmol, 1eq) were dissolved in 150-mL dichloromethane, stirred at normal temperature for reaction for 48-96h, and then vacuum filtered. Extracting the filtrate with water for three times, collecting dichloromethane phase extractive solution, adding anhydrous calcium chloride, and drying for 1-2 hr. Concentrating the extract through SiO₂Column purification gave a white solid b:

3) dissolve product b (100mg, 0.4mmol, 1eq) in 30mL acetonitrile, add phosphorus oxychloride (184mg, 1.2mmol, 3eq) and reflux at 60-85 ℃ for 12-24h under nitrogen. After the reaction is finished, cooling to room temperature, removing the solvent, dissolving the solvent and the compound a in 20-30mL of dichloromethane, and dropwise adding triethylamine at room temperature while stirring until no obvious white smoke is emitted. Stirring at room temperature for 5h, adding N, N-dimethyl-1, 3-diaminopropane (122mg, 1.2mmol, 3eq) as raw material dropwise, stirring at room temperature for 6-24h, concentrating the reaction mixture, passing through SiO₂Purifying and separating by a column, and recrystallizing in dichloromethane and petroleum ether solvent to obtain the pure compound c.

Example 2: absorption spectrum and fluorescence spectrum of Compound c

The DNA samples were purchased from Biotechnology engineering (Shanghai) Inc. as two complementary oligonucleotide strands as shown in Table 1. DNA samples were purified by HPLC, and appropriate amounts of DNA were dissolved in TE buffer (pH 7.4, 10mmol/L Tris, 1mmol/L EDTA) according to the instructions to prepare corresponding concentrations of stock solutions, which were stored at 4 ℃. When in use, the two single strands (Pro-20 and Tar-20) with the same concentration are respectively added to form double-stranded DNA (dsDNA-1).

TABLE 1

The absorption spectrum of compound c (1. mu. mol/L) and the fluorescence emission spectrum after binding to dsDNA-1(20bp, 100nmol/L) were examined in TE buffer (shown in FIG. 1). The maximum absorption peak of compound c was 467nm (blue light), and the maximum fluorescence emission peak after binding to DNA was 505nm (green light). The compound c can be excited by a light source in the 400nm-500nm region and is matched with the optical channel of the qPCR and chemiluminescence imaging instrument which are mainstream on the market at present, which shows that the dye can be well adapted to the existing instrument.

Example 3: compound c for routine nucleic acid molecule quantification

The DNA samples were as in example 2.

Comparing the structure SYBR Green I of the compound c and the compound c with N-H bond being blocked by N-propylAnd methyl-blocked SYBR Green I analogsSensitivity of fluorescent response to different concentrations of nucleic acid molecules. In TE buffer with a volume of 200. mu.L and a concentration gradient of 5nmol/L, a mixture containing dsDNA-1 at a final concentration of 0-75nmol/L and compound c, SYBR Green I (0.55X) and SYBR Green I analog at a final concentration of 0.5. mu. mol/L was prepared. The fluorescence of the above mixture was measured using a fluorescence spectrometer Fluorolog-3(HORIBA, USA), and the detected fluorescence intensity was plotted against dsDNA-1 concentration as shown in FIG. 2. First, it can be seen that almost no fluorescent signal response was detected at a dsDNA-1 concentration of 0. As dsDNA concentration increases, fluorescence is linear to DNA concentration change over a range; and the DNA concentration is higherAt high, the response becomes non-linear and the signal gradually becomes saturated. The slope of the ascending region in the linear range of change represents the sensitivity of the dye molecule to changes in DNA concentration. The results show that the sensitivity of the compound c response to 5nmol/L dsDNA-1 is significantly higher than SYBR Green I and the SYBR Green I analogues. Second, comparing the fluorescence intensity after saturation of binding of the same concentration of dye to dsDNA-1, the saturation fluorescence intensity of compound c was significantly higher than SYBR Green I and SYBR Green I analogs. The above results show that: compared with SYBR Green I and SYBR Green I analogues, the structure of the compound c has unique advantages, and the compound c has better binding capacity and higher detection sensitivity on DNA molecules.

