阅读说明：本技术 一种两亲性dna纳米胶束及其制备方法和应用 (Amphiphilic DNA nano micelle and preparation method and application thereof ) 是由 梁文斌 彭锌 卓颖 柴雅琴 袁若 于 2020-05-28 设计创作，主要内容包括：本发明属于生物荧光分析技术领域,具体涉及一种两亲性DNA纳米胶束及其制备方法和应用。该制备方法包括如下步骤：设计目标物miRNA的特异DNA发卡序列H1-5,在H3末端修饰疏水性的C12烷烃链,制备sp-H3冻干粉,再将获得的sp-H3冻干粉制成发夹结构的sp-H3分散液,最后将荧光染料加入分散液中,超声得到DNA纳米胶束。还提供了该DNA胶束纳米在无标记检测目标miRNA的应用。本发明构建的DNA胶束不仅能够快速组装成球形纳米结构,还具有较大的固载容量；同时具有选择性好,操作便捷等特点,对生物小分子的测定具有重要意义。(The invention belongs to the technical field of biological fluorescence analysis, and particularly relates to an amphiphilic DNA nano micelle and a preparation method and application thereof. The preparation method comprises the following steps: designing a specific DNA hairpin sequence H1-5 of a target miRNA, modifying a hydrophobic C12 alkane chain at the tail end of H3 to prepare sp-H3 freeze-dried powder, preparing the obtained sp-H3 freeze-dried powder into sp-H3 dispersion liquid with a hairpin structure, adding a fluorescent dye into the dispersion liquid, and performing ultrasonic treatment to obtain the DNA nano micelle. Also provides the application of the DNA micelle nano in label-free detection of target miRNA. The DNA micelle constructed by the invention not only can be rapidly assembled into a spherical nano structure, but also has larger solid-supported capacity; meanwhile, the method has the characteristics of good selectivity, convenience in operation and the like, and has important significance for measuring the biological small molecules.)

1. A preparation method of amphiphilic DNA nano-micelle is characterized by comprising the following steps:

(1) designing a specific DNA hairpin sequence H1-5 of the target miRNA, wherein the nucleotide sequence is shown as SEQ ID NO: 1-5, wherein the target miRNA is miRNA-21, and the nucleotide sequence is shown as SEQ ID NO: 6, modifying a hydrophobic C12 alkane chain at the tail end of the H3 hairpin sequence to prepare amphiphilic DNA copolymer sp-H3 freeze-dried powder;

(2) preparing dispersion liquid of sp-H3 with a hairpin structure from the sp-H3 freeze-dried powder obtained in the step (1);

(3) and (3) adding a fluorescent dye into the dispersion liquid obtained in the step (2), and performing ultrasonic treatment.

2. The method according to claim 1, wherein in the step (2), the dispersion is prepared as follows: dissolving the sp-H3 lyophilized powder in a solution containing Mg²⁺The buffer solution is incubated for 5-10min at the temperature of 95 +/-2 ℃, and then the temperature is reduced to 25 ℃ at the speed of 0.5 ℃/min and then kept for 5-10 min.

3. The method according to claim 1, wherein in the step (3), the molar ratio of the fluorescent dye to the sp-H3 having a hairpin structure in the dispersion liquid is 10: 1.

4. The method of claim 1, wherein in step (3), the fluorescent dye is a hydrophobic fluorescent dye, and the hydrophobic fluorescent dye is but not limited to perylene, cyanine, oil red or nile red.

5. The method according to claim 1, wherein the ultrasonic power in the step (3) is 30-50W for 5-10 min.

6. An amphiphilic DNA nanomicelle prepared by the method of any one of claims 1-5.

7. The method for label-free detection of target miRNA by amphiphilic DNA nano-micelle of claim 6, wherein the method comprises the following steps:

adding the amphiphilic DNA nano micelle into a mixed solution of H1, H2, H4 and H5 to form a reaction solution, adding a target object miRNA to be detected into the reaction solution for incubation, and finally detecting the fluorescence response intensity.

8. The method of claim 7, wherein the incubation temperature is 35-40 ℃ for 90-120 min.

9. The method of claim 7, wherein the amphiphilic DNA nanomicelles, H1, H2, H4 and H5 in the reaction solution are in a molar ratio of 1:1:1:1: 1.

10. The application of the amphiphilic DNA nano-micelle of claim 6 in the detection of miRNA in tumor cells.

Technical Field

The invention belongs to the field of biological fluorescence analysis, and particularly relates to an amphiphilic DNA nano micelle and a preparation method and application thereof.

Background

The DNA nanostructure attracts attention as a highly controllable emerging material in the field of nanotechnology, and has great development potential in the aspects of drug delivery, disease diagnosis, early detection of tumor markers and the like. In recent years, various DNA nanostructures, such as DNA tetrahedrons, DNA cubes, etc., are designed as nanocages for immobilizing small molecules for application in highly sensitive analytical detection. However, the DNA sequence for assembling the nanostructure usually takes a lot of time to elaborate, and the conditions for assembling the nanostructure by the long DNA chain are harsh, and the problems of long assembling time, low assembling efficiency, etc. limit the wide application of the DNA assembly-based nanomaterial. In view of these problems, it is important to construct a DNA nanostructure with simple base sequence design and high assembly efficiency.

In recent years, it has been reported that an amphiphilic block copolymer can form a nano micelle by self-assembly, and a large amount of functional small molecules can be encapsulated in the micelle, thereby being a popular research object in the fields of constructing drug delivery systems, nano reactors and the like. However, the conventional amphiphilic copolymer micelle releases inner molecules through ultraviolet or pH adjustment, and the adjustment modes not only can not directly realize specificity and quantitative detection on biological molecules, but also can influence the stability of a reaction system through long-term ultraviolet radiation or pH environment change, thereby limiting the application value to a certain extent.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an amphiphilic DNA nano-micelle. The second purpose of the invention is to provide a preparation method of the amphiphilic DNA nano-micelle, the preparation method can enable the amphiphilic DNA to be rapidly assembled into a spherical nano-structure, and the formed nano-micelle has larger solid-supported capacity. The invention also aims to provide a method for detecting target miRNA by amphiphilic DNA nano-micelle in a label-free manner, which has the characteristics of good selectivity, convenience in operation and the like. The fourth purpose of the invention is to provide an application of the amphiphilic DNA nano micelle in miRNA detection in tumor cells. In order to achieve the purpose, the invention provides the following technical scheme:

1. a preparation method of amphiphilic DNA nano-micelle comprises the following steps:

(1) designing a specific DNA hairpin sequence H1-5 of a target miRNA, wherein the nucleotide sequence is shown as SEQ ID NO: 1-5, wherein the target miRNA is miRNA-21, and the nucleotide sequence is shown as SEQ ID NO: 6, modifying a hydrophobic C12 alkane chain at the tail end of the H3 hairpin sequence to prepare amphiphilic DNA copolymer sp-H3 freeze-dried powder;

(2) preparing dispersion liquid of sp-H3 with a hairpin structure from the sp-H3 freeze-dried powder obtained in the step (1);

(3) and (3) adding a fluorescent dye into the dispersion liquid obtained in the step (2), and performing ultrasonic treatment.

As one of the preferred technical solutions: in the step (2), the dispersion is prepared by the following method: dissolving the sp-H3 lyophilized powder in a solution containing Mg²⁺The buffer solution is incubated for 5-10min at the temperature of 95 +/-2 ℃, and then the temperature is reduced to 25 ℃ at the speed of 0.5 ℃/min and then kept for 5-10 min.

As one of the preferred technical solutions: in the step (3), the molar ratio of the fluorescent dye to the sp-H3 with the hairpin structure in the dispersion liquid is 10: 1.

As one of the preferred technical solutions: the fluorescent dye is hydrophobic fluorescent dye, and the hydrophobic fluorescent dye is but not limited to perylene, cyanine, oil red or nile red.

As one of the preferred technical solutions: the ultrasonic power is 30-60W, and the time is 5-10 min.

2. An amphiphilic DNA nano micelle.

3. A method for detecting target miRNA (micro ribonucleic acid) by amphiphilic DNA nano-micelle in a label-free manner comprises the following steps:

adding the amphiphilic DNA nano micelle into a mixed solution of H1, H2, H4 and H5 to form a reaction solution, adding a target object miRNA to be detected into the reaction solution for incubation, and finally detecting the fluorescence response intensity.

As one of the preferred technical solutions: the incubation temperature is 35-40 ℃, and the incubation time is 90-120 min.

As one of the preferred technical solutions: the molar ratio of the amphiphilic DNA nano micelle, H1, H2, H4 and H5 in the reaction solution is 1:1:1: 1.

4. An application of amphiphilic DNA nano-micelle in miRNA detection in tumor cells.

The invention has the beneficial effects that:

(1) the constructed DNA nano micelle structure can be rapidly assembled through non-covalent action, so that the assembly time of the DNA nano structure is greatly shortened;

(2) the constructed DNA micelle is used as a novel nano-carrier, can quickly wrap a large number of related small molecules, and improves the immobilization capacity;

(3) the sp-H3 of the hairpin structure in the DNA nano micelle endows the DNA nano micelle with excellent specificity, accuracy and codeability, and provides a potential basis for realizing qualitative and quantitative analysis of a target object;

(4) the DNA nano-micelle has higher sensitivity, excellent selectivity and good reproducibility for detecting the target miRNA.

Drawings

FIG. 1 is a schematic diagram of the principle of label-free detection of target miRNA by DNA nanomicelle;

FIG. 2(A) is a fluorescence response curve of DNA nano-micelle to miRNA-21 standard solutions of different concentrations, and (B) is a standard curve of fluorescence signal response intensity value and logarithm of miRNA-21 concentration;

FIG. 3(A) is a result graph of the response sensitivity of the amphiphilic DNA nano-micelle to miRNA-21, and (B) is a result graph of the detection of miRNA-21 in cells.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.