阅读说明：本技术 一种aie探针及其制备方法与在荧光定量pcr方法中的应用 (AIE probe, preparation method thereof and application thereof in fluorescent quantitative PCR (polymerase chain reaction) method ) 是由 唐本忠 伍盛鋆 郭武秀 刘勇 王志明 于 2021-08-17 设计创作，主要内容包括：本发明公开了一种AIE探针及其制备方法与在荧光定量PCR方法中的应用。所述AIE探针不带荧光淬灭基团,所述AIE探针结构如下：其中,AIE表示取代或未取代的聚集诱导发光骨架,LIN表示连接基团,Oligo表示所需要检测目的条带碱基序列寡核苷酸链；AIE探针的连接基团LIN连接在Oligo的3'端或者5'端的核苷酸和AIE活性位点上。本发明的AIE探针无需额外连接淬灭基团,同时AIE材料的聚集诱导发光特性又降低了背景荧光,为难检疾病核酸物质的检出提供了一个很好的解决方案,真正做到荧光定量PCR的实时监控。(The invention discloses an AIE probe, a preparation method thereof and application thereof in a fluorescent quantitative PCR method. The AIE probe does not have a fluorescence quenching group, and the structure of the AIE probe is as follows: wherein AIE represents a substituted or unsubstituted aggregation-induced emission skeleton, LIN represents a connecting group, and Oligo represents an oligonucleotide chain of a target band base sequence to be detected; the linker LIN of the AIE probe is linked to the nucleotide at the 3 'or 5' end of the Oligo and the AIE active site. The AIE probe of the invention does not need to be additionally connected with a quenching group, and simultaneously the aggregation-induced emission characteristic of the AIE material reduces background fluorescence, thereby providing a good solution for detecting nucleic acid substances of diseases difficult to detect and really realizing real-time monitoring of fluorescence quantitative PCR.)

1. An AIE probe, wherein the AIE probe does not have a fluorescence quenching group, and the AIE probe has the following structure:

wherein AIE represents a substituted or unsubstituted aggregation-induced emission skeleton, LIN represents a connecting group, and Oligo represents an oligonucleotide chain of a target band base sequence to be detected; the linker LIN of the AIE probe is linked to the nucleotide at the 3 'or 5' end of the Oligo and the AIE active site.

2. The AIE probe of claim 1, wherein the linkage site on the nucleotide at the 3 'end or 5' end of the Oligo comprises a carboxyl, amino, thiol, and azido reactive modification group; the connecting group is a single bond or a divalent connecting group.

3. The AIE probe of claim 1, wherein the AIE molecule comprises at least one of the following structures:

in the above structural formula, R₁，R₂，R₃，R₄，R₅，R₆Each independently selected from any one of hydrogen, halogen, carbonyl, amido, phosphorus oxy, carboxyl, substituted or unsubstituted C3-C30 nitrogen heteroaryl, substituted or unsubstituted C6-C30 cyano-containing aryl, substituted or unsubstituted C3-C30 cyano-containing heteroaryl, substituted or unsubstituted C6-C30 halogen-containing aryl, substituted or unsubstituted C3-C30 halogen-containing heteroaryl, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C6-C30 aryl, and substituted or unsubstituted C3-C30 heteroaryl;

n is 1, 2, 3 and 4.

4. The AIE probe of claim 1, wherein LIN is an alkylene chain, a substituent-substituted alkylene chain, a group in which a plurality of alkylene chains or substituent-substituted alkylene chains are linked by one or more of-O-, -S-, -CONH-, -NHCO-, -COO-, -OOC-, -NH-, alkynylene, alkenylene, arylene, heteroarylene, cycloalkylene.

5. The AIE probe of claim 4, wherein the substituent-substituted alkylene chain is substituted with one or more substituents, and wherein the alkylene chain is linear or branched.

6. A method of preparing the AIE probe of any one of claims 1-5, comprising the steps of:

firstly, synthesizing Oligo according to the base sequence of the required probe;

then introducing a modification group comprising carboxyl, amino, sulfhydryl and azido on the nucleotide at the 3 'end or the 5' end of the Oligo;

finally, the AIE is subjected to condensation reaction, coupling reaction, rearrangement reaction and Click reaction with a modifying group to obtain the probe.

7. The method of claim 6, wherein the AIE has a carboxyl or azido functional group.

8. The method of claim 7, wherein the molecular structure of AIE is:

9. use of the AIE probe according to any one of claims 1 to 5 in a fluorescent quantitative PCR method.

10. The use according to claim 9, wherein the fluorescent quantitative PCR method is used for nucleic acid detection.

Technical Field

The invention relates to the field of nucleic acid detection, in particular to an AIE probe, a preparation method thereof and application thereof in a fluorescent quantitative PCR method.

Background

The qPCR technology is that a fluorescent group is added into a PCR reaction system, the whole PCR process is monitored in real time by using fluorescent signal accumulation, and finally, the total amount of an unknown gene template is analyzed by a standard curve or the template is relatively quantified by a Ct value. qPCR not only realizes qualitative to quantitative leap of PCR, but also effectively solves the problem of PCR pollution, has the characteristics of strong specificity, high automation degree and the like, and is widely applied to various fields of molecular biology research. However, as the requirements of people on the aspects of timeliness, sensitivity, stability and the like of gene detection are continuously increased, the traditional qPCR is difficult to meet the requirements of the development of the times, and needs to be innovated and broken through urgently.

The fluorescent group is a key link of the breakthrough development of qPCR, and the sensitivity and the stability of the qPCR technology are determined by the luminous property and the luminous efficiency of the fluorescent group. At present, common fluorescent groups comprise an embedded fluorescent dye (SYBR GREEN I) and a specifically combined fluorescent probe (a TaqMan probe, a molecular beacon, a scorpion probe and the like), and are widely applied to the fields of clinical disease diagnosis, pathogenic microorganism identification, food safety, scientific research and the like. SYBR GREEN I is a double-stranded DNA binding dye which is bound in the minor groove and can be specifically bound with double-stranded DNA and emit a fluorescent signal, while SYBR GREEN I dye which is not doped into the double strand does not emit any fluorescent signal, thereby ensuring complete synchronization with the increase of PCR products. The Taqman probe is an oligonucleotide probe with two ends labeled with a fluorescent group and a quenching group, when the fluorescent group is close to the quenching group, Fluorescence Resonance Energy Transfer (FRET) can occur, and the quenching group absorbs the excited fluorescence of the fluorescent group under the action of the excited light, so that the fluorescent group can not emit fluorescence; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme cleaves and degrades the probe, so that the two groups are separated, and a fluorescent signal is received. Molecular beacons, scorpion probes and the like can not be quenched by fluorescent beacon molecules, and the technical problems of complex design, difficulty in implementation, inexhaustible background fluorescence and the like are faced.

With the wide use of the current RT-qPCR, the defect of false negative or false positive for trace and even trace sample detection gradually emerges, and the main reasons are from two aspects: first, the relevance of diagnostic markers or the accuracy of design of probe molecules, usually due to the existence of silencing mechanisms or delay effects in gene control itself, leads to the possibility that the detection results will be more accurate with the help of clinical cases and more experiments, and therefore, the work in this respect has become the key point of the attack in this field in recent years; secondly, the fluorescence signal output process is affected by the luminous mechanism thereof to cause intensity distortion, and the process is mostly related to the element responsible for the luminescence, so the problem has more platform effect, and once the attack is obtained, the carrying capacity and the potential commercial value are self-evident.

The Taqman method is the mainstream method applied to qPCR at present and is the best method for specifically detecting a target gene band (Heid, C AStevens, JLIvak, K JWilliams, P M, Real time quantitative PCR. genome research. 1996; 986-94.DOI 10.1101/gr.6.10.986). However, the complexity of this strategy is also obvious, namely, the fluorescent group/quencher pair and the linkage structure between the two (such as a DNA fragment for identification) must be subjected to strict verification experiments and development schemes of differences, which increases the design difficulty, synthesis conditions and experimental cost of the probe; moreover, interference of background fluorescence cannot be completely avoided through FRET action of the fluorescent group and the quenching group, most instruments are implanted with different algorithms to reduce noise, so that detection is more accurate and stable, but certain risks and disadvantages are brought, and misjudgment results are caused (Jianwen, Yuanwen, Jianghong, Wangchen. TaqMan probe method application and research progress [ J ]. J. clinical examination journal (electronic edition), 2015,4(01): 797-. Therefore, developing a fluorescent material capable of simultaneously implementing a fluorescent switch using an identification process would essentially solve the above problems and implement technical innovation of the existing platform to form a new analysis and detection platform.

The proposal of Aggregation Induced Emission (AIE) concept essentially solves the problem of fluorescence quenching caused by Aggregation, and has attracted extensive attention in the scientific community. Most luminescent materials with AIE properties are based on an intramolecular rotation of confinement (RIR) mechanism: that is, the molecular rotation is violent in the solution state, and the excited state energy is mostly attenuated in a non-radiative mode, so that the weak fluorescence intensity is caused; in the aggregation state, the vibration transport in the molecule is blocked, the nonradiative transition form is inhibited, and the excited state energy is dissipated in the radiation form, so that the aggregate shows high-intensity fluorescence.

With intense efforts of global scientists, the luminescence quantum efficiency of some solid AIE materials has been improved to 100%, the emission wavelength has been extended from ultraviolet to near infrared, and various AIE sensors based on different turn-on principles have been developed. Because the AIE molecules have higher sensitivity under the aggregate and can realize high-selectivity recognition on certain specific cells through simple structure regulation and modification, the research of the AIE materials in the aspects of early tumor specificity detection, kinetic observation and diagnosis and treatment integration is gradually paid attention to. Although the results lay the foundation for the research of AIE-dots in specific in-vitro detection and in-vivo tracing, most of the results only stay in the basic scientific research level, and the systematic research work combined with clinical transformation application is still insufficient, particularly the optimization and the update of the mature clinical molecular diagnosis technology of qPCR. The invention just makes up for the defects, and utilizes the characteristic AIE principle of extremely characteristic to realize the innovation of qPCR technology.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides an AIE probe, a preparation method thereof and application thereof in a fluorescence quantitative PCR method. The AIE method is based on the principle of aggregation-induced emission to realize real-time monitoring of PCR reaction under the basic amplification principle of PCR. Due to the strong hydrophilicity of the oligonucleotide chain, the AIE probe does not generate fluorescence because it exhibits a very water-soluble property. When the PCR reaction is carried out, the AIE probe is degraded by enzyme digestion, and the released AIE shows extremely strong hydrophobicity, the solubility is greatly reduced, aggregation occurs, and therefore, a random fluorescent signal is generated (as shown in figure 1). The accumulation of the fluorescence signal and the amount of the DNA amplification product are in a synchronous linear relationship, and the real-time monitoring of the PCR reaction can be realized. By matching with the light stability of the AIE material, the influence on the fluorescence intensity of the AIE material after the light-induced fluorescence detection at the end of multiple cycles is very little, and the real-time monitoring of the quantity of the qPCR product is truly realized. This is a further technological innovation of signaling molecules in the field of molecular diagnostics, following the substitution of radionuclides by fluorescens.

First, the present invention solves the problem of the connection of the AIE material to the nucleotide chain (Oligo), which differs in the connection method and the AIE functional group used, because of the difference in the directionality of the 3 'and 5' ends. The present invention has realized the coupling of AIE materials to oligonucleotide chains (Oligo), summarizing the activity conditions suitable for Oligo synthesis and the synthesis scheme of AIE probes. The AIE molecule is connected to an Oligo (target band base sequence to be detected) through a proper chemical reaction to be used as an AIE-qPCR probe, and the AIE-qPCR probe is not limited to be connected to a 3 'end hydroxyl group and a 5' end phosphate or a base and a five-carbon sugar of the Oligo, and has good qPCR effect. The chemical reactions used for coupling the AIE to the Oligo include, but are not limited to, common condensation reactions, coupling reactions, rearrangement reactions, Click reactions, and the like.

The detailed technical scheme adopted by the invention is as follows:

an AIE probe, said AIE probe not carrying a fluorescence quenching group, said AIE probe having the structure:

wherein AIE represents a substituted or unsubstituted aggregation-induced emission skeleton, LIN represents a connecting group, and Oligo represents an oligonucleotide chain of a target band base sequence to be detected; the linker LIN of the AIE probe is linked to the nucleotide at the 3 'or 5' end of the Oligo and the AIE active site.

Preferably, the linkage site on the nucleotide at the 3 'end or 5' end of the Oligo comprises a carboxyl, amino, thiol and azido reactive modification group.

Preferably, the linking group is a single bond or a divalent linking group.

Preferably, the AIE molecule comprises at least one of the following structures:

in the above structural formula, R₁，R₂，R₃，R₄，R₅，R₆Each independently selected from any one of hydrogen, halogen, carbonyl, amido, phosphorus oxy, carboxyl, substituted or unsubstituted C3-C30 nitrogen heteroaryl, substituted or unsubstituted C6-C30 cyano-containing aryl, substituted or unsubstituted C3-C30 cyano-containing heteroaryl, substituted or unsubstituted C6-C30 halogen-containing aryl, substituted or unsubstituted C3-C30 halogen-containing heteroaryl, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C6-C30 aryl, and substituted or unsubstituted C3-C30 heteroaryl;

n is 1, 2, 3 and 4.

Further preferably, the AIE molecule is selected from at least one of TPE or TPE derivatives. More preferably, the structure of the AIE compound is as follows:

preferably, the LIN is an alkylene chain, a substituent-substituted alkylene chain, a group in which a plurality of alkylene chains or substituent-substituted alkylene chains are linked by one or more of-O-, -S-, -CONH-, -NHCO-, -COO-, -OOC-, -NH-, alkynylene, alkenylene, arylene, heteroarylene, cycloalkylene.

Further preferably, the substituent-substituted alkylene chain is substituted with one or more substituents, and the alkylene chain is straight or branched.

The preparation method of the AIE probe comprises the following steps:

firstly, synthesizing Oligo according to the base sequence of the required probe;

then introducing a modification group comprising carboxyl, amino, sulfhydryl and azido on the nucleotide at the 3 'end or the 5' end of the Oligo;

finally, the AIE is subjected to condensation reaction, coupling reaction, rearrangement reaction and Click reaction with a modifying group to obtain the probe.

Preferably, the AIE has a carboxyl or azido functional group.

Further preferably, the molecular structural formula of the AIE is as follows:

the AIE probe is applied to a fluorescence quantitative PCR method.

Preferably, the fluorescent quantitative PCR method is used for nucleic acid detection.

The AIE molecule is coupled with the Oligo by carboxyl activation, amino condensation and the like. The Oligo is an oligonucleotide chain with a target band base sequence to be detected, is determined according to different gene sequences to be detected, has a design principle consistent with qPCR, has fewer limiting conditions than a Taqman method, and only needs to consider the specificity. Because no quenching group is additionally connected to the Oligo, the base length limitation based on the FRET principle is avoided.

Compared with the prior art, the invention has the advantages and effects that:

the probe of the invention has simpler design, simpler synthesis and lower cost.

The invention discloses a novel AIE-qPCR technical platform based on AIE molecules, an AIE probe, a preparation method and application thereof, which just make up the problems of high background signal, difficult probe design, difficult selection of a fluorescent group and a quenching group and the like of the traditional qPCR method; the method utilizes the characteristic AIE principle, realizes innovation of qPCR technology, reduces the design difficulty of probe sequences, provides a good solution for detecting nucleic acid substances which are difficult to detect diseases, realizes better result display, enriches molecular detection means and promotes the development of the field of biotechnology.

Drawings

FIG. 1 is a reaction schematic diagram of the AIE-qPCR technology of the present invention.

FIG. 2 is a preferred AIE parent nucleus molecule of example 1.

FIG. 3 is a schematic view of the structure of the AIE probe in example 1.

FIG. 4 is a graph of mass spectral data for the AIE probe of example 1.

FIG. 5 is a graph showing the comparison of fluorescence intensity before and after the fluorescence release of the AIE probe in example 2.

FIG. 6 is the raw fluorescence curve of AIE-qPCR in example 4.

FIG. 7 is a fluorescence spectrum of the AIE-qPCR product in example 5.

FIG. 8 is an amplification curve of a reproducibility experiment of the AIE-qPCR method in example 6.

FIG. 9 is a standard curve of the AIE-qPCR method in example 7.

FIG. 10 is an amplification curve of HBV positive clinical specimens in example 8.

FIG. 11 is a schematic view of the structure of the AIE probe 2 in example 9.

FIG. 12 is the AIE parent nucleus molecule of AIE probe 3 in example 10.

FIG. 13 is a schematic view of the structure of the AIE probe 3 in example 10.

Detailed Description

The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.

The AIE probe is superior to the existing traditional fluorescent dye in fluorescence performance, has lower background and stronger signal, and has no difference from tiger in qPCR due to the characteristics, so that the AIE-qPCR technology is higher than the first floor. A large number of material molecules are accumulated in the development of AIE for years, the fluorescence performance of the material molecules is excellent, and the material molecules have the capability of modifying various reactive groups.

According to the novel AIE-qPCR method provided by the embodiment of the invention, a qPCR system comprises an AIE probe, a primer, dNTP, a substrate template and water.

The AIE probe is used as a main component of an AIE-qPCR technology and mainly used for tracing the amplification process of a target gene of qPCR reaction through the fluorescent lighting of AIE molecules from dissolved non-fluorescence to aggregation, so that the novel technology which has the same function as the traditional qPCR but different essence is realized.

In a preferred embodiment, the AIE molecule is selected from at least one of TPE and derivatives thereof.

Example 1

An AIE probe based on AIE molecule is prepared by selecting the AIE molecule from TPE and TPE-COOH (shown in figure 2) in its derivatives. Oligo is synthesized according to the required sequence, then the 5' end of Oligo is modified by Amino-C6 Linker suitable for solid phase phosphoryl triester method of Oligo, and finally TPE-COOH which has been activated by NHS is coupled to Oligo by condensation reaction with Amino. (see FIG. 3) base sequences of primers and probes for qPCR were designed based on the S gene of Hepatitis B Virus (HBV), and AIE probe 1 was synthesized with the following primer and probe sequences (5'→ 3'):

an upstream primer: CAACATCAGGATTCCTAGGACC

A downstream primer: GGTGAGTGATTGGAGGTTG

1, probe 1: AIE-LIN-CAGAGTCTAGACTCGTGGTGGACTTC

The present invention also performed mass spectrometry characterization of the AIE probe 1 (see fig. 4). Because the automatic integrated solid phase synthesizer is adopted, whether the base sequence and the modified AIE are successfully connected or not is judged, and the judgment can be carried out only by contrasting mass spectrum data of the product and the product in the last step and according to the increase or not of the molecular weight. The whole molecular weight of the probe is consistent with the structure, the molecular weight of Oligo sequence and AIE probe 1 is compared to increase the AIE and LIN parts, and the control results before and after reaction can judge that the synthesis of the AIE probe 1 is successful.

Example 2

The fluorescence release effect of the AIE probe 1 was verified by DNA hydrolase (37 ℃, 1h) under the concentration condition of qPCR reaction, and the fluorescence emission spectrum was detected by a microplate reader under the condition of optimum excitation wavelength (Ex ═ 460nm) (see fig. 5). The AIE probe 1 (probe concentration 1 μ M) in example 2 showed 20-fold increase in fluorescence after dnase action (FI 1: 10000/FI 0: 500) in free aggregate state (AIE probe 1 degraded by dnase action) and in dissolved state (AIE probe 1) with lower background fluorescence intensity per 100 μ L of well volume.

Example 3

The AIE-qPCR reaction system is optimized, the total reaction volume is 20 mu L, wherein the upstream primer and the downstream primer are respectively 0.4 mu L (the working concentration of the primers is 2 mu M), the AIE probe is 0.2 mu L (the working concentration of the probes is 1 mu M), the sample/nucleic acid template to be detected is 4 mu L, and the residual volume is filled with double distilled water. The optimal selection volume is 20. mu.L, including but not limited to 10. mu.L, 50. mu.L, 100. mu.L equal volumes.

Example 4

A plasmid containing HBV genes is designed, and the plasmid is used as a positive DNA template in a qPCR experiment, and a negative control in the qPCR experiment is double distilled water without the DNA template. A120 bp DNA product band can be amplified by using the primers and the probe designed in the example 1. (see figure 6) qPCR experiments were performed using QuantStudio 5 fluorescent quantitative PCR instrument of ABI, usa. The qPCR program was a two-step process with parameters set to: the first step is as follows: 10min at 95 ℃. The second step is that: 15s at 95 ℃; 20s at 60 ℃; 40 cycles. The fluorescent signal is detected at the end of each cycle, with the signal paths detected being: x1/m 4.

Example 5

According to the method of examples 1-4, multiple sets of experiment controls are set, so that the total reaction volume is larger than 100 μ L, the reaction product is collected after the qPCR experiment is finished, 100 μ L of the product is taken and detected by a microplate reader under the excitation wavelength of 460nm, and the fluorescence emission spectrum is used for the comparison test of 5'→ 3' enzyme cutting activity of the qPCR enzyme and DNA enzyme cutting (as shown in FIG. 7). The qPCR high-temperature cycle reaction system has little influence on the background fluorescence of the AIE probe, and the positive amplification is 2-3 times stronger than the negative amplification in peak intensity.

Example 6

The qPCR experiments were performed in 96 duplicate wells according to the methods of examples 1-4, and the results showed one hundred percent success rate of qPCR and the amplification curves showed good uniformity and stability (see fig. 8). And meanwhile, negative and positive control experiments are also carried out, the AIE-qPCR method is feasible, the AIE probe is stable in performance, the enzyme has no difference with the traditional fluorescent material in the qPCR process, and the qPCR reaction is not influenced. Meanwhile, in a negative control experiment, the temperature change of the qPCR amplification program has little influence on the AIE probe and always keeps very low background fluorescence.

Example 7

A standard curve was generated by performing AIE-qPCR experiments according to the method of examples 1-4 using plasmid template dilutions (1:10) in 5 concentration gradients as sample templates in the AIE-qPCR method, three replicate controls for each concentration gradient (see FIG. 9). The result shows that the standard curve has high regression curve fitting degree, the amplification efficiency also meets the requirements of the qPCR technology, the logarithmic growth period has good linear correlation in the qPCR amplification process, and a new method is developed for the application of the AIE material in the fluorescent quantitative PCR.

Example 8

3 HBV positive sera were selected and subjected to AIE-qPCR (see FIG. 10) according to the methods of examples 1-4, and the amplification curve showed that 3 clinical samples were positive and the cycle number was 6-12. The fluorescent quantitative PCR method based on the AIE probe can be applied to the nucleic acid detection of hepatitis B virus.

Example 9

An AIE probe based on AIE molecule is prepared by using AIE probe without fluorescence quenching group, wherein the AIE molecule is selected from TPE-COOH (shown in figure 2) in TPE derivative, and is connected to 3' end of Oligo. Amino modification was performed at the 3 'end of Oligo according to the synthetic method of example 1, and finally the condensation reaction of TPE-COOH, which had been activated with NHS, with amino group was coupled to the 3' end of Oligo (FIG. 11). The sequence of the probe 2 is as follows: CAACATCAGGATTCCTAGGACG are provided.

Example 10

An AIE probe based on AIE molecule is prepared by using AIE probe without fluorescence quenching group, wherein the AIE molecule is selected from azido methyl TPE (as shown in figure 12) in TPE derivative, and is connected to 5' end of Oligo. Amino modification is performed at the 5 'end of Oligo according to the synthetic method of example 1, condensation is performed with the carboxyl end of the connecting chain of activated carboxyl, and finally the other end of the connecting chain couples azidomethyl TPE to the 5' end of Oligo through click reaction (as shown in FIG. 13). The sequence of the probe 3 is as follows: GGTGAGTGATTGGAGGTTC are provided.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any equivalent alterations, modifications or improvements made by those skilled in the art to the above-described embodiments using the technical solutions of the present invention are still within the scope of the technical solutions of the present invention.