阅读说明：本技术 核酸纳米凝胶及其制备方法和应用 (Nucleic acid nanogel and preparation method and application thereof ) 是由 黄晋 王姣丽 王柯敏 于 2021-09-16 设计创作，主要内容包括：本发明公开了一种核酸纳米凝胶及其制备方法和应用。核酸纳米凝胶由三条带有粘性末端的核酸链通过退火杂交形成Y-motif；三条带有粘性末端的核酸链为Y1、Y2和Y3；Y1的DNA序列如SEQ ID NO.1所示；Y2的DNA序列如SEQ ID NO.2所示；Y3的DNA序列如SEQ ID NO.3所示。本发明的核酸纳米凝胶内化到细胞中,细胞内microRNA通过立足点(toehold)介导的链置换反应选择性地结合到Y1的非配对toehold区域,随后内源性高丰度能量分子ATP特异性地触发ATP适体的构象转换,形成稳定的发夹结构并从核酸纳米凝胶中解离出来,用于制备活细胞内microRNA的放大成像检测试剂盒。(The invention discloses a nucleic acid nanogel and a preparation method and application thereof. The nucleic acid nanogel is formed by annealing and hybridizing three nucleic acid chains with sticky ends to form Y-motif; the three nucleic acid chains with sticky ends are Y1, Y2 and Y3; the DNA sequence of Y1 is shown in SEQ ID NO. 1; the DNA sequence of Y2 is shown in SEQ ID NO. 2; the DNA sequence of Y3 is shown in SEQ ID NO. 3. The nucleic acid nanogel is internalized into cells, intracellular microRNA is selectively combined to an unpaired toehold region of Y1 through a foothold (toehold) mediated strand displacement reaction, then endogenous high-abundance energy molecule ATP specifically triggers the conformation transformation of an ATP aptamer, a stable hairpin structure is formed and is dissociated from the nucleic acid nanogel, and the nucleic acid nanogel is used for preparing an amplification imaging detection kit of the intracellular microRNA.)

1. A nucleic acid nanogel, wherein the nucleic acid nanogel is formed by annealing and hybridizing three nucleic acid chains with sticky ends to form Y-motif; the three nucleic acid chains with sticky ends are Y1, Y2 and Y3;

the DNA sequence of the Y1 is shown as SEQ ID NO. 1; the DNA sequence of the Y2 is shown as SEQ ID NO. 2; the DNA sequence of the Y3 is shown in SEQ ID NO. 3.

2. The nucleic acid nanogel of claim 1, wherein the 5' end of Y1 is labeled with Cy3 fluorophore; the 3' end of the Y1 was labeled with Cy5 fluorophore.

3. The nucleic acid nanogel according to claim 1 or 2, which is characterized in thatCharacterized in that two or more Y-motif are linked by a cohesive end to form- (Y-Y)_n-。

4. A method for preparing the nucleic acid nanogel according to any one of claims 1 to 3, comprising the steps of:

s1, blending Y1, Y2 and Y3 to obtain a mixed solution;

s2, heating the mixed solution at 95 ℃ for 5min, and then cooling to room temperature to obtain Y-motif.

5. The method of manufacturing according to claim 4, further comprising:

s3, blending N Y-motifs, heating at 95 ℃ for 5min, and then cooling to room temperature to obtain the nucleic acid nanogel- (Y-Y)_n-。

6. The preparation method according to claim 4 or 5, wherein S1 specifically comprises: y1, Y2 and Y3 were blended in a buffer and a magnesium chloride solution to obtain a mixed solution.

7. The method according to claim 6, wherein the concentrations of Y1, Y2 and Y3 are all 5 μ M;

and/or the buffer solution is 10mM phosphate buffer solution;

and/or the concentration of the magnesium chloride solution is 5 mM.

8. Use of the nucleic acid nanogel of any one of claims 1 to 3 for the preparation of a kit for the amplified imaging detection of microRNA in living cells.

9. The application according to claim 8, wherein the method of application is: blending the nucleic acid nanogel with a living cell, internalizing the nucleic acid nanogel into the cell, and detecting microRNA in the living cell by detecting a fluorescence resonance energy transfer signal of the nucleic acid nanogel.

Technical Field

The invention relates to the technical field of nucleic acid nanogels, in particular to a nucleic acid nanogel and a preparation method and application thereof.

Background

DNA serves as a carrier of genetic information and a protein encoder, converting the genetic code into proteins during life and further regulating a variety of cellular functions. Besides the biological function, based on the Watson-Crick base complementary pairing principle, the DNA can also be used as a general material for constructing DNA nano structures and DNA hybridization reaction networks with different sizes and shapes. In DNA nanotechnology, nucleic acid probes based on DNA nanostructures show great potential for applications in the fields of biosensing, bioimaging, drug delivery, cell biology and material manufacturing.

Nucleic acid probes have unique advantages in intracellular nucleic acid detection. However, low efficiency of cellular internalization and poor probe stability remain one of the major challenges for the application of nucleic acid probes to living cells. Although many methods for assembling nucleic acid probes by using organic or inorganic nanomaterials are reported to greatly improve the cellular internalization efficiency of the nucleic acid probes, the introduction of the organic or inorganic nanomaterials may cause inevitable toxicity to living cells and even affect the expression of target RNA in the cells. The nano structure formed by DNA self-assembly has excellent biocompatibility, good cell membrane penetrating capacity, mechanical stability and other excellent characteristics, and is widely applied to biotechnology and biomedicine. Most DNA nanostructures are inefficient and time consuming to prepare, and are expensive (because of the large amount of long-chain DNA required). In addition, the assembly efficiency of the nucleic acid probe and the nanostructure is low. Therefore, the construction of a nucleic acid nanoprobe with low cost, easy preparation, high biological safety, high endocytosis efficiency, good biocompatibility and good stability is still urgently needed.

RNA is an important nucleic acid substance, has the functions of transferring genetic information, regulating biological metabolism and reflecting cell states, can be used as a key control factor of gene expression, and plays an important role in various life processes. The microRNA is a small single-stranded non-coding RNA, and compared with other types of RNA, the microRNA has the advantages of short sequence, easy degradation, low abundance and high sequence similarity between families. Research shows that the abnormal expression of the microRNAs is closely related to various human diseases, such as the occurrence, development, metastasis, drug resistance and the like of tumors, and can be used as a biomarker for diagnosis and prognosis of diseases such as cancers. Therefore, sensitive and accurate microRNA detection provides important information for early diagnosis of diseases, prediction of disease results, treatment risk assessment and treatment response prediction. Therefore, the detection of tumor-associated microRNA is of great significance in biology and disease research. The traditional detection method of microRNA comprises the following steps: real-time fluorescent quantitative polymerase chain reaction (qRT-PCR), microarray chip, Northern blotting, and fluorescent in situ hybridization. These traditional detection methods can only be used in dead cell systems or in vitro experiments, and can only reflect the average value of a large number of cells, and cannot reflect the difference between cells, and cannot be used in situ detection of microRNA in living cells. Therefore, it is necessary to develop some in situ detection techniques for microRNA in living cells. And the common mode that one target corresponds to one signal output has low sensitivity, so that the method is not suitable for detecting low-abundance microRNA in living cells. Therefore, it is necessary to develop an effective "one-to-many signal" amplification strategy for highly sensitive in situ imaging of low abundance micrornas in living cells.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defect of low efficiency of a nucleic acid probe entering cells in the prior art, and provides a simple nucleic acid nanogel which has the advantages of excellent biocompatibility, good enzyme digestion resistance stability, high endocytosis efficiency and the like. The nucleic acid nanogel has simple preparation process and convenient operation.

In order to achieve the above object, the present invention provides a nucleic acid nanogel formed by three nucleic acid strands having cohesive ends forming Y-motif by annealing hybridization; the three nucleic acid chains with sticky ends are Y1, Y2 and Y3;

the DNA sequence of the Y1 is shown as SEQ ID NO. 1; the DNA sequence of the Y2 is shown as SEQ ID NO. 2; the DNA sequence of the Y3 is shown in SEQ ID NO. 3.

The nucleic acid nanogel as described above, wherein the 5' end of Y1 is labeled with Cy3 fluorophore; the 3' end of the Y1 was labeled with Cy5 fluorophore.

In the above nucleic acid nanogel, two or more nucleic acid nanogels are further connected through the cohesive ends of the Y-motif nucleic acid strand to form- (Y-Y)_n-。

Based on a general technical concept, the present invention provides a method for preparing the above-described nucleic acid nanogel, comprising the steps of:

s1, blending Y1, Y2 and Y3 in equal concentration to obtain a mixed solution;

s2, heating the mixed solution at 95 ℃ for 5min, and then cooling to room temperature to obtain Y-motif.

The above preparation method, further, the preparation method further comprises:

s3, blending N Y-motifs, heating at 95 ℃ for 5min, and then cooling to room temperature to obtain the nucleic acid nanogel- (Y-Y)_n-。

In the preparation method, further, the S1 specifically is:

and (3) blending equal concentrations of Y1, Y2 and Y3 in a buffer solution and a magnesium chloride solution to obtain a mixed solution.

In the preparation method, the concentration of Y1, Y2 and Y3 is 5 μ M.

In the preparation method, the buffer is 10mM phosphate buffer.

The above preparation method, further, the concentration of the magnesium chloride solution is 5 mM.

Based on a general technical concept, the invention provides an application of the method in preparing the amplification imaging detection kit for microRNA in living cells.

The above application, further, the method of the application is: the nucleic acid nanogel and the living cells are blended, the nucleic acid nanogel is internalized into the cells through the endocytosis of the living cells, and microRNA in the living cells is detected through detecting the change of a fluorescence signal of the nucleic acid nanogel.

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

(1) the invention provides a nucleic acid nanogel which is formed by three Y-motif nucleic acid chains with sticky ends through annealing and hybridization, compared with the traditional method, the nucleic acid nanogel is formed by the hybridization of three simple Y-motif (Y1, Y2 and Y3) nucleic acid chains, and the defects of complicated sequence design and large amount of long-chain DNA are avoided. The nucleic acid nanogel formed by self-assembly has higher cell internalization efficiency, good biocompatibility and stability. The nucleic acid nanogel is used for amplifying and detecting microRNA in cells by utilizing ATP (adenosine triphosphate) endogenous to the cells to drive strand displacement amplification reaction without adding any catalytic fuel. The generation of FRET-based ratio-type signals can avoid false positive signals, reduce the influence of system fluctuation, and can be widely applied to basic biomedical research, disease diagnosis and treatment.

(2) The invention provides a preparation method of nucleic acid nanogel, which is simple in preparation process and convenient to operate.

(3) The invention provides application of nucleic acid nanogel in preparation of a kit for amplifying imaging detection of microRNA in living cells, wherein the nucleic acid nanogel is internalized into cells, and specific microRNA in the cells is selectively combined to an unpaired toehold region of Y1 through a foothold (toehold) mediated strand displacement reaction. Subsequently, the endogenous high abundance of the energy molecule ATP can specifically trigger the conformational transition of the ATP aptamer, forming a stable hairpin structure and dissociating from the nanosphere. At the same time, microRNA can be released and reused in subsequent reactions. Finally, the hairpin structure brings the donor and acceptor in close proximity, resulting in high FRET fluorescence efficiency, avoiding the "false positive" signal that is generated by degradation of the nucleic acid probe by intracellular heteroenvironments.

Drawings

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.

FIG. 1 is a schematic diagram of the preparation of nucleic acid nanogel in example 1 of the invention.

FIG. 2 is a gel electrophoresis diagram of nucleic acid nanogel preparation in the first experiment of the invention.

FIG. 3 is a Zeta-size particle size plot of a nucleic acid nanogel in experiment two of the present invention.

FIG. 4 is an atomic force characterization diagram of the nucleic acid nanogel in experiment three of the present invention.

FIG. 5 is a transmission electron microscope image of the nucleic acid nanogel in experiment four of the present invention.

FIG. 6 shows gel electrophoresis analysis and Zeta-size characterization of the specific response of nucleic acid nanogel in experiment five of the present invention.

FIG. 7 shows the fluorescence resonance energy change of the specific response of the nucleic acid nanogel in the sixth experiment of the invention.

FIG. 8 is a comparison of the anti-enzymatic cleavage stability of the nucleic acid nanogel and the Y-shaped probe in experiment seven of the present invention.

FIG. 9 is a graph of MTT toxicity of MCF-7 cells in the nucleic acid nanogel of experiment eight of the invention.

FIG. 10 is a comparison of the endocytosis of nucleic acid nanogel and Y-shaped probe cells in the experiment nine of the invention.

FIG. 11 shows the optimized results of the buffer solution system of the nucleic acid nanogel in the experiment ten of the invention.

FIG. 12 shows the optimized result of the magnesium ion concentration of the nucleic acid nanogel in the eleventh experiment of the invention.

Detailed Description

The invention is further described below with reference to specific preferred embodiments, without thereby limiting the scope of protection of the invention.

Examples

The materials and equipment used in the following examples are commercially available.

Example 1:

the nucleic acid nanogel is formed by annealing and hybridizing three Y-motif nucleic acid chains with sticky ends; three Y-motif nucleic acid chains with sticky ends are Y1, Y2 and Y3;

the DNA sequence of Y1 is shown in SEQ ID NO. 1; the method specifically comprises the following steps: wherein, the bold font part is a palindromic sequence, the single underline part is an aptamer of adenosine triphosphate, and the double underline part is a non-paired toehold region.

The DNA sequence of the Y2 is shown as SEQ ID NO. 2; the method specifically comprises the following steps: AGCTCGCGCTCACGGCGAATGACTCGTACCTTCCTCCGCAATACTCCCCC are provided.

The DNA sequence of the Y3 is shown as SEQ ID NO. 3; the method specifically comprises the following steps: AGCTCGAGCTTCGACTATCAGACTGATCATTCGCCGTG are provided.

A method for preparing the nucleic acid nanogel of the embodiment, referring to fig. 1, comprises the following steps:

(1) y1, Y2 and Y3 were blended at equal concentrations (final concentration: 5. mu.M) to give a mixed solution.

(2) And heating the mixed solution at 95 ℃ for 5 minutes, and then slowly cooling to room temperature to obtain the Y-motif.

Example 2:

two nucleic acid nanogels Y-motif of example 1 are hybridized with each other through the cohesive ends (palindromic sequences) of the Y-shaped structure to form nucleic acid nanoprobes Y-Y.

A method for preparing the nucleic acid nanogel of the embodiment, referring to fig. 1, comprises the following steps:

(1) two nucleic acid nanogels of example 1 were blended at equal concentrations (final concentration of 5 μ M) to obtain a mixed solution.

(2) And heating the mixed solution at 95 ℃ for 5 minutes, and then slowly cooling to room temperature to obtain the nucleic acid nanogel Y-Y.

Example 3:

the nucleic acid nanogel disclosed by the invention is formed by pairwise hybridization of the three Y-motifs in example 1 through the cohesive ends (palindromic sequences) of a Y-shaped structure to form a nucleic acid nanoprobe Y-Y-Y.

A method for preparing the nucleic acid nanogel of the embodiment, referring to fig. 1, comprises the following steps:

(1) three Y-motif of example 1 were blended at an equal concentration (final concentration: 5. mu.M) to give a mixed solution.

(2) And heating the mixed solution at 95 ℃ for 5 minutes, and naturally and slowly cooling to room temperature to obtain the nucleic acid nanogel Y-Y-Y.

According to the method of the present invention, there may be an unlimited number of Y-motif pairs of example 1 hybridized to form a nucleic acid nanoprobe- (Y-Y)_n-。

Experiment one: gel electrophoresis graph characterization of nucleic acid nanogels

Preparing agarose solution with mass concentration ratio of 2%, and heating and dissolving in a microwave oven. And pouring 60mL of the solution into a mold, inserting a comb, standing and cooling for 1h, and then solidifying the gel. And then putting the prepared agarose gel into an electrophoresis tank, pouring 1 xTBE for waiting for sample Loading, taking 10 muL of reaction sample liquid, 2 muL of 6 xLoading buffer and 2 muL of SYBR Gold, uniformly mixing, then taking 10 muL of mixed liquid, adding 90V voltage into a gel hole, and taking a picture by using a gel imager when the blue bromophenol blue is about to move to the bottom end of the gel.

The gel imaging results are shown in fig. 2, wherein lanes 1, 2 and 3 are Y1, Y2 and Y3, lane 4 is a nucleic acid nanogel formed by annealing Y1, Y2 and Y3, and lane 4 forms a band with a larger molecular weight, indicating the successful preparation of the nanogel.

Experiment two: the nucleic acid nanogel of example 1 was examined for Zeta-size hydrated particle size.

The hydrated particle Size of the nucleic acid nanogel was measured at room temperature using Zeta-Size, the refractive medium was set to water, and the average value was plotted for three times.

As a result of measuring the particle size of the nucleic acid nanogel, as shown in FIG. 3, the particle size of the prepared nucleic acid nanogel was about 180nm (the hydrated particle size was larger).

Experiment three: the size morphology of the nucleic acid nanogel of example 1 was observed with an atomic force microscope.

A20 nM (50. mu.L) DNA sample was first prepared. The scotch tape was gently torn off the mica and a layer of mica was removed to give a fresh mica surface. Then 40. mu.L of 100mM NiCl was added dropwise₂The buffer was incubated on the mica surface for 10min, then 1mL of ultrapure water was taken to rinse the mica surface and immediately water was blown off to clean the mica surface, and the solution was blown off with an ear-washing ball. Then 20. mu.L of the DNA sample was added dropwise and incubated on the mica surface for 10 min. After incubation was complete, the solution was blown off with an ear-washing bulb. Then, 3mL of ultrapure water was immediately dropped in the middle of the mica surface, and water was immediately blown to clean the mica surface. The sample was then imaged with the air scanning mode of an atomic force microscope.

As shown in FIG. 4, the nucleic acid nanogel of example 1 had a size of about 150 nm.

Experiment four: transmission Electron microscopy of the size morphology of the nucleic acid nanogel of example 1

Dropping the nucleic acid nanogel sample prepared in example 1 on a copper net for deposition for 15min, puncturing the liquid drop, then dropping 1% uranium acetate solution for dyeing for 1min, sucking the solution on the copper net by using filter paper, air-drying, and finally carrying out transmission electron microscope shooting.

As shown in FIG. 5, the nucleic acid nanogel of example 1 was spherical and about 150nm in size.

Experiment five: the nucleic acid nanogels of example 1 were examined for their response to microRNA-21 and ATP.

Adding microRNA-21, ATP and a mixture of the microRNA-21 and the ATP into the prepared nanogel respectively, analyzing the reaction result by using 2% agarose, wherein the electrophoresis result is shown in figure 6a, and a lane 1 is nucleic acid nanogel; lane 2 is the nucleic acid nanogel added with microRNA-21; lane 3 is the nucleic acid nanogel with ATP added thereto, which is not significantly changed compared to Lane 1; and after the microRNA-21 and the ATP are added into the lane 4, the band moves down obviously, and the hydrated particle size under the reaction condition is measured at the same time, and the result is shown in FIG. 6b, and when the microRNA-21 and the ATP exist at the same time, the hydrated particle size of the nucleic acid nanogel is reduced, and the results show the disassembly of the nucleic acid nanogel.

Example 4:

the nucleic acid nanogel is formed by annealing and hybridizing three nucleic acid chains with sticky ends; the three nucleic acid chains with sticky ends are Y1, Y2 and Y3;

the DNA sequence of Y1 is shown in SEQ ID NO. 1; the method specifically comprises the following steps: cy3-AGTACGCGTACTATCAGGCAAGCTACTTACCTGGGGGAGTATTGCGGAGGAAGGTTCAACATCAGTCTGATAGTACG-Cy 5.

The DNA sequence of the Y2 is shown as SEQ ID NO. 2; the method specifically comprises the following steps: AGCTCGCGCTCACGGCGAATGACTCGTACCTTCCTCCGCAATACTCCCCC are provided.

The DNA sequence of the Y3 is shown as SEQ ID NO. 3; the method specifically comprises the following steps: AGCTCGAGCTTCGACTATCAGACTGATCATTCGCCGTG are provided.

The preparation process was identical to example 1.

Experiment six: the specific response of the nucleic acid nanogel of example 4 was examined.

In the nucleic acid nanogel of example 4, a mixture of microRNA-429 and ATP, a mixture of let-7a and ATP, a mixture of microRNA-200b and ATP, and a mixture of microRNA-21 and ATP were added, respectively, and the change in fluorescence resonance energy transfer signal (FRET) was examined.

FIG. 7 shows the fluorescence resonance energy change of the nucleic acid nanogel specific response. When other nucleic acid markers or ATP analogues are added, FRET has no obvious change, and when microRNA-21 and ATP are added simultaneously, FRET signals are obviously enhanced. The nucleic acid nanogel is shown to have good specificity and can specifically respond to microRNA-21 and ATP.

Experiment seven: the nucleic acid nanogels of example 4 were examined for stability against enzymatic cleavage.

The nucleic acid nanogel and the Y-shaped probe of example 4 were added to 10% fetal bovine serum, and transferred to a cuvette, and the temperature was maintained at 37 ℃ for 6 hours, during which the fluorescence intensity was recorded every 1 hour. The excitation wavelength is set to 530nm, fluorescence spectra in the range of 530nm and 800nm are collected, and the fluorescence intensities F of samples at 570nm and 680nm are obtained₅₇₀And F₅₈₀And take F₆₈₀Is as F₅₇₀The ratios of (A), (B), (C), (D), (E) and (D) were plotted and all experiments were repeated 3 times.

FIG. 8 is a comparison of the stability against enzymatic cleavage of nucleic acid nanogels and "Y" -shaped probes. As can be seen from the figure: with the time being prolonged, the fluorescence resonance energy signal of the nucleic acid nanogel has no obvious change, but the fluorescence resonance energy transfer signal of the Y-shaped probe is obviously reduced, and the fluorescence of the donor fluorescent group and the acceptor is far away because the Y-shaped probe is degraded by nuclease. The nucleic acid nanogel has better enzyme digestion resistance stability.

Experiment eight: the nucleic acid nanogels of example 4 were examined for MCF-7 cytotoxicity experiments.

MCF-7 cells were seeded into 96-well plates at approximately 1X 10 per well⁵And (4) cells. After 24h incubation in a cell incubator, 100. mu.L of seed with different concentrations was addedThe nucleic acid nanogel of example 4 (10, 50, 100, 200, 400nM) was incubated for 6h with medium, after which the medium was discarded and 100. mu.L of 0.5mg/mL MTT solution was added to each well and incubated for 4 h. After incubation, the wells were discarded, 150 μ L DMSO was added and shaken at room temperature for 15 min. The absorbance at 490nm was measured using a microplate reader.

FIG. 9 is a graph of the MTT toxicity of MCF-7 cells of nucleic acid nanogels. As can be seen from the figure, the survival rate of the MCF-7 cells after being incubated with the 400nM nucleic acid nanogel for 6h reaches more than 95%, which shows that the nucleic acid gel is almost non-toxic to the cells, and the excellent biocompatibility of the nucleic acid nanogel is proved.

Example 5

An application of the nucleic acid nanogel of example 4 in the amplified imaging of microRNA in living cells.

The specific reaction process is as follows: the nucleic acid nanogel is internalized into the cell and intracellular specific micrornas bind selectively to the unpaired toehold region of Y1 by foothold-mediated strand displacement (TMSD). Subsequently, endogenous high abundance ATP as energy can specifically trigger conformational transition of ATP aptamers, forming stable hairpin structures and dissociating from nanospheres. At the same time, microRNA can be released and reused in subsequent reactions. Finally, the hairpin structure brings the donor and acceptor in close proximity, resulting in high FRET efficiency, avoiding "false positive" signals due to degradation of the nucleic acid probe by intracellular heteroenvironments.

Experiment nine: the nucleic acid nanogel and the "Y" -shaped probe (which does not contain a cohesive end portion of a palindromic sequence) have endocytosis efficiency.

Inoculating MCF-7 cells into a laser confocal dish for culturing for 24h, respectively adding the prepared nucleic acid nanogel and the Y-shaped probe into the confocal dish to incubate with the MCF-7 cells for 4h, washing with PBS buffer solution for three times, and discarding the redundant probe. Finally, the cells were plated with 561nm Ar⁺And (3) observing and imaging under a laser confocal microscope. Cells were visualized under an Olympus IX-70 inverted microscope using an Olympus FluoView 500 confocal scanning system, and emission wavelengths of 570nm-620nm and 663nm-738nm were collected under 561nm excitation.

FIG. 10 is a comparison of nucleic acid nanogels and Y-shaped probes for comparing endocytosis. As shown in FIG. 10, the cells incubated with the nucleic acid nanogel can observe obvious green light and red light, while the cells incubated with the Y-shaped probe only have very weak green light, and the results show that the nucleic acid nanogel can be endocytosed well by the cells, so that the efficiency of the probe entering the cells is improved.

Example 6:

the nucleic acid nanogel is formed by annealing and hybridizing three nucleic acid chains with sticky ends; the three nucleic acid chains with sticky ends are Y1, Y2 and Y3;

the DNA sequence of Y1 is shown in SEQ ID NO. 1; the method specifically comprises the following steps: cy3-AGTACGCGTACTATCAGGCAAGCTACTTACCTGGGGGAGTATTGCGGAGGAAGGTTCAACATCAGTCTGATAGTACG-Cy 5.

The DNA sequence of the Y2 is shown as SEQ ID NO. 2; the method specifically comprises the following steps: AGCTCGCGCTCACGGCGAATGACTCGTACCTTCCTCCGCAATACTCCCCC are provided.

The DNA sequence of the Y3 is shown as SEQ ID NO. 3; the method specifically comprises the following steps: AGCTCGAGCTTCGACTATCAGACTGATCATTCGCCGTG are provided.

A method for preparing the nucleic acid nanogel of the embodiment, referring to fig. 1, comprises the following steps:

(1) y1, Y2 and Y3 were mixed in an equal concentration (final concentration: 5. mu.M) in 10mM phosphate buffer and 5mM magnesium chloride solution to obtain a mixed solution.

(2) And heating the mixed solution at 95 ℃ for 5 minutes, and then slowly cooling to room temperature to obtain the nucleic acid nanogel Y-motif.

Experiment ten: investigating the influence of the nucleic acid nanogel probe on the detection buffer system

In order to improve the signal-to-back ratio of the nucleic acid nanogel probe for detecting microRNA, concentrations of Y1, Y2 and Y3 (final concentration is 5 mu M) are respectively blended in 10mM Tris-HCl, PBS, SPSC and HEPES four buffer systems, and the rest steps are consistent with example 5. Solutions under various experimental conditions are divided into two groups, one group is added with target microRNA-21 (signal), the other group is not added with the target microRNA-21 and is used as a control (background), and the fluorescence results of different buffer systems are inspected. The excitation wavelength is set to 530nm during detection, the fluorescence spectrum in the range of 530nm and 800nm is collected,

the results are shown in FIG. 11. As can be seen from the figure, different buffer systems have certain influence on the signal-to-back ratio of detection, wherein the PBS buffer solution is favorable for improving the signal-to-back ratio of the nucleic acid nanogel probe for detecting microRNA.

Experiment eleven: investigating the influence of the magnesium ion concentration in the nucleic acid nanogel probe detection reaction system

In order to further improve the detection performance of the nucleic acid nanogel probe for detecting microRNA, concentrations (final concentration is 5 mu M) of Y1, Y2 and Y3 are blended in 10mM phosphate buffer solution and magnesium chloride solutions with concentrations of 2mM, 5mM, 10mM and 12mM respectively, and the rest steps are consistent with example 5. Solutions under various experimental conditions were divided into two groups, one group with target microRNA-21 (signal) and one group without target microRNA-21 as control (background). The excitation wavelength is set to 530nm during detection, the fluorescence spectrum in the range of 530nm and 800nm is collected,

the results are shown in FIG. 12. As can be seen from the figure, magnesium chloride solutions with different concentrations have certain influence on the detection performance, wherein the magnesium chloride solution with the concentration of 5mM is beneficial to improving the detection performance of the nucleic acid nanogel probe.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.

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