Fluorescent probe for monitoring apoptosis and preparation method and application thereof

文档序号：1083450 发布日期：2020-10-20 浏览：13次中文

阅读说明：本技术 一种用于细胞凋亡监控的荧光探针及其制备方法和应用 (Fluorescent probe for monitoring apoptosis and preparation method and application thereof ) 是由宋春元董晨张晶晶汪联辉于 2020-06-12 设计创作，主要内容包括：本发明公开一种用于细胞凋亡监控的荧光探针及其制备方法和应用,所述荧光探针包括具有三个探针结合臂的DNA四面体核酸框架纳米结构、Cy3修饰的DNA单链作为定位探针、细胞色素c探针和端粒酶探针,所述定位探针、细胞色素c探针、端粒酶探针与DNA四面体核酸框架纳米结构的三个探针结合臂通过核酸互补杂交结合,所述细胞色素c探针包含细胞色素c适配体链和细胞色素c捕获链的杂交双链,所述端粒酶探针包含端粒酶捕获链、端粒酶模板链和端粒酶染料链的杂交双链。本发明所述DNA四面体核酸框架型细胞凋亡全程监控荧光探具有灵敏度性高、稳定性好、选择性强、低毒性的特点,在细胞凋亡监控和药物评价筛选等方面表现出很好地应用性能。(The invention discloses a fluorescent probe for monitoring apoptosis and a preparation method and application thereof, wherein the fluorescent probe comprises a DNA tetrahedral nucleic acid framework nano structure with three probe combination arms, a Cy3 modified DNA single chain as a positioning probe, a cytochrome c probe and a telomerase probe, the positioning probe, the cytochrome c probe and the telomerase probe are combined with the three probe combination arms of the DNA tetrahedral nucleic acid framework nano structure through nucleic acid complementary hybridization, the cytochrome c probe comprises a hybrid double chain of a cytochrome c aptamer chain and a cytochrome c capture chain, and the telomerase probe comprises a telomerase capture chain, a telomerase template chain and a hybrid double chain of a telomerase dye chain. The DNA tetrahedral nucleic acid framework type cell apoptosis whole-process monitoring fluorescent probe has the characteristics of high sensitivity, good stability, strong selectivity and low toxicity, and shows good application performance in the aspects of cell apoptosis monitoring, drug evaluation and screening and the like.)

1. A fluorescent probe for monitoring apoptosis is characterized by comprising a DNA tetrahedral nucleic acid framework nano structure with three probe binding arms, a Cy3 modified DNA single chain serving as a positioning probe, a cytochrome c probe and a telomerase probe, wherein the positioning probe, the cytochrome c probe and the telomerase probe are combined with the three probe binding arms of the DNA tetrahedral nucleic acid framework nano structure through nucleic acid complementary hybridization, the cytochrome c probe comprises a hybridized double chain of a cytochrome c aptamer chain and a cytochrome c capture chain, and the telomerase probe comprises a hybridized double chain of a telomerase capture chain, a telomerase template chain and a telomerase dye chain.

2. The fluorescent probe for apoptosis monitoring according to claim 1, wherein the DNA tetrahedral nucleic acid framework nanostructure with three probe binding arms is formed by hybridization of six single-stranded nucleotides through base complementary pairing with each other, the three probe binding arms are located in the middle of three edges in the tetrahedral structure, and the base sequences of the six single-stranded nucleotides are respectively as follows:

ssDNA A：

ssDNA B：

ssDNA C：

ssDNA D：

ssDNA E：

ssDNA F：

3. the fluorescent probe for apoptosis monitoring according to claim 1, wherein the nucleotide base sequence of the localization probe is as follows:

4. the fluorescent probe for apoptosis monitoring according to claim 1, wherein the nucleotide base sequences of the cytochrome c aptamer chain and cytochrome c capture chain are as follows:

cytochrome c aptamer chain:

cytochrome c capture chain:

5. the fluorescent probe for apoptosis monitoring according to claim 1, wherein the nucleotide base sequences of the hybridized double strands of the telomerase capture strand, the telomerase template strand and the telomerase dye strand are as follows:

telomerase capture strand: 5 '-BHQ 1-AACCCTAACCCTAACTCTGCTCGACGGATTATGTTAAGTAACTCTAGAAT-3'

Telomerase template strand: 5'-AATCCGTCGAGCAGAGTT-3'

Telomerase dye chain: 5 '-AGGGTTAGGGTT-FAM-3'.

6. The method for preparing a fluorescent probe for apoptosis monitoring as claimed in any one of claims 1 to 5, comprising the steps of:

1) construction of DNA tetrahedral nucleic acid framework nanostructures: mixing ssDNA A, ssDNA B, ssDNA C, ssDNA D, ssDNA E and ssDNA F with equal amount in PBS buffer solution, annealing and cooling to obtain a DNA tetrahedral nucleic acid framework structure;

2) construction of cytochrome c probes: mixing equal amounts of cytochrome c capture chain and cytochrome c aptamer in PBS buffer, annealing and cooling;

3) construction of telomerase Probe: mixing the equal amount of the telomerase capture chain, the telomerase template chain and the telomerase dye chain in a PBS buffer solution, annealing and finally cooling;

4) and uniformly mixing the DNA tetrahedral nucleic acid framework structure, the cytochrome c probe, the telomerase probe and the positioning probe, and carrying out incubation reaction to form a cytochrome c and telomerase binary detection probe, namely the fluorescent probe for monitoring apoptosis.

7. The method for preparing a fluorescent probe for apoptosis monitoring according to claim 6, wherein in the steps 1), 2) and 3), the annealing reaction conditions are specifically: 10min at 95 ℃.

8. The method for preparing a fluorescent probe for apoptosis monitoring according to claim 6, wherein in the steps 1), 2) and 3), the cooling reaction conditions are specifically as follows: the reaction was carried out at 37 ℃ for 1 hour.

9. Use of the fluorescent probe for apoptosis monitoring according to any one of claims 1 to 5 in cytochrome c detection, telomerase activity detection or apoptosis overall process monitoring.

10. Use of the fluorescent probe for apoptosis monitoring according to any one of claims 1 to 5 in drug efficacy evaluation and drug screening.

Technical Field

The invention belongs to the field of functional nano materials and biological detection, and particularly relates to a fluorescent probe for monitoring apoptosis as well as a preparation method and application thereof.

Background

Cytochrome c, which contains metalloproteins, is located in the mitochondrial membrane space and is released into the blood under pathological conditions. Cytochrome c release by mitochondria is a key active step in activating cell death pathways, and cytochrome c leakage is generally considered as an early event of apoptosis and indicates the beginning of apoptosis, so that detection of cytochrome c has important significance for monitoring early apoptosis of cells. Telomerase is a ribonucleoprotein reverse transcriptase responsible for maintaining cell telomere length. In the late stage of apoptosis, telomerase is released from the cell nucleus outwards along with the breakage of the nuclear membrane, and thus can be used as a late-cell marker. In addition, telomerase is expressed in almost all malignant cells and has become a promising biomarker. Therefore, accurate and efficient implementation of telomerase in situ imaging is a very essential medical diagnosis. At present, no probe can realize combined detection of early and late markers of cells. In order to monitor the whole process of apoptosis, real-time and in-situ detection of cytochrome c and telomerase is required.

There are many methods currently used for cytochrome c or telomerase detection, respectively, such as electrochemical, raman, and fluorescence spectroscopy. Among these developed methods, the fluorescence method has the characteristics of high sensitivity, simplicity, low instrument cost, and the like, so that the fluorescence-based method exhibits significant advantages in the aspect of application prospects. However, the current method can only be used for detecting a single marker in cytochrome c or telomerase, and reports for simultaneously detecting probes are not available, so that the detection of the whole apoptosis process is difficult to realize. In addition, the joint detection of multiple markers also improves the reliability of detection and ensures the accuracy of detection.

DNA can be self-assembled according to the base complementary pairing principle, and the space structure has higher controllability and precision, so that the DNA nano-material with various forms can be easily assembled, and a plurality of unique advantages are shown: (1) the nanometer material based on the DNA composition is easy to penetrate cell membranes with negative electricity; (2) compared with the problem that other nano materials generally have cytotoxicity, the DNA nano materials have almost no cytotoxicity; (3) the material can better resist the ribozyme action and has high stability; (4) the material has rich functional modification sites, and can modify biomolecules or fluorescent dyes and the like according to different requirements. Currently, DNA nanotechnology, which uses DNA as a basic element, is of great interest. Different from a complex DNA polyhedral structure, the functionalized DNA tetrahedral nano material has simple self-assembly and low cost, and is one of the most ideal biological carriers.

Disclosure of Invention

The purpose of the invention is as follows: the invention combines a plurality of double-stranded probes to construct a cytochrome c and telomerase double-element detection probe based on a DNA tetrahedron, realizes the monitoring of the whole apoptosis process, can evaluate and screen the effect of apoptosis drugs based on the detection, and has wide application prospect.

The fluorescent probe can simultaneously detect cytochrome c and telomerase, and the method comprises the following steps: firstly, hybridizing six DNA single strands to obtain a DNA tetrahedral nucleic acid framework, and hybridizing a cytochrome c aptamer and a cytochrome c capture chain to obtain a cytochrome c probe; and hybridizing the telomerase capture chain, the telomerase template chain and the telomerase dye chain to obtain the telomerase probe. After the preparation is finished, the DNA tetrahedral nucleic acid framework, the cytochrome c probe, the telomerase probe and the positioning probe are uniformly mixed and hybridized in equal quantity, the apoptosis fluorescent probe with three signal emissions of FAM, Cy3 and Cy5 is constructed, and the simultaneous detection of cytochrome c and telomerase can be realized.

The invention also aims to solve the technical problem of providing a preparation method of the fluorescent probe for monitoring apoptosis. The method comprises the steps of hybridizing six DNA single strands to obtain a DNA tetrahedral nucleic acid framework, and hybridizing a cytochrome c aptamer and a cytochrome c capture chain to obtain a cytochrome c probe; and hybridizing the telomerase capture chain, the telomerase template chain and the telomerase dye chain to obtain the telomerase probe. After the preparation is finished, the DNA tetrahedral nucleic acid framework, the cytochrome c probe, the telomerase probe and the positioning probe are uniformly mixed and hybridized in equal quantity, the apoptosis fluorescent probe with three signal emissions of FAM, Cy3 and Cy5 is constructed, and the simultaneous detection of cytochrome c and telomerase can be realized.

The invention also aims to solve the technical problem of providing the application of the fluorescent probe for monitoring apoptosis in the whole process monitoring of apoptosis.

The invention finally solves the technical problem of providing the application of the fluorescent probe for monitoring apoptosis in drug effect evaluation and drug screening.

The technical scheme is as follows: in order to solve the technical problem, the invention provides a fluorescent probe for monitoring apoptosis, which comprises a DNA tetrahedral nucleic acid framework nanostructure with three probe binding arms, a Cy3 modified DNA single chain as a positioning probe, a cytochrome c probe and a telomerase probe, wherein the positioning probe, the cytochrome c probe and the telomerase probe are combined with the three probe binding arms of the DNA tetrahedral nucleic acid framework nanostructure through nucleic acid complementary hybridization, the cytochrome c probe comprises a hybrid double chain of a cytochrome c aptamer chain and a cytochrome c capture chain, and the telomerase probe comprises a hybrid double chain of a telomerase capture chain, a telomerase template chain and a telomerase dye chain.

Wherein, the DNA tetrahedral nucleic acid framework nano-structure with three probe binding arms is formed by six nucleotide single strands through mutual base complementary pairing hybridization, the three probe binding arms are positioned in the middle of three edges in the tetrahedral structure, and the base sequences of the six nucleotide single strands are respectively as follows:

ssDNA A：

ssDNA B：

ssDNA C：

ssDNA D：

ssDNA E：

ssDNA F：

wherein the nucleotide base sequence of the positioning probe is as follows:

wherein the nucleotide base sequences of the cytochrome c aptamer chain and the cytochrome c capture chain are as follows:

cytochrome c aptamer chain:

cytochrome c capture chain:

wherein the nucleotide base sequences of the hybrid double strands of the telomerase capture strand, the telomerase template strand and the telomerase dye strand are as follows:

telomerase capture strand:

telomerase template strand:

telomerase dye chain:

the invention also discloses a preparation method of the fluorescent probe for monitoring apoptosis, which comprises the following steps:

1) construction of DNA tetrahedral nucleic acid framework nanostructures: mixing ssDNA A, ssDNA B, ssDNA C, ssDNAD, ssDNA E and ssDNA F with equal amount in PBS buffer solution, annealing and cooling to obtain a DNA tetrahedral nucleic acid framework structure;

2) construction of cytochrome c probes: mixing equal amounts of cytochrome c capture chain and cytochrome c aptamer in PBS buffer, annealing and cooling;

In the steps 1), 2) and 3), the PBS buffer is 2 XPBS buffer, the concentration of the 2 XPBS, namely phosphate buffer, is preferably 20mM, and the pH is preferably 7.4.

Wherein, in the steps 1), 2) and 3), the annealing reaction conditions are specifically as follows: 10min at 95 ℃.

Wherein, in the steps 1), 2) and 3), the cooling reaction conditions are specifically as follows: the reaction was carried out at 37 ℃ for 1 hour.

The invention also comprises the application of the fluorescent probe for monitoring apoptosis in cytochrome c detection, telomerase activity detection or whole apoptosis monitoring.

The invention also comprises the application of the fluorescent probe for monitoring apoptosis in drug effect evaluation and drug screening.

The invention also comprises the application of the fluorescent probe for monitoring apoptosis in the screening process of tumor treatment drugs.

The invention also discloses an application of the apoptosis whole-process monitoring fluorescent probe constructed based on the DNA tetrahedral nucleic acid framework in detecting cytochrome c, wherein the cytochrome c detection comprises the following specific steps:

and (3) detecting cytochrome c: mu.L of the apoptosis probe prepared in the above step was diluted to 90. mu.L with 1 XPBS buffer, 10. mu.L of cytochrome c of various concentrations was added, and then the fluorescence intensity of the solution was measured at an excitation wavelength of 640 nm. With the increase of the added concentration, the red fluorescence intensity of Cy5 is continuously enhanced, the cytochrome c concentration and the Cy5 fluorescence signal present good linearity in the range of 20nM-2 MuM, the detection limit is 85.93nM, and the method can be used for high-efficiency detection of cytochrome c.

Wherein the concentration of the 1 XPBS buffer solution is preferably 10mM, and the pH is preferably 7.4.

The invention also provides an application of the apoptosis whole-course monitoring fluorescent probe constructed based on the DNA tetrahedral nucleic acid framework in detecting the activity of telomerase, wherein the telomerase detection comprises the following specific steps:

detecting telomerase activity: mu.L of the apoptosis probe prepared in the above step was diluted to 90. mu.L with 1 XPBS buffer, 10. mu.L of telomerase (HeLa cell lysate) of various concentrations was added, and the fluorescence intensity of the solution was measured at an excitation wavelength of 494 nm. With the increase of the added concentration, the green fluorescence intensity of the FAM is continuously enhanced, the telomerase concentration and the FAM fluorescence signal show good linearity in the range of 0.625-70 cells/mu L, the detection limit is 1.54 cells/mu L, and the method can be used for high-sensitivity detection of the telomerase.

Wherein the concentration of the 1 XPBS buffer solution is preferably 10mM, and the pH is preferably 7.4.

The invention also provides application of the apoptosis whole-course monitoring fluorescent probe constructed based on the DNA tetrahedral nucleic acid framework in monitoring apoptosis, wherein the apoptosis monitoring step specifically comprises the following steps:

monitoring of apoptosis process: 10000 HeLa cells are added into a confocal dish, the cells adhere to the wall after overnight growth, the cells are washed three times by 1 XPBS, 1ml of 50nM is added and dissolved in DMEM culture solution for incubation for 2 hours, 2 microliter of 1mM STS is added, the confocal dish is immediately placed under a confocal fluorescence microscope, and Cy3 representing a probe position signal, a Cy5 signal representing a cytochrome c signal and a FAM signal representing a telomerase signal in the cells are observed in real time. The Cy3 signal was excited by 559nm laser, the Cy5 signal was excited by 635nm laser, and the FAM signal was excited by 488nm laser.

The invention also provides application of the apoptosis probe constructed based on the DNA tetrahedron in the aspects of drug evaluation and screening, and the drug evaluation and screening steps are as follows:

drug evaluation and screening: 10000 HeLa cells are added into a confocal dish, the cells adhere to the wall after the cells grow overnight, the cells are washed three times by 1 XPBS, 1ml of 50nM is added and dissolved in DMEM culture solution for incubation for 2 hours, then anticancer drugs with the final concentration of 2 MuM are added for incubation for 2 hours, the confocal dish is placed under a confocal fluorescence microscope, and Cy5 signals representing cytochrome c signals and FAM signals representing telomerase signals in the cells are observed. Similarly, the Cy3 signal was excited with a 559nm laser, the Cy5 signal was excited with a 635nm laser, and the FAM signal was excited with a 488nm laser.

Wherein the Cy3 has a probe localization effect.

Wherein the Cy5 or red fluorescence has a cytochrome c monitoring effect and has a characteristic of specifically responding to cytochrome c.

The FAM or green fluorescence has a telomerase monitoring effect and has the characteristic of specifically responding to telomerase.

The reaction mechanism is as follows: the six DNA single strands A, B, C, D, E, F of the invention are hybridized by simple annealing to obtain a multi-arm DNA tetrahedral nucleic acid framework. By the same method, the cytochrome c Capture Chain (CC) modified with the quencher BHQ-2 can be hybridized with the cytochrome C Aptamer (CA) modified with the dye Cy5 to obtain the cytochrome c probe, and the fluorescent signal of Cy5 is quenched by BHQ-2. Similarly, a telomerase capture chain (TC) modified with a quencher BHQ-1, a telomerase template chain (TP) and a telomerase dye chain (TD) modified with a dye FAM are hybridized to obtain a telomerase probe, and the fluorescence of the FAM close to the BHQ-1 is quenched. And then, incubating the DNA tetrahedral nucleic acid framework, the cytochrome c probe and the telomerase probe with a fluorescence localization chain (FT) to form the DNA tetrahedral nucleic acid framework type apoptosis whole-process monitoring fluorescent probe. Cy3 on the fluorescent mapping strand (FT) allows the probe to be traced and the probe location to be accurately located within the cell. When cytochrome c is present, cytochrome C Aptamer (CA) on the apoptosis probe will bind to cytochrome c and detach from the probe, and the dye Cy5, which was quenched, is restored. When telomerase exists, a telomerase template chain (TP) can be extended under the action of the telomerase, a telomerase dye chain (TD) is replaced, and the originally quenched dye FAM is recovered. In conclusion, the DNA tetrahedral nucleic acid framework type apoptosis whole-process monitoring fluorescent probe can realize combined detection on cytochrome c and telomerase, and realize apoptosis monitoring and drug evaluation screening based on the detection.

The DNA tetrahedral nucleic acid framework type apoptosis whole-process monitoring fluorescent probe has the advantages of good selectivity and simultaneous detection of multiple substances, when cytochrome c exists in a solution, the combination of cytochrome c aptamer and cytochrome c is separated from a cytochrome c capturing chain, and the distance between Cy5 on the cytochrome c aptamer and BHQ-2 on the cytochrome c capturing chain is increased, so that the recovery of red fluorescent signals is realized. The intensity of Cy5 fluorescence emission gradually increased with increasing cytochrome c concentration; similarly, when telomerase exists in the solution, the telomerase template chain extends under the action of the telomerase to replace the telomerase dye chain from the telomerase capture chain, so that FAM on the telomerase dye chain is far away from BHQ-1 on the telomerase capture chain, and the recovery of a green fluorescent signal is realized. The fluorescence emission intensity of FAM gradually increased with increasing telomerase concentration. After the probe enters the cell, a bright probe positioning signal existing in the cell can be observed, then an apoptosis inducer STS is added, the early apoptosis is accompanied by the leakage of cytochrome c from mitochondria, a red fluorescence signal corresponding to the cytochrome c can be observed firstly, and a green fluorescence signal at the late apoptosis stage belonging to telomerase is gradually enhanced along with the time lapse. The monitoring of the whole process of apoptosis is realized through the fluorescence signals of the two markers.

Has the advantages that: compared with the prior art, the invention has the following advantages: the invention firstly synthesizes a DNA tetrahedral nucleic acid framework with three capture segments, a cytochrome c probe with cytochrome c response capability, a telomerase probe with telomerase response capability and a fluorescent positioning probe with positioning capability, and then connects three active components to the DNA tetrahedral nucleic acid framework by utilizing a base complementary pairing mode to construct a novel apoptosis monitoring fluorescent probe. The probe has three fluorescent signals of Cy3, Cy5 and FAM, can accurately position the probe position in a cell, can realize quantitative analysis of cytochrome c and telomerase, realizes simultaneous detection of the cytochrome c and the telomerase, realizes monitoring of the whole apoptosis process based on the quantitative analysis, and can evaluate the curative effect of tumor treatment drugs according to the apoptosis condition. The apoptosis fluorescent probe has the advantages of high sensitivity, good stability, strong selectivity, low toxicity and the like.

Drawings

FIG. 1 is a schematic diagram of the operation of the fluorescent probe for apoptosis monitoring of the present invention;

FIG. 2 is a PAGE gel electrophoresis characterization construction and its working mechanism of the fluorescent probe for apoptosis monitoring of the present invention;

FIG. 3 is a graph showing the behavior of fluorescent probes for apoptosis monitoring of cytochrome c and telomerase according to the present invention;

FIG. 4 is a representation of the specificity of the fluorescent probes for apoptosis monitoring of the present invention;

FIG. 5 is a graph showing the stability of the fluorescent probe for apoptosis monitoring of the present invention;

FIG. 6 is a comparison of the resistance of the fluorescent probe for apoptosis monitoring of the present invention to enzymatic degradation with a conventional double-stranded probe;

FIG. 7 is a fluorescent probe real-time in situ fluorescence imaging of apoptosis for apoptosis monitoring of the present invention;

FIG. 8 is fluorescence imaging of apoptosis induced by different concentrations of STS by the fluorescent probe for apoptosis monitoring of the present invention;

FIG. 9 is fluorescence imaging of multiple drug-induced apoptosis with the fluorescent probe for apoptosis monitoring of the present invention;

FIG. 10 is fluorescence imaging of apoptosis induced by different concentrations of EGCG using the fluorescence probe for apoptosis monitoring of the present invention;

FIG. 11 is fluorescence imaging of photothermally induced apoptosis with the fluorescent probes for apoptosis monitoring of the present invention.

Detailed Description

As mentioned above, apoptosis is important for the growth, development and aging of organisms. However, the inventor finds that most apoptosis probes can only detect and image one apoptosis marker, a single marker has certain limitation on apoptosis, and at present, one probe capable of monitoring the whole apoptosis process is still lacked.

In view of the above, the invention develops a fluorescent probe for monitoring apoptosis, which is constructed by 6 DNA single strands and has three capturing chains, wherein the three capturing chains are connected with a cytochrome c probe, a telomerase probe and a fluorescence positioning chain. The fluorescence of the fluorescent probe is quenched, the fluorescence signal of Cy5 can be recovered only in the presence of cytochrome c, the fluorescence signal of FAM can be recovered only in the presence of telomerase, and the monitoring of the apoptosis process and the screening of tumor treatment drugs can be realized based on the combined detection of the two markers respectively corresponding to the early stage and the late stage of apoptosis.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. In addition, molecular biological methods which are not described in detail in the examples are all conventional methods in the field, and specific operations can be referred to molecular biological guidelines or product specifications. The DNA sequence and the probe sequence used by the invention are as follows:

ssDNA A：