CN111825734A - Two-branch DNA tetrahedral nano structure and synthetic method and application thereof - Google Patents

Two-branch DNA tetrahedral nano structure and synthetic method and application thereof Download PDF

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CN111825734A
CN111825734A CN202010707821.4A CN202010707821A CN111825734A CN 111825734 A CN111825734 A CN 111825734A CN 202010707821 A CN202010707821 A CN 202010707821A CN 111825734 A CN111825734 A CN 111825734A
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CN111825734B (en
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郝京诚
王雅静
刘淑雅
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Shandong University
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Abstract

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.

Description

Two-branch DNA tetrahedral nano structure and synthetic method and application thereof
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.
Example 1:
in the experiment, a four-branch DNA tetrahedron nanostructure formed by hybridizing four oligonucleotide chains with equal length is also constructed, and the molecular weight of the tetrahedron is the same as that of two-branch DNA tetrahedron nanostructures. The sequence of the four-branch tetrahedra was also designed by the DNA sequence design software, equimer. The sequence of the four component chains of the four-branch tetrahedron is as follows:
Stand 1:
AAACTACTCCTCGAAGTGATTTGTACCGTCTTGATAGGGCG GGACCCGGGATAGCATATGGGTTTGCCCGGATCGAGACCCCTCA ATTCGGGAGG
Stand 2:
ACCCATATGCTATCCCGGGTCCCGCCCTATCTATTTGCGTGA TCGCATCACTACCAGACGGACTTAAAAGGGGAATCCCTGCCAC GTGAATGCGG
Stand 3:
AGACGGTACAAATCACTTCGAGGAGTAGTTTTTTATTCGGT ATGGTTATGCCTTACGCATGTTTGTCCGTCTGGTAGTGATGCGAT CACGCAAAT
Stand 4:
AACATGCGTAAGGCATAACCATACCGAATAATCCTCCCGAA TTGAGGGGTCTCGATCCGGGCATCCGCATTCACGTGGCAGGGAT TCCCCTTTTA
the invention customizes corresponding deoxynucleotide chains from biological companies according to the sequence designed by software, and is used for synthesizing two-branch and four-branch DNA tetrahedral nano-structures. In the process of constructing the DNA tetrahedron, the invention uses an experimental method of temperature rise annealing. First, the present invention is applied to a sample containing TEM buffer (10mM Tris,1mM EDTA,10mM MgCl)2pH7.6) was added to the corresponding DNA component strands in an equivalent stoichiometric ratio, ensuring that the final concentration of each DNA strand was 0.1. mu.M. Thereafter, the temperature was raised to 95 ℃ and held for about 5 minutes, and finally the centrifuge tube was transferred to a holding vessel containing about 1.5L of water, and the system was allowed to slowly cool to room temperature over a period of 48 hours.
Characterization results
The invention uses 2.5% agarose gel electrophoresis and atomic force microscopy to characterize the two-and four-branched DNA tetrahedron.
1. Agarose gel electrophoresis result analysis
FIG. 2 shows the results of gel electrophoresis of four-branched DNA tetrahedrons. Lane 1 is the four-branched tetrahedral Stand 1 strand, Lane 2 is the product of the four-branched tetrahedral Stand 1 strand and Stand 2 strand, Lane 3 is the product of the four-branched tetrahedral Stand 1 strand, Stand 2 strand and Stand 3, Lane 4 is the four-branched DNA tetrahedral nanostructure, and the M band is 50 bpDNAsder. According to the gel electrophoresis experimental result, the invention can preliminarily verify that the four-branch DNA tetrahedron is successfully constructed by the invention.
FIG. 3 shows the results of gel electrophoresis of two-branched DNA tetrahedrons. Four-branched DNA tetrahedron nanostructures are located in lanes 4 and 5, DNA tetrahedron 2branches A in lane 3, and DNA tetrahedron 2branches B in lane 8. The two component strands of DNA tetrahedron 2branches A and 2branches B are located in lanes 1, 2 and 6, 7, respectively, and the M band is 50 bpDNAsder. As can be seen from the gel electrophoresis chart, the bands of the two-branch tetrahedron and the four-branch tetrahedron are positioned at the same horizontal position of the gel electrophoresis chart. According to the gel electrophoresis experimental result, the invention can preliminarily verify that the two-branch DNA tetrahedron is successfully constructed by the invention.
FIG. 4 is the results of stability testing of four-and two-branched DNA tetrahedrons. The invention uses agarose gel electrophoresis to compare and research the stability of two-branch and four-branch DNA tetrahedron. The four panels in FIG. 4 are the results of agarose gel electrophoresis experiments after 1 day, 1 week, 2 weeks and 3 weeks of sample placement, respectively. Among them, the four-branched DNA tetrahedron nanostructure is shown in lane 1, DNA tetrahedron 2branches a is shown in lane 2, and DNA tetrahedron 2branches B is shown in lane 3. By comparing gel electrophoresis images at different stages, the invention can show that the two-branch DNA nano structure constructed by the invention has better structural stability within three weeks.
2. Analysis of atomic force microscope characterization results
The invention uses an atomic force microscope to carry out shape representation on the DNA tetrahedral nano structure constructed by the invention, and the imaging substrate is a mica sheet. According to the invention, firstly, polylysine is used for modifying mica sheets, then a proper amount of samples are dropwise added to a mica substrate modified by lysine, the mica substrate is dried after standing for a period of time, and then the appearance of the mica substrate is scanned by using an atomic force microscope Bruker BioScopeResolve.
FIGS. 5, 6 and 7 are the results of the shape scanning of DNA tetrahedron 2branches A, DNA tetrahedron 2branches B and four-branch DNA tetrahedron, respectively. As can be seen from the shape scanning image, the two-branch DNA tetrahedrons have more regular particle sizes, the longitudinal size of the tetrahedron is about 20nm and is slightly larger than the expected size of 10nm, and the invention considers that the two-branch DNA tetrahedrons are caused by the broadening effect of the AFM needle point. In the experimental results, the height of the sample was about 2nm, which is slightly higher than the height of the DNA strand, and this is considered to be caused by the strong force of the substrate against the sample and the loss of water from the sample. The imaging result of the atomic force microscope can well prove that the invention successfully synthesizes two-branch DNA tetrahedral structures with different topological structures.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
SEQUENCE LISTING
<110> Shandong university
<120> two-branch DNA tetrahedral nano structure and synthetic method and application thereof
<130>2020
<160>8
<170>PatentIn version 3.5
<210>1
<211>287
<212>DNA
<213> Artificial sequence
<400>1
atcgtctata gtaagttttt cctaacgcag gttgttttcg cgttacttta tagcggattt 60
tcatttggat caaatatgag taggtcacgt atctattcgg atcctaggct caggatctgg 120
gtatccatta gcacattcaa tctccgttca ggggctcggt tgaaaatccg ctataaagta 180
acgcgaaaac atcacatctg atccgactgt ttgtctctct tcattagata cgtgacctac 240
tcatatttga tccaatccga gcccctgaac ggagattgaa tgtgcta 287
<210>2
<211>95
<212>DNA
<213> Artificial sequence
<400>2
cctgcgttag gaaaaactta ctatagacga tttggatacc cagatcctga gcctaggatc 60
cgattgaaga gagacaaaca gtcggatcag atgtg 95
<210>3
<211>255
<212>DNA
<213> Artificial sequence
<400>3
ggtcgctgtc gaaaggcagt ttcctagcaa ttttcgcacg gtggagagtc cgtcttaacc 60
gccttgccgt ccgactggat gttcagttcc tcaaatgctg tgtaggtctg acgcaaagat 120
cgtacattat tgctaggaaa ctgcctttcg acagcgacct gggttttgcc cttgttcagg 180
ccatgcagtc attttgagga actgaacatc cagtcggacg gcatggaagc tcccatgacc 240
ataggtgaat aagct 255
<210>4
<211>127
<212>DNA
<213> Artificial sequence
<400>4
atgtacgatc tttgcgtcag acctacacag cttgactgca tggcctgaac aagggcaaaa 60
ccctagctta ttcacctatg gtcatgggag cttcctggcg gttaagacgg actctccacc 120
gtgcgaa 127
<210>5
<211>95
<212>DNA
<213> Artificial sequence
<400>5
aaactactcc tcgaagtgat ttgtaccgtc ttgatagggc gggacccggg atagcatatg 60
ggtttgcccg gatcgagacc cctcaattcg ggagg 95
<210>6
<211>95
<212>DNA
<213> Artificial sequence
<400>6
acccatatgc tatcccgggt cccgccctat ctatttgcgt gatcgcatca ctaccagacg 60
gacttaaaag gggaatccct gccacgtgaa tgcgg 95
<210>7
<211>95
<212>DNA
<213> Artificial sequence
<400>7
agacggtaca aatcacttcg aggagtagtt ttttattcgg tatggttatg ccttacgcat 60
gtttgtccgt ctggtagtga tgcgatcacg caaat 95
<210>8
<211>95
<212>DNA
<213> Artificial sequence
<400>8
aacatgcgta aggcataacc ataccgaata atcctcccga attgaggggt ctcgatccgg 60
gcatccgcat tcacgtggca gggattcccc tttta 95

Claims (10)

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:
Figure FDA0002594870640000011
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.
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