Example 4: thermal stability of Compound c after binding to nucleic acid molecules

DNA samples were prepared as in example 2, and the DNA usage sequences are shown in Table 2.

TABLE 2

Protocol for thermal stability determination: samples containing 1. mu. mol/L of Compound c and 100nmol/L of DNA were mixed with 20. mu.L of Tris-HCl buffer (containing Mg at pH 8.3)²⁺) Holding at 95 ℃ for 15s followed by annealing; after incubation at 45 ℃ for 15min, StepOnePlus was used^TMThe qPCR instrument (ABI, usa) collects fluorescence signals (including 45 ℃) every +0.5 ℃; the temperature was raised to 95 ℃ and the incubation was carried out for 30s (the conditions for the thermal stability test of SYBR Green I were identical to those of Compound c). The fluorescence intensity of complexes formed between compound c and SYBR Green I and the different DNA samples described above was plotted against temperature (fig. 3A, 4A, and 5A), and the melting curves of the different DNA samples were plotted after taking the negative reciprocal of the slope at each point (fig. 3B, 4B, and 5B). The test result shows that: on one hand, the decrease speed of the fluorescence intensity (a relatively flat change area before the fluorescence intensity change is steeply reduced) in the temperature rise process after the compound c prepared in the embodiment 1 is combined with DNA is obviously slower than that of SYBR Green I, which shows that the thermal stability of a compound formed by the compound c and the DNA is obviously improved compared with that of the SYBR Green I. Another one isAspect comparison of the corresponding abscissas (i.e.melting temperature values, T) at the peaks of the melting curves of different DNA samples_mValue), detected T of Compound c_mThe value is also larger than SYBR Green I (shown in Table 3), and the compound c is proved to have good binding capacity and thermal stability in combination with DNA.

TABLE 3

Example 5: sequence Selectivity of Compound c

DNA samples were prepared as in example 2, and the DNA usage sequences are shown in Table 4.

TABLE 4

In a volume of 1mL and a pH of 7.4TE buffer, a DNA sample (AT-1) containing 10% adenine (A) -thymine (T) and a DNA sample (AT-2) containing 100% adenine (A) -thymine (T) were prepared AT a concentration of 100nmol/L, respectively, and a compound c was added thereto AT a final concentration of 1. mu. mol/L. After 10min incubation, the fluorescence intensity of the complex formation of compound c with two different DNA samples was determined using fluorescence spectrometer Fluorolog-3(HORIBA, usa), to which the sequence selectivity test conditions of SYBR Green I for comparison were kept consistent, the results are shown in fig. 6. The difference of the signal response of the compound c to the A-T sequence preference is only 15 percent and is obviously lower than SYBR Green I (40 percent), which indicates that the compound c has very low sequence preference to nucleic acid molecules and is suitable for the conventional detection of the nucleic acid molecules.

Example 6: application of compound c in qPCR amplification

Using the compound c prepared in example 1 as a fluorescent dye, StepOnePlus was used^TMqPCR instrument (ABI, usa) amplified the invA gene in salmonella. The primer sequences for qPCR amplification were as follows:

an upstream primer: 5'-TCCCTTTCCAGTACGCTTCG-3'

A downstream primer: 5'-TCTGGATGGTATGCCCGGTA-3'

Obtaining a template gene: salmonella DNA was extracted using a bacterial DNA extraction Kit (TIANAmp Bacteria DNA Kit, cat # DP302, TIANGEN, Beijing) according to the protocol.

qPCR conditions: the assay fluorescence intensity was set at 55 ℃ for 30s after holding at 95 ℃ for 20s, followed by extension at 72 ℃ for 30s for 30-40 cycles.

Selecting PCR kit (TaKaRa Taq)^TMHS Low DNA, cat No.: R090A, TaKaRa, Japan) was prepared in a volume of 20. mu.L according to the instructions, including Taq^TMHS Low DNA (2 ×), 10 μ L; 0.4. mu.L of each of the upstream primer and the downstream primer (containing DNA at final concentrations of 50nmol/L, 100nmol/L, 200nmol/L, and 300nmol/L, respectively); compound c or SYBR Green I for comparison, 2. mu.L (final concentrations of 0.5. mu. mol/L, 1. mu. mol/L, 2. mu. mol/L and 4. mu. mol/L, respectively); template gene, 2 μ L; sterile water was added to make up to a final volume of 20. mu.L.

1) Comparison of Signal intensity of Compound c with SYBR Green I in qPCR

The other conditions are kept consistent, the qPCR fluorescence amplification curve (fluorescence intensity versus cycle number) with only the concentration of the compound c being changed is shown in the left graph of FIG. 7, the SYBR Green I with different concentrations for comparison is shown in the right graph of FIG. 7, and the result shows that the change amplitude and the intensity of the fluorescence signal monitored by the compound c with different concentrations are higher than those of the SYBR Green I with the corresponding concentrations. In addition, at higher concentrations, SYBR Green I showed a more pronounced concentration-inhibiting effect, with C appearing compared to Compound C at the same concentration_TThe values lag (about 1 cycle). Experiments prove that compared with SYBR Green I, the compound c can be used for real-time amplification detection of nucleic acid in a larger concentration range, and can realize more sensitive detection.

2) Comparison of amplification curves of Compound c and SYBR Green I in qPCR

qPCR amplification curves with only change in primer concentration (ordinate. DELTA.R) keeping other conditions consistent (concentrations of Compound c and SYBR Green I are both 1. mu. mol/L)_nNumber of cycles to abscissa) as shown in the upper graph of fig. 8As shown, different concentrations of SYBR Green I for comparison are shown in the lower graph of fig. 8. The results show that: the compound c synthesized by the invention has lower amplification background, and has no obvious concentration dependence compared with SYBR Green I at low primer concentration (50 nmol/L).

3) Compound c monitoring PCR amplicon melting temperature

SYBR Green I is reported to be an advantageous tool for monitoring the amplification product (i.e., the melting temperature of the amplicon) after completion of the qPCR amplification. Determination of the melting temperature of the amplicon provides information on the presence or absence of non-specific amplification and the identity of the amplicon. Accordingly, the melting curve of the amplicon monitored with compound c of the invention (upper panel in fig. 9) was also determined with varying primer concentrations to test the effect of monitoring the melting peak of the amplicon with compound c and compared to SYBR Green I (lower panel in fig. 9). First, the results of the two amplicon melting temperature tests have good correlation, and the measurement results are consistent. Secondly, the test baseline for compound c was lower compared to SYBR Green I, indicating that compound c provided in example 1 of the present invention enables detection of amplicon melting temperature.

Example 7: comparative experiment of gel staining effect

Gel staining is reported to be currently achieved mainly by two means: (1) before separation, adding the dye into the DNA mixed solution in advance, carrying out electrophoretic separation, and then carrying out fluorescence imaging, namely a pre-dyeing method; (2) after the electrophoresis separation of the DNA mixed solution, placing the gel into a fluorescent dye aqueous solution or buffer solution for soaking for a period of time, and then carrying out fluorescence imaging, namely a post-dyeing method; both modes of use are very common. The effect of the compound c prepared in example 1 (final concentration of 1. mu. mol/L) and SYBR Green I (final concentration of 1. mu. mol/L, 1.1X) on the pre-staining method (FIG. 10) and the post-staining method (FIG. 11), respectively, was compared, using DNA samples of 25-500bp DNA molecular weight standard Marker (cat # B600303, BBI, USA) according to the following instructions: 10ng, 5ng, 2ng, 1ng and 0.5 ng.

