CN109884007B

CN109884007B - Integrated synchronous DNA nano device and living cell multi-target imaging application and imaging method thereof

Info

Publication number: CN109884007B
Application number: CN201910123442.8A
Authority: CN
Inventors: 赵永席; 薛静; 陈锋; 黄平
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2019-02-18
Filing date: 2019-02-18
Publication date: 2020-10-27
Anticipated expiration: 2039-02-18
Also published as: CN109884007A

Abstract

The invention discloses an integrated synchronous DNA nano device and living cell multi-target imaging application and an imaging method thereof, belonging to the technical field of living cell imaging. The invention relates to an integrated synchronous DNA nano device formed by gold nanoparticle (AuNP) loaded three sets of DNA chains, which is triggered by microRNA and/or telomerase in a living cell, driven by human 8-hydroxyguanine DNA glycosylase (hOGG1) and human source depurination/depyrimidinization (AP) endonuclease APE 1(APE1) to release fluorescent signals, and realizes the simultaneous imaging of multiple targets of the living cell. The integrated synchronous DNA nano device integrates all reaction modules, can avoid the difference of movement of separation modules in cytoplasm with different cell-entering efficiency and diffusion limitation, does not need to supply any substance by cell exogenous, and can simultaneously realize the accurate imaging analysis of multiple targets in living cells.

Description

Integrated synchronous DNA nano device and living cell multi-target imaging application and imaging method thereof

Technical Field

The invention belongs to the technical field of living cell imaging, and particularly relates to an integrated synchronous DNA nano device, and living cell multi-target imaging application and an imaging method thereof.

Background

Various biological molecules in living cells and corresponding interaction networks cooperatively regulate various metabolic processes in organisms. Therefore, the method can simultaneously monitor a plurality of intracellular biological targets and acquire comprehensive information, and has important significance in practical application such as biochemical analysis, clinical diagnosis and the like. Deoxyribonucleic acid (DNA) is a black horse in a probe construction material due to the outstanding advantage that the DNA follows the base complementary pairing principle and is accurately programmable. The DNA-based nano device has flexible and diversified structures and is widely applied to multi-target monitoring and imaging.

The existing DNA nano device comprises a molecular robot, a molecular motor, a DNA walking machine and the like, and can be theoretically applied to monitoring the expression difference of multiple targets in living cells simultaneously. However, most of the devices are composed of a plurality of separated modules, and different modules have different cell-entering efficiencies, so that intracellular chemometrics information is lost, signal differences are caused, accurate monitoring imaging is interfered, and misjudgment is generated. In addition, researchers have found that the diffusion coefficients of macromolecules and nanoscale structures in mammalian cell cytoplasm and other solutions differ by orders of magnitude. Therefore, the co-entry of a plurality of modules, cell-exogenous energy substances or nucleic acid probes into living cells complicates the reaction system and limits the reaction rate. Meanwhile, the DNA nano-devices lack a signal calibration module, cannot respond to heterogeneity among single cells, and have poor anti-interference capability of complex cytoplasmic environment, so that the imaging result is unreliable, and the research and application in the single cell field are limited.

Disclosure of Invention

The invention aims to provide an integrated synchronous DNA nano device, living cell multi-target imaging application and an imaging method thereof, which can overcome the defects of the prior art that the cell entrance efficiency of a separation module structure is different, the reaction kinetics is limited due to the limitation of intracytoplasmic diffusion, the signal calibration function is lacked, and the like.

The invention is realized by the following technical scheme:

the invention discloses an integrated synchronous DNA nano device, which comprises three sets of DNA chains modified on the surface of a gold nanoparticle AuNP, wherein each set of DNA chain consists of two DNA chains: one is single-stranded DNA in which the process of motion occurs, i.e., SD strand; the other is a stem-loop structure DNA chain with a specific chemical labeling base, namely an HD chain, wherein the annular part of the HD chain comprises 8-oxygen-7-hydrogen deoxyguanine, the 3 '-end sulfhydryl group is connected to the surface of the gold nanoparticle AuNP through a covalent bond, and the 5' -end is modified with fluorescent molecules FAM, Cy5 or TAMRA.

Preferably, the SD chain comprises an R-SD chain, a T-SD chain and a C-SD chain;

16 bases at the 5 'end of the R-SD strand and 16 bases at the 3' end of the Block DNA strand are complementary to form a double strand; when the target object microRNA-21 exists, 22 basic groups of the microRNA-21 are complemented with 22 basic groups of a Block DNA chain to form a double chain through a strand displacement reaction, and an R-SD chain is released;

a T-SD chain, wherein the 5' end of the T-SD-S chain (T-SD-S is a telomerase extension primer chain) is a telomerase primer, and when a target telomerase exists, a TTAGGG repetitive sequence is extended out to form the T-SD chain;

the C-SD strand, the 5' end partial base is complementary to the loop partial base of the C-HD strand, the movement of which is triggered directly by the enzymes hOGG1 and APE 1.

Further preferably, 14 bases of the 5' -end of the R-SD strand after release are complementary to 14 bases of the loop portion of the corresponding R-HD strand to form a double strand; the 14 bases at the 5' end of the T-SD strand are complementary to the 14 bases of the corresponding loop part of the T-HD strand to form a double strand; the 14 bases at the 5' -end of the C-SD strand are complementary to the 14 bases of the loop portion of the corresponding C-HD strand to form a double strand.

Further preferably, after the SD strand and the HD strand form a DNA double strand, the damaged base oG site on the HD strand can be acted by the enzyme hOGG1 to form an apurinic/apyrimidinic AP site, and then fluorescence is released after the enzyme is cut by APE 1.

The invention also discloses application of the integrated synchronous DNA nano device in living cell imaging.

Further, the integrated synchronous DNA nano-device can be used for imaging single detection or multiple detections in multiple targets in situ in different living cell samples.

The invention also discloses a living cell multi-target imaging method based on the integrated synchronous DNA nano device, which comprises the following steps:

1) introducing the integrated synchronous DNA nano device into living cells by a co-culture method;

2) the living cells introduced with the integrated synchronous DNA nano device are incubated and cultured for 4h at 37 ℃, and then signals of different fluorescence channels of the living cells are observed through a laser confocal fluorescence microscope, so that the simultaneous and synchronous accurate imaging of various targets is realized.

Compared with the prior art, the invention has the following beneficial technical effects:

the invention relates to an integrated synchronous DNA nano device formed by loading three sets of DNA chains on gold nanoparticles (AuNP). The integrated structure integrates all the reaction modules, can avoid the difference of movement of the separation modules in cytoplasm with different cell-entering efficiency and diffusion limitation, does not need to supply any substance exogenously to cells, and can simultaneously realize the accurate imaging analysis of multiple targets in living cells. The concrete advantages are as follows:

1) the integrated synchronous DNA nano device integrates the DNA probes specifically designed for various target objects and the DNA probes reflecting the activity of the tool enzyme in the cell on the surface of the gold nano particle, and the integrated structure synchronously enters the cell without being influenced by factors such as cell entering efficiency difference and the like, thereby improving the imaging accuracy.

2) The synchronous DNA nano device can utilize the integrated probe to realize the simultaneous monitoring of different targets in living cells and realize the deep and accurate research of the synergistic action of multiple targets.

3) The integrated synchronous DNA nano device is triggered by a specific target object in a living cell and driven by a cell endogenous enzyme, energy substances, reaction factors and the like are not required to be supplied by the cell exogenously, and the integrated synchronous DNA nano device has a simple reaction system and high reaction efficiency.

4) The DNA nano device has a reaction rate in the viscous cytoplasm higher than that in a separation module reaction mode (differential step cell-dissociation-diffusion-assembly-enzyme digestion), reaction kinetics is accelerated, and multi-target imaging sensitivity is improved.

5) The DNA motor provided by the invention comprises a signal calibration module in an integrated structure, can reflect signal differences caused by non-target objects in different cell samples, and realizes accurate imaging.

6) The integrated DNA nano device is composed of gold nanoparticles and DNA, has low cytotoxicity, and can realize simultaneous monitoring of multiple targets in cells by incubation with the cells.

Drawings

FIG. 1 is a schematic diagram showing the difference between the reaction processes of the integrated synchronous DNA nanodevice of the present invention and the prior art separation module DNA nanodevice in the cytoplasm of a viscous cell;

FIG. 2 is a comparison of the reaction kinetics of integrated synchronous DNA nanodevices and separation module probes under diffusion limiting conditions; wherein A is the action principle of the integrated synchronous DNA nano device; b is the reaction rate comparison of two DNA probes in diffusion limited environments with different viscosities; c, analyzing the single-particle fluorescence change of the two DNA nano devices by using an STED (stimulated emission depletion) fluorescence microscope;

FIG. 3 is a reaction kinetics study of an integrated synchronous DNA nanodevice; wherein, A is the reaction in the particle and the reaction between non-particles which proves that the DNA nanometer device; b is the influence of different SD chain modification densities on the reaction kinetics of the DNA nano device; c and D are detection range and specificity respectively;

FIG. 4 shows miR-21 monitoring results of different designs of integrated synchronous DNA nano-devices in living cells (HeLa); wherein A is the imaging result and the corresponding flow result of the DNA nano devices with different designs in a cell sample; b is the imaging result of a designed DNA nano device in cell samples with different APE1 activities;

FIG. 5 shows the simultaneous imaging of multiple targets in three samples of cells with different APE1 activities (HeLa) by the integrated synchronized DNA nano-device.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

Referring to fig. 1, a schematic diagram of the working in living cells comparing the integrated synchronous DNA nano-device and the separation module DNA nano-device provided by the present invention is shown in fig. 1, which compares the difference of the reaction processes of the integrated synchronous DNA nano-device and the separation module DNA nano-device in the cytoplasm of viscous cells, and it can be seen that the integrated DNA nano-device is cellular simultaneously, the endogenous tools of cell, i.e., chogg 1 and APE1, diffuse rapidly in the cytoplasm, and the integrated synchronous DNA nano-device reacts synchronously in situ, and the separation module probes have different cell-entering efficiencies and need to improve exogenous substances, and the reaction process is complex and has low efficiency.

Referring to fig. 2, the reaction process of the integrated synchronous DNA nano-machine is described in detail, and the reaction rates of the two DNA nano-devices of the integration module and the separation module under the diffusion limitation condition of different viscosities are monitored by using a real-time fluorescence intensity curve and a STED fluorescence microscope, and the result shows that the reaction rate of the integrated synchronous DNA nano-device of the present invention is higher than that of the reaction mode of the separation module under the viscosity diffusion limitation condition, which illustrates that the reaction rate of the integrated DNA nano-device of the present invention is significantly higher than that of the reaction process of the separation module.

Referring to fig. 3, reaction parameters of the miR-21-triggered integrated DNA nano-device are optimized, and the detection range and specificity thereof are tested, wherein a is to prove that the DNA nano-device is an intra-particle reaction, and a non-inter-particle reaction; b is the influence of different SD chain modification densities on the reaction kinetics of the DNA nano device; c and D are detection range and specificity respectively. Optimal reaction conditions can be obtained for subsequent living cell multi-target imaging.

The invention will be further explained and illustrated with reference to two embodiments:

example 1: and imaging the single target miR-21 in the living cells by using different designed integrated synchronous DNA nano devices.

A cervical cancer cell HeLa cell line is taken as a basic model, and 20 mu L of integrated synchronous DNA nano device probe is added into a HeLa cell (0.2mL, 1 multiplied by 10) pre-cultured in an eight-hole confocal culture dish⁶mL^-1) And incubated at 37 ℃ for 4 hours. After PBS washing, imaging was performed using a laser confocal fluorescence microscope. In target miR-21 positive cells, miR-21 replaces a Block chain sealing R-SD to release a single-chain R-SD, 14 bases at the 5' end of the single-chain R-SD are complementary with 14 bases of a corresponding R-HD chain loop part to form a double chain, wherein a damaged base oG site on the R-HD chain can be acted by an enzyme hOGG1 to form an apurinic/apyrimidinic AP site, and FAM fluorescence is released after the enzyme digestion by APE 1.

In addition, a DNA probe reflecting the activity of the intracellular tool enzyme, namely a C-SD chain with a signal calibration function and 14 bases of a corresponding cyclic part of a C-HD chain are also complemented into a double chain, wherein a damaged base oG site on the C-HD chain can be acted by an enzyme hOGG1 to form an AP site, and after the AP is cut by APE1, TAMRA fluorescence is released for the analysis and research of intracellular 'tool enzymes' hOGG1 and APE 1.

The result is shown in fig. 4, and it can be seen from the figure that the fluorescence intensity of the specific integrated DNA nano device is much higher than that of the negative control, and the same result is verified by the statistical data corresponding to the flow experiment; in addition, the DNA nanometer device with the signal calibration function can reflect the activity difference of two tool enzymes in the cell, and realize accurate imaging.

Example 2: and simultaneously imaging multiple targets in different living cells by using the integrated synchronous DNA nano device.

Taking a HeLa cell line of a cervical cancer cell as a basic model, and taking 20 mu L of integrated synchronous DNA nano-device probeHeLa cells (0.2mL, 1X 10) pre-cultured in eight-well confocal plates were added⁶mL^-1) And incubated at 37 ℃ for 4 hours. After PBS washing, imaging was performed using a laser confocal fluorescence microscope. When the target object miR-21 exists, a double strand is formed through strand displacement reaction and Block DNA strand complementation, and an R-SD strand is released; T-SD-S chain, the 5' primer is extended into the T-SD chain in the presence of the target telomerase. The 14 bases at the 5' end of the R-SD chain, the T-SD chain and the C-SD chain are complementary with the 14 bases of the corresponding cyclic part of the R-HD chain, the T-HD chain and the C-HD chain to form a double strand; after three sets of SD chains and HD chains form a DNA double chain, a damaged base oG site on the HD chain can be acted by enzyme hOGG1 to form an apurinic/apyrimidinic AP site, and then the AP 1 is subjected to enzyme digestion to release corresponding fluorescent signals FAM, Cy5 or TAMRA. TAMRA fluorescence was used for analytical studies of intracellular "tool enzymes" hOGG1 and APE 1.

The result is shown in fig. 5, and it can be seen from fig. 5 that the integrated synchronous DNA nano-device can realize simultaneous imaging of two target substances miR-21 and telomerase in different cell samples. In the cells treated by the telomerase inhibitor EGCG, the fluorescence of Cy5 is weakened, and the fluorescence signals of the other two cells are basically unchanged; the triple fluorescence signal was significantly reduced in the cell samples treated with the tool enzyme APE1 inhibitor. The result shows that the integrated DNA nano device can realize simultaneous and accurate imaging of multiple targets in living cells.

The DNA sequences used in the two examples above are shown in Table 1 below:

TABLE 1

Note: (1) underlined letters represent DNA damaging bases;

(2) italicized letters represent mismatched bases of miR-21.

The embodiments given above are preferable examples for implementing the present invention, and the present invention is not limited to the above-described embodiments. Any non-essential addition and replacement made by the technical characteristics of the technical scheme of the invention by a person skilled in the art belong to the protection scope of the invention.

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Claims

1. The integrated synchronous DNA nano device is characterized by comprising three sets of DNA chains modified on the surface of a gold nanoparticle AuNP, wherein each set of DNA chain consists of two DNA chains: one is single-stranded DNA in which the process of motion occurs, i.e., SD strand; the other is a stem-loop structure DNA chain with a specific chemical labeling base, namely an HD chain, wherein the annular part of the HD chain comprises 8-oxo-7-hydrodeoxyguanine, the 3 '-end sulfhydryl is connected to the surface of the gold nanoparticle AuNP through a covalent bond, and the 5' -end is modified with fluorescent molecules FAM, Cy5 or TAMRA;

the SD chain comprises an R-SD chain, a T-SD chain and a C-SD chain;

the 5' end of the T-SD-S chain is a telomerase primer, and when the telomerase exists in a target object, a TTAGGG repetitive sequence is extended out to form the T-SD chain;

the C-SD strand, the 5' end partial base is complementary to the loop partial base of the C-HD strand, the movement of which is triggered by the enzymes hOGG1 and APE 1;

the 14 bases at the 5' end of the released R-SD chain are complementary to the 14 bases of the corresponding cyclic part of the R-HD chain to form a double chain; the 14 bases at the 5' end of the T-SD strand are complementary to the 14 bases of the corresponding loop part of the T-HD strand to form a double strand; the 14 bases at the 5' -end of the C-SD strand are complementary to the 14 bases of the loop part of the corresponding C-HD strand to form a double strand;

after the SD chain and the HD chain form a DNA double chain, a damaged base oG site on the HD chain can be acted by an enzyme hOGG1 to form an apurinic/apyrimidinic AP site, and then fluorescence is released after the APE1 enzyme digestion.

2. Use of the integrated synchronized DNA nanodevice of claim 1 for living cell imaging.

3. The use of claim 2, wherein the integrated simultaneous DNA nanodevices are capable of imaging single or multiple detection of multiple targets in situ in different living cell samples.

4. The living cell multi-target imaging method based on the integrated synchronous DNA nano-device of claim 1, characterized by comprising the following steps: