阅读说明：本技术 基于非天然核酸纳米镊子的特异性检测技术在活细胞mRNA检测中的检测方法及其应用 (Detection method of specificity detection technology based on non-natural nucleic acid nano tweezers in living cell mRNA detection and application thereof ) 是由 李喆 彭劲 于涵洋 于 2019-10-28 设计创作，主要内容包括：本发明公开了基于非天然核酸纳米镊子的特异性检测技术在活细胞mRNA检测中的检测方法及其应用。首先非天然核酸单链通过碱基互补配对形成打开的纳米镊子结构,其中一条单链的5’与3’端分别标记荧光基团。然后利用非天然核酸稳定性高的特点,在细胞内使用纳米镊子检测mRNA分子；当存在目标mRNA分子时,纳米镊子的单链部分将与之进行互补配对,从而关闭纳米镊子,使荧光基团之间发生能量共振转移,通过光学信号的变化达到检测效果。最后加入燃料单链使纳米镊子恢复打开状态,使之可以进行下一轮检测。本发明高灵敏度的细胞无损检测。基于荧光共振能量转移的纳米镊子,具有极高的检测灵敏度,检测速度快,且具有良好的生物相容性,可直接进入细胞进行无损检测。(The invention discloses a detection method and application of a specificity detection technology based on non-natural nucleic acid nano tweezers in living cell mRNA detection. Firstly, an unnatural nucleic acid single strand forms an open nano-tweezers structure through base complementary pairing, wherein the 5 'end and the 3' end of one single strand are respectively marked with a fluorescent group. Then, the characteristic of high stability of the non-natural nucleic acid is utilized, and the mRNA molecules are detected in the cells by using nano tweezers; when the target mRNA molecule exists, the single-chain part of the nano-tweezers is subjected to complementary pairing with the target mRNA molecule, so that the nano-tweezers are closed, energy resonance transfer is generated between fluorescent groups, and the detection effect is achieved through the change of optical signals. Finally, adding a fuel single chain to restore the opening state of the nano-tweezers, so that the nano-tweezers can be subjected to the next round of detection. The invention relates to a cell nondestructive test with high sensitivity. The nanometer tweezers based on fluorescence resonance energy transfer have extremely high detection sensitivity, high detection speed and good biocompatibility, and can directly enter cells for nondestructive detection.)

1. The detection method of the specificity detection technology based on the non-natural nucleic acid nano tweezers in the detection of living cell mRNA is characterized by comprising the following steps:

(1) designing and synthesizing non-natural nucleic acid nano tweezers;

(2) detecting target mRNA in living cells by using nano tweezers;

(3) fluorescence spectroscopy was performed.

2. The method for detecting the mRNA of the living cells by the non-natural nucleic acid nanotweezer-based specific detection technology according to claim 1, wherein the detection method comprises the following steps: in the step (1), the construction of the different non-natural nucleic acid nano-tweezers designs three non-natural nucleic acid single strands: two ends of the central chain are marked by two fluorescent groups capable of generating energy resonance transfer, and the other two single chains respectively extend out of a plurality of base sequences for carrying out base complementary pairing with target mRNA; can be realized by changing the chemical species of the non-natural nucleic acid according to different detection environment requirements.

3. The method for detecting the mRNA of the living cells by the non-natural nucleic acid nanotweezer-based specific detection technology according to claim 2, wherein the detection method comprises the following steps: in the step (1), the first step of the method,

step 1: two methoxy-modified non-natural nucleic acid single strands and one fluorophore-modified single strand are respectively mixed in a proportion of 1: 1: 1, 10. mu.L of 10 XTAE-Mg was added²⁺Buffer solution, Mg in the buffer solution²⁺The concentration is 12.5mol/L, the pure water is supplemented to the final volume of 100 mu L, the oscillation is uniform, and the specific volume can be set according to the requirementFilling, and finally TAE-Mg²⁺The buffer solution is 1X;

step 2: and (3) placing the mixed solution into a PCR instrument, annealing at the speed of 0.1 ℃/10s from 95 ℃ to 20 ℃, assembling into a 1 mu M methoxyl modified nano-tweezers structure, and placing at 4 ℃ for later use.

4. The method for detecting the mRNA of the living cells by the non-natural nucleic acid nanotweezer-based specific detection technology according to claim 1, wherein the detection method comprises the following steps: in the step (2), the target mRNA is detected, and when the target mRNA exists in the system, the nano-tweezers are changed from the original open state to the closed state; when mRNA molecules which are completely complementary and paired do not exist in the system, the structure of the nano tweezers can not be changed, so that the high specificity of detection is ensured.

5. The method for detecting the mRNA of the living cells by the non-natural nucleic acid nanotweezer-based specific detection technology according to claim 1, wherein the detection method comprises the following steps: in Step (2), Step 1: viable cells cultured in a 96-well plate were washed three times with PBS buffer, and then 20. mu.L of assembled methoxy-modified nanotweezer and 180. mu.L of culture medium (10% serum + 89% high-sugar culture medium + 1% antibody) were added at 37 ℃ with 5% CO₂For 3 hours, allowing it to enter the cells and bind to the target mRNA;

step 2: within this system with nanotweezer: the fuel chain is 1: 1 molar ratio, 5% CO at 37 ℃ in the fuel chain₂And (3) allowing the cells to enter and bind to the target mRNA, and displacing the target mRNA from the nanotweezer so that the nanotweezer returns to the initial open state for the next round of detection.

6. The method for detecting the mRNA of the living cells by the non-natural nucleic acid nanotweezer-based specific detection technology according to claim 1, wherein the detection method comprises the following steps: in the step (3), in the fluorescence spectrum analysis, when the nano tweezers are changed from the original open state to the closed state, the two carried fluorescent groups are close to each other, the fluorescence energy resonance transfer is increased, and the detection purpose is achieved through the change of the optical signal.

7. The method for detecting the mRNA of the living cells by the non-natural nucleic acid nanotweezer-based specific detection technology according to claim 6, wherein the detection method comprises the following steps: in the step (3), placing the methoxy-modified nano-tweezers, the co-incubated product of the nano-tweezers and the living cells and the incubated product added with the fuel single chain under an enzyme-labeling instrument for detection, setting the excitation wavelength to be 513nm, using the living cells and the culture solution as a zero-setting group, repeating the three experiments, and taking an average value; when target mRNA exists in cells, the fluorescence resonance transfer phenomenon can be observed in the system, and after the fuel chain is added, the fluorescence resonance transfer phenomenon disappears, so that the nano tweezers can be restored to an initial opening state.

8. Use of the methoxy-modified non-natural nucleic acid nanotweezer of claim 1 for monitoring diabetes.

9. The non-natural nucleic acid nanotweezer-based specific detection technique of any one of claims 1-8 and its use for the detection of mRNA of living cells.

10. The non-natural nucleic acid nanotweezer-based specific detection technique and its use in the detection of mRNA of living cells according to claim 9, characterized in that: the change of different target mRNAs can be dynamically monitored in real time according to the requirement; the assembled non-natural nucleic acid single strand can be chemically modified to have specific performance and adapt to intracellular detection; the detection steps are as follows:

(1) co-culturing the non-natural nucleic acid nano tweezers and the cells to be detected, wherein the non-natural nucleic acid nano tweezers enter the cells and are combined with target mRNA;

(2) the combination of the non-natural nano tweezers and the target mRNA changes the non-natural nano tweezers from an open state to a closed state to trigger fluorescence energy resonance transfer;

(3) and observing the fluorescence change before and after the cell reaction on a fluorescence detection platform.

Technical Field

The invention relates to the technical field of detection analysis and biology, in particular to a detection method and application of a non-natural nucleic acid nano-tweezers-based specific detection technology in living cell mRNA detection.

Background

mRNA is one of important biological macromolecules in organisms, and the expression level, stability and temporal-spatial distribution of mRNA in cells are closely related to the functions of mRNA. Real-time detection of mRNA expression changes will greatly facilitate disease diagnosis and increase of monitoring levels, thus leading to more and more extensive research interests.

At present, researchers mainly extract and purify mRNA in cell lysates, and then quantitatively analyze the expression of mRNA at the cellular level by methods such as polymerase chain reaction and the like. These methods do not allow for the comprehensive acquisition of information about the vital activities of living cells, since they require the destruction of the cells. In recent years, molecular beacons have been widely used for detection of mRNA in living cells. It is easy to construct and characterize, and does not require disruption of the cell or recombinant gene to detect mRNA expression in the cell. However, molecular beacons are easily degraded by enzymes or other components in the body, resulting in an increased distance between the fluorescent dye and the quencher molecule, thereby generating false signals and failing to dynamically and reversibly detect mRNA in real time.

In the field of DNA nanotechnology, artificially designed and synthesized DNA single strands are used to assemble various two-dimensional and three-dimensional structures. The DNA nano structure has a complex and fine programmable pattern and good site addressability, and has wide application value in the fields of drug delivery, molecular calculation, nano optical devices and the like. In the field of biomolecule detection using DNA nanostructures, researchers have mainly: 1. the AFM probe is used for realizing direct visualization on the combination of the DNA nano structure and the biomolecule in vitro; 2. fluorescence imaging was performed intracellularly using fluorescent DNA probes. Compared with a molecular beacon, the DNA nano structure has stronger enzyme cutting resistance and protection performance; however, since its structure is constructed from natural nucleic acids, it is still susceptible to degradation by in vivo components.

Compared with natural nucleic acid, the non-natural nucleic acid has similar base complementary pairing ability, thus having specific recognition function for target nucleic acid chain; meanwhile, the non-natural nucleic acid has better characteristics of enzyme digestion resistance, acid resistance, heat resistance and the like, so that the non-natural nucleic acid has higher stability in cells. The combination of the non-natural nucleic acid and the nano structure can solve the problems that the natural nucleic acid is easy to degrade in cells and the like, thereby realizing the purpose of long-term dynamic monitoring of mRNA in living cells.

At present, a detection method and application of a specific detection technology based on non-natural nucleic acid nano tweezers with high sensitivity in living cell mRNA detection are lacked.

Disclosure of Invention

The invention aims to provide a detection method of a specific detection technology based on non-natural nucleic acid nano-tweezers with high sensitivity in living cell mRNA detection and application thereof.

The technical scheme of the invention is as follows: the invention discloses a detection method of a specificity detection technology based on non-natural nucleic acid nano tweezers in living cell mRNA detection, which comprises the following steps:

(1) designing and synthesizing non-natural nucleic acid nano tweezers;

(2) detecting target mRNA in living cells by using nano tweezers;

(3) fluorescence spectroscopy was performed.

Further, in the step (1), the construction of the different non-natural nucleic acid nano-tweezers designs three non-natural nucleic acid single strands: two ends of the central chain are marked by two fluorescent groups capable of generating energy resonance transfer, and the other two single chains respectively extend out of a plurality of base sequences for carrying out base complementary pairing with target mRNA; can be realized by changing the chemical species of the non-natural nucleic acid according to different detection environment requirements.

Further, in Step (1), Step 1: two methoxy-modified non-natural nucleic acid single strands and one fluorophore-modified single strand are respectively mixed in a proportion of 1: 1: 1, and 10L of 10 XTAE-Mg is added²⁺Buffer solution (Mg)²⁺The concentration is 12.5mol/L), supplementing ultrapure water to the final volume of 100L, and shaking uniformly;

step 2: and (3) placing the mixed solution into a PCR instrument, annealing at the speed of 0.1 ℃/10s from 95 ℃ to 20 ℃, assembling into a 1M methoxyl modified nano-tweezers structure, and placing at 4 ℃ for later use.

Further, in the step (2), the target mRNA is detected, and when the target mRNA exists in the system, the nano-tweezers are changed from the original open state to the closed state; when mRNA molecules which are completely complementary and paired do not exist in the system, the structure of the nano tweezers can not be changed, so that the high specificity of detection is ensured.

Further, in Step (2), Step 1: viable cells cultured in a 96-well plate were washed three times with PBS buffer, and then 20L of assembled methoxy-modified nanotweezer and 180L of culture medium (10% FBS + 89% DMEN + 1% P/S) were added at 37 ℃ with 5% CO₂And incubated for 3 hours to allow it to enter the cells and bind to the target mRNA.

Step 2: within this system with nanotweezer: the fuel chain is 1: 1 molar ratio, 5% CO at 37 ℃ in the fuel chain₂And (3) allowing the cells to enter and bind to the target mRNA, and displacing the target mRNA from the nanotweezer so that the nanotweezer returns to the initial open state for the next round of detection.

Further, in the step (3), in the fluorescence spectrum analysis, when the nano-tweezers are changed from the original open state to the closed state, the two carried fluorescent groups are close to each other, the fluorescence energy resonance transfer is increased, and the detection purpose is achieved through the change of the optical signal.

Further, in the step (3), placing the methoxy-modified nano-tweezers, the co-incubated product of the nano-tweezers and the living cells, and the single-chain-added incubated product with the fuel under an enzyme-labeling instrument for detection, setting the excitation wavelength to be 513nm, using the living cells and the culture solution as a zero-setting group, repeating the three experiments, and taking an average value; when target mRNA exists in cells, the fluorescence resonance transfer phenomenon can be observed in the system, and after the fuel chain is added, the fluorescence resonance transfer phenomenon disappears, so that the nano tweezers can be restored to an initial opening state.

The application of the methoxy modified non-natural nucleic acid nano-tweezers provided by the invention in monitoring diabetes mellitus.

The invention relates to a specificity detection technology based on non-natural nucleic acid nano tweezers and application thereof in living cell mRNA detection.

Furthermore, the change of different target mRNAs can be dynamically monitored in real time according to the requirement; the assembled non-natural nucleic acid single strand can be chemically modified to have specific performance and adapt to intracellular detection; the detection steps are as follows:

(1) co-culturing the non-natural nucleic acid nano tweezers and the cells to be detected, wherein the non-natural nucleic acid nano tweezers enter the cells and are combined with target mRNA;

(2) the combination of the non-natural nano tweezers and the target mRNA changes the non-natural nano tweezers from an open state to a closed state to trigger fluorescence energy resonance transfer;

(3) and observing the fluorescence change before and after the cell reaction on a fluorescence detection platform.

Has the advantages that: the invention relates to a cell nondestructive test with high sensitivity. The nanometer tweezers based on fluorescence resonance energy transfer have extremely high detection sensitivity, high detection speed and good biocompatibility, and can directly enter cells for nondestructive detection. The method based on the non-natural nucleic acid nano-tweezers realizes dynamic monitoring of target mRNA in living cells, and the technology has the following advantages:

(1) multiple target mRNA sequences can be detected. The detection of various target mRNAs can be realized by changing the base sequence of the non-natural nucleic acid single strand so as to construct nano tweezers with various different sequences. Other nanotweezers are susceptible to hydrolysis in vivo and produce false signals, and cannot be reused.

(2) High stability. The non-natural nucleic acid has the advantages of enzyme cutting resistance, acid resistance, heat resistance and the like, so that the problems that the nucleic acid nano tweezers are easy to degrade in cells and the like are solved.

(3) The detection can be repeated. After one round of detection is finished, the fuel single chain is added into the system to perform a displacement reaction with the target mRNA, and the target mRNA is replaced from the nano-tweezers, so that the nano-tweezers are returned to the open state from the closed state to wait for the next detection.

(4) The method provides an effective way for dynamic monitoring of diseases, does not aim at a specific target mRNA, can design a plurality of non-natural nucleic acid nanostructures to detect different target mRNAs according to requirements, and aims to dynamically monitor a plurality of mRNAs in the same cell at the same time in the future.

(5) Firstly, an unnatural nucleic acid single strand forms an open nano-tweezers structure through base complementary pairing, wherein the 5 'end and the 3' end of one single strand are respectively marked with a fluorescent group. Then, the characteristic of high stability of the non-natural nucleic acid is utilized, and the mRNA molecules are detected in the cells by using nano tweezers; when the target mRNA molecule exists, the single-chain part of the nano-tweezers is subjected to complementary pairing with the target mRNA molecule, so that the nano-tweezers are closed, energy resonance transfer is generated between fluorescent groups, and the detection effect is achieved through the change of optical signals. Finally, adding a fuel single chain to restore the opening state of the nano-tweezers, so that the nano-tweezers can be subjected to the next round of detection. The method can effectively detect various target mRNAs and can monitor the expression level of the genes at the cellular level in real time. The high stability of the non-natural nucleic acid successfully solves the problem that the natural nucleic acid is easy to degrade in cells. The method can be used in the research fields related to disease monitoring such as diabetes, cancer and the like.

Drawings

The following will be further explained in conjunction with the attached drawings, in which:

FIG. 1 is a structural design diagram of the non-natural nucleic acid nanotweezer according to the present invention;

FIG. 2 is a schematic diagram of the reversible reaction of the non-natural nucleic acid nanotweezer of the present invention for detecting target mRNA;

FIG. 3 is a schematic diagram of detection of glucose transporter GLUT-4 in cells by using the methoxy-modified nanotweezer of the present invention (the presence/absence of target mRNA corresponds to the spectrum).

Detailed Description

The invention is further illustrated by the following examples. It should be understood that these examples are illustrative and exemplary of the present invention, and are not intended to limit the scope of the present invention in any way.

The invention discloses a detection method of a specificity detection technology based on non-natural nucleic acid nano tweezers in living cell mRNA detection, which comprises the following steps:

(1) designing and synthesizing non-natural nucleic acid nano tweezers;

(2) detecting target mRNA in living cells by using nano tweezers;

(3) fluorescence spectroscopy was performed.

In the step (1), the construction of the different non-natural nucleic acid nano-tweezers designs three non-natural nucleic acid single strands: two ends of the central chain are marked by two fluorescent groups capable of generating energy resonance transfer, and the other two single chains respectively extend out of a plurality of base sequences for carrying out base complementary pairing with target mRNA; can be realized by changing the chemical species of the non-natural nucleic acid according to different detection environment requirements.

Step 1: two methoxy-modified non-natural nucleic acid single strands and one fluorophore-modified single strand are respectively mixed in a proportion of 1: 1: 1, and 10L of 10 XTAE-Mg are added²⁺Buffer solution (Mg)²⁺The concentration is 12.5mol/L), supplementing ultrapure water to the final volume of 100L, and shaking uniformly;

step 2: and (3) placing the mixed solution into a PCR instrument, annealing at the speed of 0.1 ℃/10s from 95 ℃ to 20 ℃, assembling into a 1M methoxyl modified nano-tweezers structure, and placing at 4 ℃ for later use.

In the step (2), the target mRNA is detected, and when the target mRNA exists in the system, the nano-tweezers are changed from the original open state to the closed state; when mRNA molecules which are completely complementary and paired do not exist in the system, the structure of the nano tweezers can not be changed, so that the high specificity of detection is ensured.

Step 1: viable cells cultured in a 96-well plate were washed three times with PBS buffer, and then 20L of assembled methoxy-modified nanotweezer and 180L of culture medium (10% FBS + 89% DMEN + 1% P/S) were added at 37 ℃ with 5% CO₂And incubated for 3 hours to allow it to enter the cells and bind to the target mRNA.

Step 2: within this system with nanotweezer: the fuel chain is 1: 1 molar ratio, 5% CO at 37 ℃ in the fuel chain₂Is incubated for 3 hours under conditions to allow it to enter the cells and to interact with the targetAnd (3) mRNA is combined, the target mRNA is replaced from the nano-tweezers, and the nano-tweezers are returned to the initial opening state for the next round of detection.

In the step (3), in the fluorescence spectrum analysis, when the nano tweezers are changed from the original open state to the closed state, the two carried fluorescent groups are close to each other, the fluorescence energy resonance transfer is increased, and the detection purpose is achieved through the change of the optical signal.

Placing methoxy-modified nano-tweezers, co-incubated products of the nano-tweezers and living cells and incubated products added with fuel single chains under an enzyme-labeling instrument for detection, setting the excitation wavelength to be 513nm, using the living cells and culture solution as a zero-setting group, repeating the three experiments, and taking an average value; when target mRNA exists in cells, the fluorescence resonance transfer phenomenon can be observed in the system, and after the fuel chain is added, the fluorescence resonance transfer phenomenon disappears, so that the nano tweezers can be restored to an initial opening state.