As can be seen from the comparison of the effect of the pre-dyeing method (FIG. 10) and the post-dyeing method (FIG. 11), the compound c prepared in the example 1 of the invention has the same effect as SYBR Green I in both dyeing modes, and compared with SYBR Green I in the pre-dyeing method, the compound c has no obvious tailing phenomenon in the dyeing result and has cleaner bands. In addition, the effect of compound c (final concentration of 1. mu. mol/L) compared with SYBR Green I (final concentration of 1. mu. mol/L, 1.1X) on the amplicon samples produced in example 6 was compared between the pre-staining and the post-staining (FIG. 12), and the results show that compound c is comparable to SYBR Green I in both pre-staining and post-staining in the practical application process. And more obviously, compared with the tailing phenomenon which is not generated by the SYBR Green I compound c pre-staining of an actual sample, the band is cleaner and clearer, and the different DNA fragments are more obviously distinguished.

Example 8: intrinsic stability of Compound c

The temperature range of qPCR is typically 55 ℃ to 105 ℃ and the qPCR reagents are stored at-20 ℃ to 4 ℃. In addition, the nucleic acid molecules need to be in an environment of pure aqueous solution or buffer solution. This requires that the dye molecules be stable in water or buffer and also stable at temperatures ranging from 55 ℃ to 105 ℃ and from-20 ℃ to 4 ℃.

1) Thermal stability

Tris-HCl buffer (Mg-containing) at pH 8.3 in a volume of 20. mu.L containing compound c at a final concentration of 10. mu. mol/L²⁺) In the system (a), after the compound c is subjected to 40 thermal cycles of 95 ℃ for 20s, 55 ℃ for 30s and 72 ℃ for 30s, as shown in FIG. 13, the compound c still keeps stable (the fluorescence intensity of the test is set at 55 ℃), the signal is not obviously reduced, and the durability of the compound c in qPCR is confirmed.

2) Stability in solution

Tris-HCl buffer (Mg-containing) pH 8.3 at 3mL volume containing compound c or SYBR Green I (1.1 ×) at a final concentration of 1 μmol/L²⁺) Or in a pure water system, the signal changes of the compound c and SYBR Green I at the maximum absorption wavelength of each compound c and SYBR Green I are monitored every 12-24h, wherein the pure water is shown as figure 14A, and the buffer solution is shown as figure 14B. The results show that: SYBR Green I in pure water is extremely unstable compared to buffering, which is consistent with the reported phenomenon that there is already nearly half of the molecular decomposition after 72 h; are combined with each other after the same timeSubstance c was more stable than SYBR Green I both in pure water and in buffer. More importantly, the compound c is very stable in pure water, which indicates that the storage environment of the compound c is not as harsh as SYBR Green I, and when a gel staining method adopts a post-staining method, the compound c can be directly prepared by using the pure water without additionally preparing a buffer solution, so that the use is more convenient.

Example 9: comparison of Synthesis costs of Compound c with SYBR Green I

Currently, SYBR Green I is the most widely applied dye in the market at present and has relatively high cost performance. However, according to the analysis of the SYBR Green I synthetic pathway (FIG. 15), the compound c has low requirements on reaction conditions, many sources for raw materials, no need of self-synthesis and low price (reagent price inquiry website: https:// www.bidepharm.com; https:// www.chemicalbook.com/product index. aspx); only three-step reaction is needed, intermediate product purification is not needed, heating is not needed in key step reaction, the yield is higher, and the experiment shows that the performance of the compound c is superior to that of SYBR Green I.

More importantly, in practical nucleic acid molecule detection, dye cost is the most important ring affecting the overall cost of the assay. Although SYBR Green I is a highly sensitive nucleic acid dye, the price is high, the official price is about 1 ten thousand yuan (1 mL) (10000x), and the cost of the reagent per se is too high, particularly when the SYBR Green I is put into large-scale nucleic acid detection and gel electrophoresis imaging. As proved by a gel staining experiment in example 7, the compound c has the staining effect equivalent to that of SYBR Green I and even has better effect, but the synthesis cost of the compound c is far lower than that of the SYBR Green I. This indicates that compound c can greatly reduce the production cost matched therewith while maintaining high sensitivity.

SEQUENCE LISTING

<110> Sichuan university

<120> nucleic acid fluorescent dye and preparation and application thereof

<130> 2021.8.2

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA