阅读说明：本技术 一种目标物催化诱导自组装dna网状纳米材料的制备与应用 (Preparation and application of target object catalytic induction self-assembly DNA (deoxyribonucleic acid) net-shaped nano material ) 是由 许钬 叶晓晶 贾力 于 2019-11-30 设计创作，主要内容包括：本发明公开了一种目标物催化诱导自组装DNA网状纳米材料的制备与应用。首次提出将回文序列嵌入到发卡探针,进而miRNA诱导催化自组装形成DNA网状纳米材料。细胞内的miRNA-21或miRNA-31,触发一对含回文序列的发夹探针HP1/HP2或HP3/HP4的级联杂交反应,从而自组装形成多个信号输出的网状纳米材料。本发明不依赖酶的使用,无需制备复杂的纳米材料,只涉及两条DNA探针,避免了复杂、耗时的DNA链设计过程,也节省了成本。本发明所构建的DNA自组装网状纳米材料能用于活细胞内生物标志物miRNA-21或miRNA-31的成像,为疾病的早期诊断和药物研发提供潜在工具。(The invention discloses preparation and application of a target object catalytic induction self-assembly DNA reticular nano material. Embedding a palindromic sequence into a hairpin probe is firstly proposed, and then miRNA induces catalytic self-assembly to form a DNA reticular nano material. The miRNA-21 or miRNA-31 in the cell triggers a cascade hybridization reaction of a pair of hairpin probes HP1/HP2 or HP3/HP4 containing palindromic sequences, so that the reticular nano-material with a plurality of signal outputs is formed by self-assembly. The invention does not depend on the use of enzyme, does not need to prepare complex nano materials, only relates to two DNA probes, avoids the complex and time-consuming DNA chain design process and also saves the cost. The DNA self-assembly mesh-shaped nanometer material constructed by the invention can be used for imaging of biomarkers miRNA-21 or miRNA-31 in living cells, and provides a potential tool for early diagnosis of diseases and drug research and development.)

1. A target object catalytic induction self-assembly DNA net-shaped nanometer material is characterized in that: the self-assembly DNA reticular nano material comprises a pair of hairpin probe DNA containing palindromic sequences and miRNA corresponding to the hairpin probe DNA.

2. The target substance catalytic-induced self-assembly DNA reticular nano-material of claim 1, which is characterized in that: the hairpin probe DNA containing the palindromic sequence is HP1/HP2, and miRNA corresponding to the hairpin probe DNA is miRNA-21;

the nucleotide sequence of the HP1 is as follows:

5’-GATCGCGATCTCAACAT-FAM-CAGTCTGATAAGCTAACATCGTAGCTTATCAGACTG-BHQ-1-3’；

the nucleotide sequence of the HP2 is as follows:

5’-AGTTCGAACTT-Cy3-AGCTTATCAGACTGATGTTGATTTTCAGTCTGATAAGCT-BHQ-2-ACGATGT-3’，

the nucleotide sequence of the MiRNA-21 is as follows: 5'-UAGCUUAUCAGACUGAUGUUGA-3' are provided.

3. The target substance catalytic-induced self-assembly DNA reticular nano-material of claim 1, which is characterized in that: the hairpin probe DNA containing the palindromic sequence is HP3/HP4, and miRNA corresponding to the hairpin probe DNA is miRNA-31;

the nucleotide sequence of the HP3 is as follows: 5 '-GATCGCGATCAGCTAT(FAM) GCCAGCATCTTGCCTACATCGAGGCAAGATGCTGG (BHQ-1) -3';

the nucleotide sequence of the HP4 is as follows: 5'-AGTTCGAACTAGGCAAGATGCTGGCATAGCTTTTGCCAGCATCTTGCCTCGATGT-3', respectively;

the nucleotide sequence of the MiRNA-31 is as follows: 5'-AGGCAAGAUGCUGGCAUAGCU-3' are provided.

4. A preparation method of a target object catalytic induction self-assembly DNA reticular nano material is characterized by comprising the following steps: firstly, diluting a pair of hairpin probe DNAs containing palindromic sequences to 10 mu M respectively, annealing at 90 ℃ respectively, and gradually cooling to 25 ℃ to form a hairpin structure for later use; then 1 mul of each annealed hairpin probe DNA, miRNA corresponding to the hairpin probe DNA with a certain concentration and 17 mul of tris-buffer are mixed and reacted for 2 hours at normal temperature.

5. The method for preparing the target substance catalytic induced self-assembly DNA reticular nano-material according to claim 4, wherein: the concentration of the miRNA corresponding to the hairpin probe DNA is as follows: 50 nM.

6. The method for preparing the target substance catalytic induced self-assembly DNA reticular nano-material according to claim 4, wherein: the preparation method of the tris-buffer comprises the following steps: 25mM tris-HCl, 100 mM NaCl, 50 mM KAc, 10 mM MgAc₂， 1 mM DTT，pH 8.2。

7. The target of claim 1, wherein the target catalyzes and induces self-assembly of DNA-network nanomaterials for imaging of living cell biomarkers miRNA.

8. A diagnostic reagent comprising a target catalytically induced self-assembled DNA-network nanomaterial of claim 1.

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a preparation method and application of a target object catalytic induction self-assembly DNA (deoxyribonucleic acid) net-shaped nano material.

Background

Currently, several conventional techniques for miRNA analysis have been developed, including Northern blot, Fluorescence In Situ Hybridization (FISH), and Polymerase Chain Reaction (PCR). However, the need to immobilize cells or lyse large numbers of cells prior to analysis in these techniques results in cumbersome, time consuming, and low sensitivity, which greatly limits their application in biomedicine. In addition, Molecular beacons (Molecular beacons) developed by scientists produce false positive signals due to their susceptibility to non-specific degradation by nucleases (e.g., DNase) in the intracellular environment. To address the above issues, we constructed DNA-network nanostructures with certain viscosity to enable in situ imaging of target mirnas in living cells.

Disclosure of Invention

The invention aims to solve the problems and provides a preparation method and application of a target substance catalytic-induced self-assembly DNA reticular nano material. A pair of hairpin probes containing palindromic sequences are induced, catalyzed and self-assembled to form the DNA reticular nano material by miRNA, the preparation method is simple, convenient and quick, and the cost is saved. The DNA reticular nano material can be used for imaging tumor biomarkers, realizes visible, sensitive, specific and stable imaging of target miRNA in cells, and is further used for development of clinical diagnosis kits.

In order to achieve the purpose, the invention adopts the following technical scheme:

a target object catalytic induction self-assembly DNA reticular nano material comprises a pair of hairpin probe DNA containing palindromic sequences and miRNA corresponding to the hairpin probe DNA.

The hairpin probe DNA containing the palindromic sequence is HP1/HP2, and miRNA corresponding to the hairpin probe DNA HP1/HP2 is miRNA-21; or the pair of hairpin probe DNA containing the palindromic sequence is HP3/HP4, and miRNA corresponding to the hairpin probe DNA HP3/HP4 is miRNA-31;

the nucleotide sequence of the HP1 is as follows: 5 '-GATCGCGATCTCAACAT-FAM-CAGTCTGATAAGCTAACATCGTAGCTTATCAGACTG-BHQ-1-3'; wherein GATCGCGATC is a palindromic sequence; TCAACAT (FAM) CAGTCTGATAAGCTA is miRNA-21 recognition sequence; CAGTCTGATAAGCTA is complementary to the TAGCTTATCAGACTG sequence;

the nucleotide sequence of the HP2 is as follows: 5 '-AGTTCGAACTT-Cy 3-AGCTTATCAGACTGATGTTGATTTTCAGTCTGATAAGCT-BHQ-2-ACGATGT-3', wherein AGTTCGAACT is a palindromic sequence; DNA sequences corresponding to T-Cy3-AGCTTATCAGACTGATGTTGA and miRNA-21; the T-Cy3-AGCTTATCAGACTGA is complementary to the TCAGTCTGATAAGCT-BHQ-2-A sequence;

the nucleotide sequence of the MiRNA-21 is as follows: 5'-UAGCUUAUCAGACUGAUGUUGA-3' are provided.

The nucleotide sequence of the HP3 is as follows: 5 '-GATCGCGATCAGCTAT-FAM-GCCAGCATCTTGCCTACATCGAGGCAAGATGCTGG-BHQ-1-3'; wherein GATCGCGATC is a palindromic sequence; AGCTAT-FAM-GCCAGCATCTTGCCT is a miRNA-31 recognition sequence, CCAGCATCTTGCCT is complementary with AGGCAAGATGCTGG sequence;

the nucleotide sequence of the HP4 is as follows: 5'-AGTTCGAACTAGGCAAGATGCTGGCATAGCTTTTGCCAGCATCTTGCCTCGATGT-3', wherein AGTTCGAACT is a palindromic sequence; AGGCAAGATGCTGGCATAGCT DNA sequence corresponding to miRNA-31; AGGCAAGATGCTGGC is complementary to the GCCAGCATCTTGCCT sequence;

the nucleotide sequence of the MiRNA-31 is as follows: 5'-AGGCAAGAUGCUGGCAUAGCU-3' are provided.

A preparation method of a target object catalytic induction self-assembly DNA reticular nano material comprises the following steps: firstly, a pair of hairpin probe DNAs (HP 1/HP2 or HP3/HP 4) containing palindromic sequences are respectively diluted to 10 mu M, and are respectively annealed at 90 ℃ and gradually cooled to 25 ℃ to form a hairpin structure for later use; then, 1. mu.L each of the hairpin probe DNAs (HP 1/HP2 or HP3/HP 4) annealed as described above, and a miRNA (miRNA-21 or miRNA-31) corresponding to the hairpin probe DNA at a given concentration were mixed with 17. mu.L of tris-buffer, and reacted at room temperature for 2 hours.

The concentration of miRNA corresponding to the hairpin probe DNA is as follows: 50 nM.

The preparation method of the tris-buffer comprises the following steps: 25mM tris-HCl, 100 mM NaCl, 50 mM KAc, 10 mM MgAc₂ and 1 mM DTT，pH 8.2。

The target object catalytic induction self-assembly DNA reticular nano material is used for imaging of biomarker miRNA in living cells.

A detection reagent containing target substance catalytic induction self-assembly DNA reticular nanometer material.

The invention has the advantages that:

(1) the preparation method of the DNA reticular nano material is simple, convenient and quick, and the DNA reticular nano material can be obtained only by mixing three polymers in the raw material components at room temperature for reaction for 2 hours.

(2) The raw material of the DNA reticular nano material comprises a pair of hairpin probes HP1 and HP2 containing palindromic sequences, or HP3 and HP 4; the invention firstly proposes that a palindromic sequence is embedded into the 5 end of a hairpin probe, and then miRNA-21 or miRNA-31 is induced, catalyzed and self-assembled to form a DNA reticular nano structure. In the absence of any enzyme, miRNA-21 or miRNA-31 in the cell triggers a cascade hybridization reaction of HP1 and HP2 or HP3 and HP4, so that a plurality of signal output reticular nanostructures are formed through self-assembly.

(3) The preparation of the DNA reticular nano material is independent of the use of enzyme, and the preparation of complex nano material is not needed, so that the stability is good, and the method is different from the traditional DNA origami self-assembly method, only two DNA probes are involved in the method, so that the complicated and time-consuming DNA chain design process is avoided, and the cost is saved; the DNA self-assembly mesh nano material constructed by the invention can be used for imaging of biomarker miRNA in living cells, and provides a potential tool for early diagnosis of diseases and drug research and development.

Drawings

FIG. 1 is a representation of AFM preparation of DNA network nano-material.

FIG. 2 is a graph of the viscosity test of the DNA network nano-material. Left side of fig. 2: is a material viscosity schematic diagram; fig. 2 right side top left small picture: adding loading dye into the synthesized reticular nano material, and drawing by using a glass rod; fig. 2 right large view: and (4) viscosity test without loading dye.

FIG. 3 is a diagram of feasibility of intracellular miRNA-21 imaging. DAPI: fluorescence of cells stained with DAPI nuclei; FAM: fluorescence of cells without nuclear staining; merged: nuclear staining and fluorescence overlay of cells without nuclear staining.

FIG. 4 is a feasibility map of intracellular miRNA-31. DAPI: fluorescence of cells stained with DAPI nuclei; FAM: fluorescence of cells without nuclear staining; merged: nuclear staining and fluorescence overlay of cells without nuclear staining.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the following examples are only examples of the present invention and do not represent the scope of the present invention defined by the claims.

Example 1

A target object catalytic induction self-assembly DNA reticular nano material comprises the following raw materials: three oligomers, HP1, HP2 and miRNA-21; wherein the content of the first and second substances,

the nucleotide sequence of HP1 is:

5 '-GATCGCGATCTCAACAT-FAM-CAGTCTGATAAGCTAACATCGTAGCTTATCAGACTG-BHQ-1-3', wherein GATCGCGATC is a palindromic sequence; TCAACAT (FAM) CAGTCTGATAAGCTA is miRNA-21 recognition sequence; CAGTCTGATAAGCTA is complementary to the TAGCTTATCAGACTG sequence;

the nucleotide sequence of HP2 is:

5 '-AGTTCGAACTT-Cy 3-AGCTTATCAGACTGATGTTGATTTTCAGTCTGATAAGCT-BHQ-2-ACGATGT', wherein AGTTCGAACT is a palindromic sequence; DNA sequences corresponding to T-Cy3-AGCTTATCAGACTGATGTTGA and miRNA-21; the T-Cy3-AGCTTATCAGACTGA is complementary to the TCAGTCTGATAAGCT-BHQ-2-A sequence;

the nucleotide sequence of MiRNA-21 is: 5'-UAGCUUAUCAGACUGAUGUUGA-3', respectively;

the preparation method comprises the following steps: firstly, respectively diluting HP1 and HP2 to 10 mu M, annealing at 90 ℃, and gradually cooling to 25 ℃ to form a hairpin structure for later use; then 1. mu.L of annealed HP1, 1. mu.L of annealed HP2, 50 nM miRNA-21 were mixed with 17. mu.L of tris-buffer and reacted at ambient temperature for 2 hours.

The preparation method of the tris-buffer comprises the following steps: 25mM tris-HCl, 100 mM NaCl, 50 mM KAc, 10 mM MgAc₂ and 1 mM DTT，pH 8.2。

Example 2

A target object catalytic induction self-assembly DNA reticular nano material comprises the following raw materials: three oligomers, HP3, HP4 and miRNA-31; wherein the content of the first and second substances,

the nucleotide sequence of HP3 is: 5 '-GATCGCGATCAGCTAT-FAM-GCCAGCATCTTGCCTACATCGAGGCAAGATGCTGG-BHQ-1-3', wherein GATCGCGATC is a palindromic sequence; AGCTAT (FAM) GCCAGCATCTTGCCT is miRNA-31 recognition sequence, CCAGCATCTTGCCT is complementary with AGGCAAGATGCTGG sequence;

the nucleotide sequence of HP4 is: 5'-AGTTCGAACTAGGCAAGATGCTGGCATAGCTTTTGCCAGCATCTTGCCTCGATGT-3', wherein AGTTCGAACT is a palindromic sequence; AGGCAAGATGCTGGCATAGCT DNA sequence corresponding to miRNA-31; AGGCAAGATGCTGGC is complementary to the GCCAGCATCTTGCCT sequence;

the nucleotide sequence of MiRNA-31 is: 5'-AGGCAAGAUGCUGGCAUAGCU-3'

The preparation method comprises the following steps: firstly, respectively diluting HP3 and HP4 to 10 mu M, annealing at 90 ℃, and gradually cooling to 25 ℃ to form a hairpin structure for later use; then, 1. mu.L of annealed HP3, 1. mu.L of annealed HP4, 50 nM miRNA-31 were mixed with 17. mu.L of tris-buffer and reacted at room temperature for 2 hours.

The preparation method of the tris-buffer comprises the following steps: 25mM tris-HCl, 100 mM NaCl, 50 mM KAc, 10 mM MgAc₂ and 1 mM DTT，pH 8.2。

Example 3

And (3) morphology testing: taking 10 muL of the self-assembled DNA reticular nano material prepared in the embodiment 1, titrating the self-assembled DNA reticular nano material onto a clean and freshly torn mica sheet, placing the self-assembled DNA reticular nano material for 30 minutes at normal temperature, washing the self-assembled DNA reticular nano material for 4 times by using 100 muL of water, and then washing the self-assembled DNA reticular nano material by using N₂And (5) drying. The morphology was then tested and AFM images were taken by the Nanoscope AFM System (Bruker, Germany) in tapping mode, with the following parameters for the silicon cantilever: drive frequency =70 kHz, spring rate = 0.4N/m.

Blank group: HP 1: HP 2: miRNA-21= 1: 1: the resulting material was prepared at a ratio of 0.

The results of the topography test are shown in FIG. 1. The results in FIG. 1 show that: in contrast to the blank group, the self-assembled DNA network nano-material prepared in example 1 is observed to have a network structure in the shape under AFM.

And (3) viscosity test: the dosage of HP1, HP2 and miRNA-21 is enlarged by 10 times. mu.L of HP1 (100. mu.M), 1. mu.L of HP2 (100. mu.M), 500 nM miRNA-21 were mixed with 17. mu.L of tris-buffer and incubated overnight at ambient temperature. The DNA net-like nanomaterial is picked up by a glass rod and pulled upwards.

The results of the tack test are shown in FIG. 2, which shows that: the DNA reticular nano material is drawn by the glass rod and is found to have certain adhesion, which shows that the self-assembled DNA reticular nano material has certain viscosity and has certain advantages for in-situ imaging of cells.

Example 4

Transfection experiments were performed according to the protocol: firstly, mixing HP1 (2 muL, 10 muM), HP2 (2 muL, 10 muM) and non-serum Opti-MEM to 200 muL, standing for 10 minutes at normal temperature, then adding 10 muL of liposome-3000, and incubating for 10 minutes to prepare a DNA mixture; HEK-293 cells, Hela cells and MCF-7 cells were cultured and grown in 12-well plates, and when the cells grew to 80%, the above DNA mixture was added, and after 2 hours, the cells were washed 3 times with PBS, and then in Opti-MEM medium containing serum in the presence of 5% CO₂Incubating in an incubator at 37 ℃ for 12 hours; finally, cells were fixed with 4% (w/v) paraformaldehyde for 20 min; finally, nuclei were exposed to DAPI dye for 5 min; fluorescence imaging was then scanned by A1R confocal laser scanning microscope. The excitation wavelengths used by the DAPI and FAM labeled mesh nanomaterials are 405 nm and 488 nm, respectively.

The results of intracellular imaging are shown in FIG. 3. FIG. 3 shows the results: when miRNA-21 is highly expressed in MCF-7 cells, the fluorescence in the cells is strongest, the fluorescence is inferior to that of HeLa cells expressed in miRNA-21, and the fluorescence signal of HEK-293 cells with low miRNA-21 expression is lowest. Indicating that this method is feasible for miRNA-21 imaging.

Example 5

Transfection experiments were performed according to the protocol. Firstly, HP3 (2 muL, 10 muM), HP4 (2 muL, 10 muM) and non-serum Opti-MEM are mixed to 200 muL, and the mixture is kept stand for 10 minutes at normal temperature. Then, 10 μ L of liposome-3000 was added and incubated for 10 minutes to prepare a DNA mixture. Hela cells and MCF-7 cells were grown in 12-well plates, and when the cells reached about 80%, the DNA mixture was added, and after 2 hours, the cells were treated with PBSAfter 3 washes, the cells were incubated with Opti-MEM containing serum in a medium containing 5% CO₂Incubate at 37 ℃ for 12 hours in an incubator. Finally, the cells were fixed with 4% (w/v) paraformaldehyde for 20 minutes. Finally, nuclei were exposed to DAPI dye for 5 min. Fluorescence imaging was then scanned by A1R confocal laser scanning microscope. The excitation wavelengths used by the DAPI and FAM labeled mesh nanomaterials are 405 nm and 488 nm, respectively.

The results of intracellular imaging are shown in FIG. 4. FIG. 4 shows the results: when miRNA in MCF-7 cells is low expressed, the fluorescence in the cells is weak, and when miRNA in HeLa cells is high expressed, the fluorescence signal in the cells is obviously improved, which indicates that the method is feasible for miRNA imaging.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> Minjiang academy

<120> preparation and application of target object catalytic induction self-assembly DNA mesh nano material

<130> 6

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 53

<212> DNA

<213> HP1

<400> 1

gatcgcgatc tcaacatcag tctgataagc taacatcgta gcttatcaga ctg 53

<210> 2

<211> 57

<212> DNA

<213> HP2

<400> 2

agttcgaact tagcttatca gactgatgtt gattttcagt ctgataagct acgatgt 57

<210> 3

<211> 22

<212> RNA

<213> MiRNA-21

<400> 3

uagcuuauca gacugauguu ga 22

<210> 4

<211> 51

<212> DNA

<213> HP3

<400> 4

gatcgcgatc agctatgcca gcatcttgcc tacatcgagg caagatgctg g 51

<210> 5

<211> 55

<212> DNA

<213> HP4

<400> 5

agttcgaact aggcaagatg ctggcatagc ttttgccagc atcttgcctc gatgt 55

<210> 6

<211> 21

<212> RNA

<213> MiRNA-31

<400> 6

aggcaagaug cuggcauagc u 21