阅读说明：本技术 二分支dna四面体纳米结构及其合成方法与应用 (Two-branch DNA tetrahedral nano structure and synthetic method and application thereof ) 是由 郝京诚 王雅静 刘淑雅 于 2020-07-21 设计创作，主要内容包括：本发明属于纳米生物材料领域,涉及二分支DNA四面体纳米结构及其合成方法与应用。通过化学方法合成了两种拓扑结构不同的二分支DNA四面体纳米结构,通过琼脂糖凝胶电泳和原子力显微镜对两种结构的合成情况进行了验证并对两种结构的稳定性进行了对比探究。琼脂糖凝胶电泳结果和原子力显微镜表征结果表明,我成功构筑出了这两种二分支DNA四面体纳米结构。(The invention belongs to the field of nano biological materials, and relates to a two-branch DNA tetrahedral nano structure and a synthetic method and application thereof. Two kinds of two-branch DNA tetrahedral nano-structures with different topological structures are synthesized by a chemical method, the synthesis conditions of the two kinds of structures are verified by agarose gel electrophoresis and an atomic force microscope, and the stability of the two kinds of structures is contrasted and researched. Agarose gel electrophoresis results and atomic force microscope characterization results show that I successfully constructs the two-branch DNA tetrahedral nano-structures.)

1. A two-branch DNA tetrahedron nanostructure, wherein each side of the DNA tetrahedron is formed by hybridization of a DNA strand of 31 deoxynucleotide bases to its complement, and an unpaired thymine T is designed at the vertex of the tetrahedron.

2. The two-branch DNA tetrahedral nanostructure of claim 1, wherein an angle between adjacent edges of the tetrahedron is 60 °.

3. The two-branch DNA tetrahedron nanostructure of claim 1, wherein the DNA tetrahedron has a structure as shown in 2branches a or 2branches B:

4. the two-branched DNA tetrahedral nanostructure of claim 3, wherein the DNA tetrahedra 2branchesA has the sequence:

Stand 1:

ATCGTCTATAGTAAGTTTTTCCTAACGCAGGTTGTTTTCGCGTTACTTTATAGCGGATTTTCATTTGGATCAAATATGAGTAGGTCACGTATCTATTCGGATCCTAGGCTCAGGATCTGGGTATCCATTAGCACATTCAATCTCCGTTCAGGGGCTCGGTTGAAAATCCGCTATAAAGTAACGCGAAAACATCACATCTGATCCGACTGTTTGTCTCTCTTCATTAGATACGTGACCTACTCATATTTGATCCAATCCGAGCCCCTGAACGGAGATTGAATGTGCTA

Stand 2:

CCTGCGTTAGGAAAAACTTACTATAGACGATTTGGATACCCAGATCCTGAGCCTAGGATCCGATTGAAGAGAGACAAACAGTCGGATCAGATGTG。

5. the two-branched DNA tetrahedral nanostructure of claim 3, wherein the DNA tetrahedron 2branchesB has the sequence:

Stand 1:

GGTCGCTGTCGAAAGGCAGTTTCCTAGCAATTTTCGCACGGTGGAGAGTCCGTCTTAACCGCCTTGCCGTCCGACTGGATGTTCAGTTCCTCAAATGCTGTGTAGGTCTGACGCAAAGATCGTACATTATTGCTAGGAAACTGCCTTTCGACAGCGACCTGGGTTTTGCCCTTGTTCAGGCCATGCAGTCATTTTGAGGAACTGAACATCCAGTCGGACGGCATGGAAGCTCCCATGACCATAGGTGAATAAGCT

Stand 2:

ATGTACGATCTTTGCGTCAGACCTACACAGCTTGACTGCATGGCCTGAACAAGGGCAAAACCCTAGCTTATTCACCTATGGTCATGGGAGCTTCCTGGCGGTTAAGACGGACTCTCCACCGTGCGAA。

6. the preparation method of the two-branch DNA tetrahedral nano structure is characterized by comprising the following steps:

designing a DNA single strand constituting a DNA tetrahedron;

and mixing the DNA single chains in a buffer solution, and carrying out heating annealing treatment to obtain the double-branch DNA tetrahedral nano structure.

7. The method for preparing a two-branch DNA tetrahedral nanostructure of claim 6, wherein the buffer is TEM buffer.

8. The method for preparing a two-branch DNA tetrahedral nanostructure of claim 6, wherein the final concentration of each DNA single strand is 0.1 μ M.

9. The method for preparing a two-branch DNA tetrahedral nano-structure according to claim 6, wherein the heating annealing comprises the following specific steps: the temperature was raised to 95 ℃ and held for 5 minutes, and then the system was slowly cooled to room temperature over 48 hours.

10. Use of the two-branch DNA tetrahedral nanostructure of any one of claims 1-5 in biological detection, in vivo imaging, gene vector or drug delivery.

Technical Field

The invention belongs to the field of nano biological materials, and particularly relates to synthesis of two-branch DNA tetrahedral nano structures with different topological structures.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

DNA is a long-chain polymer whose basic building block is a deoxynucleotide monomer. Such deoxynucleotide monomers consist of three covalently linked moieties: nitrogenous bases, deoxyribose, and phosphate backbone. In double-stranded DNA, two DNA strands are arranged in antiparallel, and bases at opposite positions on the two DNA backbones arranged in antiparallel interact with each other to form Watson Crick hydrogen bonds, wherein two hydrogen bonds are present in the A and T base pairs, and 3 hydrogen bonds are present in the G and C base pairs. Under normal physiological conditions, the most common double-stranded DNA is the B-type, right-handed DNA duplex, which has a duplex width of 2.2-2.6nm and a length of 3.36nm per spiral cycle, with 10.5 base pairs per spiral cycle. In addition, when the external conditions are changed, for example, the water content in the vicinity of the skeleton is decreased, the configuration of the DNA strand is also changed, and right-hand type DNA double helix A and left-hand type double helix Z DNA, which are more compact than B-type DNA, are more common in organisms.

The DNA molecule has the characteristics of good programmability, predictable stability and easy synthesis and modification, which makes the DNA molecule a very ideal self-assembly material. The hollow structure of the frame-shaped DNA polyhedron is very similar to the existing structure of the natural world such as virus, etc., can encapsulate chemical micromolecules, drug molecules and other functional materials, and has very wide application prospect. Numerous researchers have explored the applications of DNA tetrahedron in biomedicine and material science, but the most widely used DNA tetrahedron is the four-branch type DNA tetrahedron nanostructure synthesized by one-step method in Turberfield group. In the construction process of the four-branch tetrahedron, the chemical equivalent ratio of four DNA component chains is required to be accurately controlled to be 1: 1: 1: 1, the experimental operation has great difficulty. To investigate the success of constructing a completed DNA tetrahedron using only one DNA strand, researchers designed and constructed a nanoscale DNA tetrahedron structure synthesized from a single 286 base DNA strand and replicated this tetrahedron by in vivo molecular cloning techniques. But the inventor finds that: the tetrahedral structure of DNA constructed in this study is not compact, limited by the polarity of the DNA strand, and a parallel DNA double helix portion must be present on one side of the tetrahedron.

Disclosure of Invention

In order to overcome the problems, the invention synthesizes two double-branch DNA tetrahedral nano-structures with different topological structures by a chemical method, verifies the synthesis conditions of the two structures by agarose gel electrophoresis and an atomic force microscope and contrasts and explores the stability of the two structures. The agarose gel electrophoresis result and the atomic force microscope characterization result show that the invention successfully constructs the two-branch DNA tetrahedral nano-structures. Meanwhile, by performing topology analysis on DNA tetrahedrons and combining the polarity of DNA chains, the invention considers that the minimum branch number of the wire frame type DNA regular tetrahedron with a compact structure is two branches.

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

in a first aspect of the invention, a two-branch DNA tetrahedron nanostructure is provided, each side of the DNA tetrahedron being formed by hybridization of a 31 deoxynucleotide base DNA strand to its complement, and an unpaired thymine T being designed at the vertex of the tetrahedron.

The invention proves that the minimum branch number required for constructing the DNA tetrahedron with compact and symmetrical structure is two branches, and the synthetic steps of the DNA tetrahedron are effectively simplified.

In a second aspect of the present invention, there is provided a method for preparing a two-branch DNA tetrahedral nanostructure, comprising:

designing a DNA single strand constituting a DNA tetrahedron;

and mixing the DNA single chains in a buffer solution, and carrying out heating annealing treatment to obtain the double-branch DNA tetrahedral nano structure.

In a third aspect of the invention, there is provided the use of any of the two-branched DNA tetrahedral nanostructures described above in biological detection, in vivo imaging, gene vector or drug delivery.

The invention has the beneficial effects that:

(1) the invention synthesizes two-branch DNA tetrahedral nano-structures with different topological structures by a chemical method, verifies the synthesis condition of the two structures by agarose gel electrophoresis and an atomic force microscope and contrasts and explores the stability of the two structures. Agarose gel electrophoresis results and atomic force microscope characterization results show that the invention successfully constructs the two-branch DNA tetrahedral nano-structures, and has compact structure and good stability.

(2) The method is simple, strong in practicability and good in application prospect.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a diagram of the folding path of two divergent DNA tetrahedral deoxynucleotide strands;

FIG. 2 is a graph showing the results of gel electrophoresis of a four-branched DNA tetrahedron in example 1 of the present invention;

FIG. 3 is a graph showing the results of gel electrophoresis of the two-branched DNA tetrahedron of example 1 of the present invention;

FIG. 4 is a graph showing the results of stability testing of four-and two-branched DNA tetrahedrons in example 1 of the present invention;

FIG. 5 is a graph showing the results of the shape scanning of the DNA tetrahedron 2branches A in example 1 of the present invention.

FIG. 6 is a graph showing the results of the shape scanning of the DNA tetrahedron 2branches B in example 1 of the present invention.

FIG. 7 is a graph showing the results of the shape scanning of the four-branched DNA tetrahedron in example 1 of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

A two-branch DNA tetrahedron nanostructure, each edge of the DNA tetrahedron being formed by hybridization of a 31 deoxynucleotide base DNA strand to its complement, an unpaired thymine T being designed at the vertex of the tetrahedron.

The folding pathways of the deoxynucleotide chains of the two-branched DNA tetrahedra are shown in FIG. 1. Each side of the two DNA tetrahedrons is formed by hybridizing a DNA chain with 31 deoxynucleotide bases and a complementary sequence thereof, and an unpaired thymine T designed at the vertex of the tetrahedron endows the DNA chain with enough folding flexibility, so that an included angle of 60 degrees can be formed between adjacent sides of the tetrahedron. After annealing, the two component strands are folded according to a predetermined path to form a DNA tetrahedral structure with each side being a DNA double helix.

The invention uses DNA sequence design software Uniquimer to design the sequences of the two-branch DNA tetrahedrons with different topological structures.

The sequence of DNA tetrahedron 2branches A is:

Stand 1:

ATCGTCTATAGTAAGTTTTTCCTAACGCAGGTTGTTTTCGCG TTACTTTATAGCGGATTTTCATTTGGATCAAATATGAGTAGGTCA CGTATCTATTCGGATCCTAGGCTCAGGATCTGGGTATCCATTAGC ACATTCAATCTCCGTTCAGGGGCTCGGTTGAAAATCCGCTATAA AGTAACGCGAAAACATCACATCTGATCCGACTGTTTGTCTCTCT TCATTAGATACGTGACCTACTCATATTTGATCCAATCCGAGCCCC TGAACGGAGATTGAATGTGCTA

Stand 2:

CCTGCGTTAGGAAAAACTTACTATAGACGATTTGGATACCC AGATCCTGAGCCTAGGATCCGATTGAAGAGAGACAAACAGTCG GATCAGATGTG

the sequence of DNA tetrahedron 2branches B is:

Stand 1:

GGTCGCTGTCGAAAGGCAGTTTCCTAGCAATTTTCGCACGG TGGAGAGTCCGTCTTAACCGCCTTGCCGTCCGACTGGATGTTCA GTTCCTCAAATGCTGTGTAGGTCTGACGCAAAGATCGTACATTA TTGCTAGGAAACTGCCTTTCGACAGCGACCTGGGTTTTGCCCTT GTTCAGGCCATGCAGTCATTTTGAGGAACTGAACATCCAGTCGGACGGCATGGAAGCTCCCATGACCATAGGTGAATAAGCT

Stand 2:

ATGTACGATCTTTGCGTCAGACCTACACAGCTTGACTGCAT GGCCTGAACAAGGGCAAAACCCTAGCTTATTCACCTATGGTCAT GGGAGCTTCCTGGCGGTTAAGACGGACTCTCCACCGTGCGAA

the invention also provides a preparation method of the double-branch DNA tetrahedral nano structure, which comprises the following steps:

designing a DNA single strand constituting a DNA tetrahedron;

and mixing the DNA single chains in a buffer solution, and carrying out heating annealing treatment to obtain the double-branch DNA tetrahedral nano structure.

The specific type of buffer is not particularly limited in this application and in some embodiments, the buffer is a TEM buffer to allow for efficient dispersion of the DNA.

The reaction rate increases with the increase in the concentration of DNA single strands, but an excessively high concentration of DNA strands increases the mismatching rate of bases and decreases the specificity. Thus, in some embodiments, the final concentration of each DNA single strand is 0.1 μ M to increase the efficiency of the reaction.

In some embodiments, the temperature-raising annealing comprises the following specific steps: the temperature was raised to 95 ℃ and held for 5 minutes, and then the system was slowly cooled to room temperature over 48 hours. After annealing, the two component strands are folded according to a predetermined path to form a DNA tetrahedral structure with each side being a DNA double helix.

The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.