阅读说明：本技术 一种双链dna结合荧光染料及其制备与应用 (Double-stranded DNA (deoxyribonucleic acid) combined fluorescent dye as well as preparation and application thereof ) 是由 徐祎春 崔雷 韩峻松 袁箐 周佳菁 苏军 周欣怡 于 2019-09-17 设计创作，主要内容包括：本发明公开了一种双链DNA结合荧光染料,该染料包含有式ⅰ所示结构或其立体异构体。本发明还公开了上述染料的制备方法及用途。本发明通过叔胺连接两个荧光团,且荧光团易于π-π堆积错位,降低两个分子结合前的荧光强度,获得更强的扩增信号,从而提高了双链DNA结合荧光染料的灵敏性和稳定性,降低了其细胞毒性和对PCR的抑制性。(The invention discloses a double-stranded DNA binding fluorescent dye, which comprises a structure shown as a formula i or a stereoisomer thereof. The invention also discloses a preparation method and application of the dye. According to the invention, the tertiary amine is used for connecting the two fluorophores, and the fluorophores are easy to be subjected to pi-pi stacking dislocation, so that the fluorescence intensity before the combination of the two molecules is reduced, and a stronger amplification signal is obtained, thereby improving the sensitivity and stability of the double-stranded DNA combined fluorescent dye, and reducing the cytotoxicity and the inhibition on PCR.)

1. A double-stranded DNA-binding fluorochrome, characterized in that the molecular structure of the fluorochrome comprises a structure according to formula i:

（ⅰ）。

2. the double-stranded DNA-binding fluorescent dye according to claim 1, wherein the molecular structural formula of the fluorescent dye is:

wherein X is halogen.

3. The double-stranded DNA binding fluorescent dye according to claim 2, wherein X is Br or I.

4. The double-stranded DNA binding fluorescent dye according to claim 1, wherein the stereoisomer has a structure represented by formula (iii) or formula (iv):

（ⅲ）

（ⅳ）。

5. a method for preparing a double-stranded DNA binding fluorescent dye according to any one of claims 1 to 3, comprising the steps of:

1) reacting 2-methylbenzothiazole with 1, 2-diiodoethane to obtain an intermediate I:

2) reacting the intermediate I with methylamine to obtain an intermediate II:

3) and (3) reacting the intermediate II with p-aminobenzaldehyde to obtain the double-stranded DNA combined fluorescent dye with the structure shown in the formula III:

6. the process according to claim 5, wherein step 1), 2-methylbenzothiazole is slowly added dropwise to an ethanol solution of 1, 2-diiodoethane at 60 ℃, after completion of the addition, the reaction is carried out at reflux temperature for 90min, and then the solvent is removed and the intermediate I is crystallized from acetone.

7. The method as claimed in claim 5, wherein the step 2) adopts a tube-sealing reaction method, n-butanol is used as a solvent, the reaction temperature is 135 ℃, and the methylamine is a methanol solution of methylamine.

8. The method of claim 7, wherein 1.5 equivalents of an acid-binding agent is further added to the reaction system of step 2).

9. The method as claimed in claim 5, wherein the reaction in step 3) adopts acetic acid and piperidine as catalysts, and the volume ratio of the acetic acid to the piperidine is 1: 1-1: 4.

10. Use of the double stranded DNA binding fluorescent dye according to any one of claims 1 to 4 for real time quantitative PCR detection of nucleic acids.

Technical Field

The invention relates to the field of nucleic acid dyes, in particular to a novel double-stranded DNA (deoxyribonucleic acid) combined fluorescent dye.

Background

Double-stranded DNA (dsDNA) is most typically in combination with a fluorescent dye, molecular ethidium bromide, which has been used for nucleic acid staining in agarose gel electrophoresis.

Double-stranded DNA binding fluorescent dyes are equally applicable for detecting real-time amplification of nucleic acids, such as PCR. The corresponding amplification product can be identified by DNA binding fluorescent dye, and when the dye interacts with double-stranded nucleic acid, the dye is excited with proper wavelength to emit corresponding fluorescent signal. In the PCR reaction process, as long as double-stranded DNA exists in a sample to be detected, the dye can be selectively combined, and simultaneously, a fluorescent signal can be detected. When double-stranded DNA dissociates, the signal decreases rapidly. The decay of this signal is also suitable for monitoring the fluorescence intensity versus temperature-time curve.

The double-stranded DNA binding fluorescent dye can also be used for real-time PCR detection. In this case, SYBRGreen I is the most frequently used fluorescent dye. SYBRGreen I is widely applied due to high efficiency and low toxicity, but cannot be used for common PCR buffer solution per se, and additional reagents such as DMSO, DBA and the like are required to be added; meanwhile, although the PCR inhibition effect can be reduced by increasing the concentration of MgCl2, the concentration-dependent inhibition effect still exists, and thus SYBRGreen I is limited in multiplex PCR applications. It was found that when double PCR amplification was performed, only one product of the dissolution curve could be detected, but electrophoretic analysis clearly showed two products. Therefore, although SYBRGreen I is used in a wide range, it still has disadvantages such as a small available concentration range and a large sequence binding bias.

In addition, many existing nucleic acid dyes have high toxicity, mainly because dye molecules can easily penetrate through cell membranes, so that the dye molecules can be combined with nucleic acid DNA in cells, gene mutation is caused, and certain carcinogenicity is achieved.

Disclosure of Invention

One of the technical problems to be solved by the invention is to provide a double-stranded DNA-conjugated fluorescent dye which has good sensitivity and stability, low cytotoxicity and small inhibition on PCR.

In order to solve the above technical problems, the double-stranded DNA binding fluorescent dye of the present invention has a molecular structure comprising a structure represented by formula i or a stereoisomer thereof:

（ⅰ）

preferably, the molecular structural formula of the fluorescent dye is as follows:

wherein X is a halogen, such as Br or I.

The stereoisomer of formula i is predominantly ZE isomeric:

（ⅲ）

（ⅳ）

the invention also provides a preparation method of the double-stranded DNA combined fluorescent dye. The method mainly comprises the following steps:

1) reacting 2-methylbenzothiazole with 1, 2-diiodoethane to obtain an intermediate I:

2) reacting the intermediate I with methylamine to obtain an intermediate II:

3) and (3) reacting the intermediate II with p-aminobenzaldehyde to obtain the double-stranded DNA combined fluorescent dye with the structure shown in the formula III:

。

the 1, 2-diiodoethane as the raw material in the above step 1) may be replaced by other o-dihaloethanes, for example, 1, 2-dibromoethane.

The tube sealing reaction method is preferably adopted in the step 2), an acid-binding agent can be added into the reaction system, the acid-binding agent can be diisopropylethylamine, and the addition amount is preferably 1.5 equivalents.

The reaction in the step 3) preferably adopts a mixed solution of acetic acid and piperidine as a catalytic system, and the volume ratio of the acetic acid to the piperidine is preferably 1: 1-1: 4.

The invention also provides the application of the double-stranded DNA binding fluorescent dye, and the double-stranded DNA binding fluorescent dye can be used for real-time quantitative PCR detection of nucleic acid.

The double-stranded DNA combined fluorescent dye is connected with two fluorophores through tertiary amine, and N, N' -dimethylbenzene in the fluorophores is easy to form strong pi-pi accumulation, so that a molecular cluster-like structure is formed, and the fluorescence intensity of the dye is reduced; after the dye is combined with nucleic acid, because the double bond connecting arm of benzothiazole and N, N' -dimethylbenzene is easy to be limited and fixed by the nucleic acid structure, the whole fluorophore structure is rigidized, and conjugation is convenient to form, so that a stronger amplification signal can be obtained, the sensitivity and stability of the double-stranded DNA combined fluorescent dye are improved, and the cytotoxicity and the inhibition on PCR are reduced. In addition, the double-stranded DNA binding fluorescent dye can be directly used in common PCR buffer solution without adding additional reagents such as DMSO, DBA and the like.

Drawings

FIG. 1 is a nuclear magnetic resonance spectrum of intermediate II of example 1 of the present invention;

FIG. 2 shows the NMR spectrum of the final product of example 1 of the present invention (compound of formula III);

FIGS. 3 to 6 are graphs showing the results of the amplification curves of the compound of formula III prepared in example 1 of the present invention for real-time fluorescent quantitative PCR;

FIG. 7 is a graph showing the results of a real-time fluorescent quantitative PCR amplification curve using a contrast agent SYBR;

FIGS. 8 to 11 are graphs showing the results of a standard curve for PCR quantification of the compound of formula III prepared in example 1 of the present invention;

FIG. 12 is a graph showing the results of a standard curve for PCR quantification using a comparative reagent SYBR.

FIG. 13 is a diagram showing the results of agarose gel electrophoresis of a control EB;

FIG. 14 is a diagram showing the result of agarose gel electrophoresis using GelRed as a control;

FIG. 15 is a graph showing the results of agarose gel electrophoresis of a compound of formula III prepared in example 1 of the present invention;

FIG. 16 is a comparative SYBR Safe cytotoxicity test chart;

FIG. 17 is a graph showing the safety of cytotoxicity of the compound of formula III prepared in example 1 of the present invention.

Detailed Description

In order to more specifically understand the technical content, characteristics and effects of the present invention, the technical solution of the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments.