CN114045283A

CN114045283A - DNA nano structure, construction method and application

Info

Publication number: CN114045283A
Application number: CN202111287563.XA
Authority: CN
Inventors: 黄震; 吕家臻
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-11-02
Filing date: 2021-11-02
Publication date: 2022-02-15

Abstract

The invention belongs to the technical field of nano-structures, and discloses a DNA nano-structure, a construction method and application, wherein the construction method comprises the following steps: DNA nanostructures are formed by folding scaffold DNA strands using RNA and modified RNA. The invention utilizes the short RNA chain as a clamp to guide the formation of the DNA nano structure, and RNA and modified RNA (2' -Z-RNA) fold the bracket DNA chain to form the DNA nano structure, thereby enriching the construction method of the DNA nano structure. The invention uses the short RNA clip to efficiently assemble the DNA scaffold chain into a designed shape and structure, and provides an architectural platform for folding various complex DNA nano materials by using the strategy of folding DNA by the RNA clip.

Description

DNA nano structure, construction method and application

Technical Field

The invention belongs to the technical field of nanostructures, and particularly relates to a DNA nanostructure, a construction method and application.

Background

Currently, RNA self-assembly is widespread, such as RNA quadrilateral, tetrahedral, k-turn, pRNA-3WJ, viral IRES model, because of their high diversity, long RNA strands are difficult to predict. In contrast, DNA strands are programmable and can be folded artificially. Based on Watson-Crick base pairing, DNA is undoubtedly the most promising molecule for self-assembly capability. DNA can self-assemble into 2D and 3D nanostructures (e.g., DNA triangles, DNA tetrahedrons, and other recombinant DNA geometries under reasonable design), which makes them more potentially useful in a variety of applications. A long DNA scaffold can be folded by DNA staple strands into different nanostructures, i.e. DNA origami. With the help of calculation, DNA paper folding technology is found, and DNA nano-structure is developed into a top-down construction method, such as DNA expression, DNA box and the like. Programmed DNA nanobodies are of great interest in the study of nanostructures, molecular instrumentation applications, and nanomedicine, and RNA scaffold strands folded by DNA or RNA staple strands are also used to develop these applications.

DNA nanostructures formed due to DNA scaffold and DNA staple chains are often accompanied by nanostructure configuration errors (e.g., polymerization at high concentrations), low yields, and low efficiencies. In addition, the cost of RNA is relatively high, and when preparing a large DNA nanostructure, a segment of DNA needs to be introduced to participate in pairing, and the assembly efficiency of the corresponding large DNA nanostructure is low. These challenges are a major impediment in DNA nanostructure and nanotechnology applications. Therefore, a new method for constructing DNA nanostructures is needed.

Through the above analysis, the problems and defects of the prior art are as follows: because the cost of RNA is relatively high and a section of DNA needs to be introduced to participate in pairing, the assembly efficiency for preparing a large DNA nano structure is low.

The difficulty in solving the above problems and defects is: the large DNA nanostructure requires longer DNA and RNA strands, the cost of preparing longer DNA and RNA single strands is higher and the purity is relatively low, and it is also more easily degraded by nuclease, and the longer nucleic acid strand will more easily generate base mismatch, further restricting the assembly efficiency of the large DNA nanostructure.

The significance of solving the problems and the defects is as follows: the 2' -OMe-RNA is introduced to help improve the stability of the DNA-RNA nanostructure, and the obtained high-purity large DNA nanostructure can promote the wide application of the DNA-RNA nanostructure in the fields of novel materials, drug delivery systems, molecular sensors, cell imaging and the like.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a DNA nano structure, a construction method and application, and particularly relates to a method for guiding the formation of the DNA nano structure by an RNA clip.

The invention is realized by a method for constructing a DNA nano structure, which comprises the following steps:

DNA nanostructures are formed by folding scaffold DNA strands using RNA and modified RNA.

Further, the modified RNA is 2' -OMe-RNA.

Further, the method for constructing the DNA nanostructure further comprises the following steps:

using the outer strand as an RNA clip, and folding the DNA scaffold strand containing 3-5 folding units into a triangle, a quadrangle and a pentagon respectively; wherein, the DNA nano-structure formed by folding one RNA clip comprises two oligonucleotide components with high assembly efficiency.

and folding the DNA scaffold chain by using RNA with a special structure as a clamp to obtain the DNA nano structure.

and changing the number of the RNA clip binding sites of the DNA support chain to obtain DNA nano-structures with different shapes.

and folding the DNA scaffold by using the 2' -OMe modified RNA as a clamp to obtain the DNA nanostructure.

by introducing a piece of DNA into the RNA-folded DNA nanostructure, a larger DNA nanostructure is obtained.

folding DNA by using a DNA-RNA hybrid chain obtained by replacing RNA with partial DNA as a clamp to obtain a DNA nano structure; and/or DNA nanostructures obtained by stably folding DNA scaffold strands by extended DNA clip strands.

Another objective of the present invention is to provide a novel DNA nanostructure constructed by the method for constructing a DNA nanostructure, wherein the general formula of the novel DNA nanostructure is:

the invention also aims to provide the application of the novel DNA nano structure in molecular instruments and nanomedicine.

By combining all the technical schemes, the invention has the advantages and positive effects that: the method for constructing the DNA nanostructure, provided by the invention, uses a short RNA chain as a clamp to guide the formation of the DNA nanostructure, and RNA and modified RNA (2' -OMe-RNA) can fold a bracket DNA chain to form the DNA nanostructure, so that the method for constructing the DNA nanostructure is enriched.

The invention can efficiently assemble DNA scaffold chains into designed shapes and structures by using short RNA clips (including modified RNA), and the short RNA is an ideal bracket clip of DNA self-assembly nano materials because the short RNA is low in price and has better chemical stability after 2-methyl modification. The invention provides an architectural platform for folding various complex DNA nano materials by using the strategy of folding DNA by using the RNA clamp, and the DNA nano structure formed by folding the RNA clamp comprises two oligonucleotide components with high assembly efficiency, so that the construction of the DNA nano structure is easy.

The invention uses RNA with special structure as clamp to fold DNA support chain to obtain DNA nanometer structure; the number of the RNA clip binding sites of the DNA support chain is changed, so that DNA nano-structures with different shapes can be obtained; the 2' -OMe modified RNA is used as a clamp to fold the DNA support to obtain a DNA nano structure with higher efficiency and better stability; by introducing a piece of DNA into the RNA-folded DNA nanostructure, larger DNA nanostructures can be obtained.

Under the condition of conventional RNA self-assembly, when the RNA chain is longer, a complex structure which is difficult to predict is easily formed, and the design and the modification of the complex structure are not facilitated, so that the problem is well avoided by utilizing the short RNA chain to fold the long DNA chain; in the conventional DNA origami or DNA self-assembly nanostructure, the assembly efficiency is low due to the insufficient diversity of DNA structure, so that more DNA strands are needed to complete the assembly, and the more DNA strands also easily cause the wrong assembly of DNA, which finally causes the assembly efficiency to be low. The invention skillfully utilizes the characteristics of structural diversity of RNA and easy programmability of DNA, and utilizes two nucleic acid chains to form a DNA nano structure with higher purity.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram of a novel DNA nanostructure provided by an embodiment of the present invention.

FIG. 2 is a schematic diagram of DNA squares that can be folded by an RNA clip according to an embodiment of the present invention.

FIG. 3 is a schematic diagram of triangular, square and pentagonal nanostructures of RNA clip folded DNA provided by an embodiment of the invention.

FIG. 4 is a schematic diagram of the design and electron microscopy imaging of enlarged triangular, square and pentagonal DNA nanostructures provided by embodiments of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a DNA nano structure, a construction method and application thereof, and the invention is described in detail with reference to the accompanying drawings.

The construction method of the DNA nano structure provided by the embodiment of the invention comprises the following steps: DNA nanostructures are formed by folding scaffold DNA strands using RNA and modified RNA.

The technical solution of the present invention will be further described with reference to the following explanation of terms.

In the present invention, DNA nanostructure (dnananostructured): artificial nucleic acid structures designed based on complementary pairing of nucleic acid bases. RNA clamps (RNA clamps) have a short RNA strand with a specific structure that can be mated and assembled with a DNA scaffold strand to form a specific shape.

The technical solution of the present invention is further described below with reference to specific examples.

To solve the problems of the prior art, the present invention uses short RNA clips rather than DNA staple strands, taking advantage of the structural diversity and double-strand stability of RNA. In fact, short RNA strands with specific structures still allow RNA-DNA interaction and stability, unlike double-stranded DNA staple strands, short RNA clips have stronger binding capacity and more diverse conformation, and can in principle guide DNA scaffold strands into a variety of shapes. The invention can efficiently assemble DNA scaffold chains into designed shapes and structures by using short RNA clips (including modified RNA), and the short RNA is an ideal bracket clip of DNA self-assembly nano materials because the short RNA is low in price and has better chemical stability after 2-methyl modification.

The present invention first reports that RNA clips can efficiently fold DNA scaffold strands into many shapes, such as DNA triangles, quadrilaterals, and pentagons, while the corresponding DNA cannot be efficiently completed. This RNA clamp is an IRES domain 2 α from the hepatitis C virus RNA genome, which is spliced by two overlapping sequences into a corner, which can assemble into an RNA quadrilateral under X-ray crystal confirmation. In experimental design, the outer strand is used as an RNA clamp, and DNA scaffold strands (containing 3-5 folding units) are folded into triangles, quadrangles and pentagons respectively. These DNA nanostructures have been successfully assembled into larger DNA nanostructures, some polygonal nanostructures (DNA triangles, quadrilaterals, pentagons) being designed into the building cornerstone of more complex nanostructures. The invention provides an architectural platform for folding various complex DNA nano materials by using the strategy of folding DNA by using the RNA clamp, and the DNA nano structure formed by folding the RNA clamp comprises two oligonucleotide components with high assembly efficiency, so that the construction of the DNA nano structure is easy.

The structure of the invention is schematically shown in FIG. 1, and RNA and modified RNA (2' -OMe-RNA) can fold scaffold DNA strands to form DNA nanostructures.

The invention uses RNA with special structure as clamp to fold DNA support chain to obtain DNA nanometer structure; the number of the RNA clip binding sites of the DNA support chain is changed, so that DNA nano-structures with different shapes can be obtained; the 2' -OMe modified RNA is used as a clamp, so that DNA can be folded to obtain a DNA nano structure with higher efficiency and better stability; by introducing a piece of DNA into the RNA-folded DNA nanostructure, larger DNA nanostructures can be obtained.

Under the condition of conventional RNA self-assembly, when the RNA chain is longer, a complex structure which is difficult to predict is easily formed, and the design and the modification of the complex structure are not facilitated, so that the problem is well avoided by utilizing the short RNA chain to fold the long DNA chain;

in the conventional DNA origami or DNA self-assembly nanostructure, the assembly efficiency is low due to the insufficient diversity of DNA structure, so that more DNA strands are needed to complete the assembly, and the more DNA strands also easily cause the wrong assembly of DNA, which finally causes the assembly efficiency to be low. The invention skillfully utilizes the characteristics of structural diversity of RNA and easy programmability of DNA, and utilizes the DNA nano structure with higher purity which can be obtained by two nucleic acid chains.

The alternative scheme of the invention is as follows: (1) because RNA and 2' -OMe-RNA have better structural diversity, DNA-RNA hybrid strand obtained by replacing RNA with partial DNA can be used as a clamp to fold DNA to obtain a DNA nano structure; (2) also, since the diversity is insufficient when short DNA is used as a clip, the DNA scaffold strand cannot be stably folded, and the DNA nanostructure can be obtained by stably folding the DNA scaffold strand by the extended DNA clip strand.

The RNA outer strand (15R) plays a key role in RNA quadrilateral stable assembly, and the invention explores whether the corresponding DNA strand can also form a DNA quadrilateral. The present invention replaces the inner strand (10R) and the outer strand (15R) of the RNA quadrilateral with the corresponding DNAs (10D and 15D), respectively, allowing the inner strands (10R and 10D) to assemble with the outer strands (15R and 15D). As shown by non-denaturing polyacrylamide gel electrophoresis (PAGE; FIG. 2a), RNA quadrangles (10R +15R) were successfully assembled, but DNA quadrangles (10D +15D) were poorly assembled and DNA quadrangles (10R +15D) were not formed. However, in the case of 15R, DNA-RNA quadrilateral (10D +15R) assembly was successful. These results indicate that 15R has better diversity and binding affinity in nano-assembly compared to the corresponding DNA. Therefore, the invention finds an RNA outer strand with better diversity, can be used as an RNA clamp and can fold a DNA strand into various nano structures.

FIG. 2DNA squares can be formed by folding RNA clips. 15R: RNA square outer strand RNA (15 nt); 10R: inner strand RNA (10nt) of RNA squares; 15D: 15R corresponding DNA; 10D: 10R corresponding DNA; 20D: 10D DNA in duplicate; 40D: 10D quadruple DNA. The assembly of these nanostructures was analyzed by non-denaturing gels. (a) Assembly of inner chains (10R and 10D) and outer chains (15R and 15D); (b) the DNA scaffolds (10D, 20D and 40D) were folded with RNA clips (15R).

To investigate whether RNA clips can fold DNA into nanostructures, the present invention designed some DNA strands, including the corresponding DNA inner strand (10D, a folding unit) and its repetitive DNA strand with 2-, 4-fold units, 20D and 40D, respectively. Interestingly, the present inventors found that such RNA clips can fold 10D, 20D and 40D into DNA quadrangles, which also improves the DNA quadrangle assembly efficiency (FIG. 2 b).

To further investigate whether RNA clips can fold DNA strands into nanostructures, the present invention designed DNA strands with 3-and 5-fold units, 30D and 50D, respectively. The present inventors found that RNA clips were assembled with 30D, 40D, and 50D to form triangular, quadrilateral, and pentagonal DNA nanostructures, respectively (fig. 3 a). In particular, all nanostructures folded efficiently in the presence of 2-methylated counterpart RNA clips, and the folding of DNA strands was more efficient than 15R in the triangle (98.5% vs 95.1%) quadrilateral (96.0% vs 91.2%) and pentagon (84.3% vs 62.1%). The results show that the assembly efficiency increases in the order of DNA pentagons (theoretical angle: 108 ℃), quadrilaterals (90 ℃) and triangles (60 ℃), which demonstrates that the RNA gripper (15R) is more inclined to form a compact structure of small angles (60 ℃), but the corresponding DNA (15D) cannot fold into any nanostructure (FIG. 3 c). Unlike most RNA-DNA hybrid nanostructures with 4 oligonucleotides, each RNA clip fold forms a DNA nanostructure that includes only two oligonucleotides, which balances the diversity and dual stability of RNA clips, but DNA staple chains do not compromise both.

FIG. 3 triangular, square and pentagonal nanostructure of the RNA clip fold DNA, confirmed by denaturing PAGE and MS studies. 15O: 15R corresponding 2' -methylated RNA; in the triangular (98.5% vs 95.1%), square (96.0% vs 91.2%) and pentagonal (84.3% vs 62.1%) nano-shapes, the 15O folded DNA scaffold chain effect was superior to 15R; 30D: DNA with 10D triplicates; 50D: DNA with 10D quintic repeats; P10D: phosphorylated 10D at the 5' end; P30D: phosphorylated 30D at the 5' end; P40D: 5' phosphorylation 40D; P50D: 5' phosphorylation 50D; C30D: cyclized 30D; C40D: cyclized 40D; C50D: cyclized 50D. (a) (b) and (c) native PAGE analysis of assembly of triangles, squares and pentagons of DNA after folding at 15R, 15O and 15D. (d) Denaturing PAGE analysis of T4 DNA ligase-ligated triangles (30D +15R), squares (10D +15R, 40D +15R) and pentagons (50D +15R), (e) ESI-MS analysis of triangle, square and pentagon assemblies. The folded DNA triangles, squares and pentagons were ligated together using T4 DNA ligase and the ligated triangles, squares and pentagons were then detected using ESI-MS.

TABLE 1 ESI-MS data for circular DNA scaffold strands

Because 15R can fold the nanomaterial and form a gap, C30D, C40D, and C50D are ligated together with T4 DNA ligase in the presence of the 5-phosphorylated DNA scaffold strand to form a circularized single-stranded DNA strand. Can be detected under denaturing PAGE based on the mobility of these circularised DNA strands (FIG. 3 d). For PAGE analysis, circularized P30D, P40D, and P50D can be synthesized with ssDNA circularizing ligase as positive markers (C30D, C40D, C50D), and these nanostructures can be further confirmed under electrospray mass spectrometry (ESI-MS, fig. 3 e). The results were consistent with ESI-MS data by denaturing PAGE analysis, with or without attached folded nanostructures (fig. 3d and fig. 3 e).

In detail, 5-terminal phosphorylation of 10D, 30D, 40D and 50D gave P10D, P30D, P40D and P50D in that order. After assembly ligation with TADNA ligase, P10D +15R formed C30D and C40D and was simultaneously detected by MS (fig. 3d and 3 e). No assembly and connection phenomena of P20D and P30D were observed due to the presence of P10D +15R, and it is suspected that the triangles and quadrilaterals are formed first and then connected to form C30D and C40D. As shown (fig. 3e, table 1), the assembly reaction of 3-unitDNA (P30D +15R) formed a folded P30D, after which T4 DNA ligase ligation produced circularized 30D (C30D); the assembly reaction of 4-unitDNA (P40D +15R) formed a folded P40D, followed by ligation to form circularized 40D (C40D) (FIG. 3e, Table 1)); the assembly reaction of 5-unitDNA (P50D +15R) yielded folded P50D, which was then ligated to yield circularized 50D (C50D) (FIG. 3e, Table 1). In order to investigate whether circular nano-DNAs could be formed without 15R clips, the present invention performed 15R-free ligation of these phosphorylated scaffolds (P10D, P30D, P40, P50D) as negative controls. As expected by the present invention, the presence of circular DNA was not observed, indicating that their formation requires the priming of RNA clips.

In addition, in order to confirm and observe the circular DNA nanostructure folded by the 15R clip, the present invention employs an Atomic Force Microscope (AFM), but their shapes are difficult to confirm because they are too small in size (about 4 nm). In addition, the present invention increases the size of nanostructures to observe them and validate the construction strategy of DNA nanostructures guided by the RNA clip of the present invention. Thus, the present invention assembles a larger-sized polygon by inserting each side of a triangle, a quadrangle and a pentagon with a 21-bp double-stranded DNA (21D; FIG. 4 a). The expanded DNA triangle consists of 93-nt DNA (93D) and has three identical binding

units

21D and 15R. The expanded DNA quadrilateral contained 124-nt DNA (124D) with four identical 21D and 15R binding units, the expanded DNA pentagon containing 155-nt DNA (155D) with five identical 21D and 15R binding units (FIG. 4 a). As shown on native PAGE (fig. 4b), the 15R clip successfully guided the formation of nanostructures (enlarged DNA triangles, quadrilaterals and pentagons), whereas in native PAGE the enlarged quadrilaterals had a more detailed assembly process and finally the enlarged triangles, quadrilaterals and pentagons were imaged and confirmed by negative staining EM (fig. 4 c). The method of the invention provides a feasible strategy for constructing the DNA nanostructure by using the specific and diversified RNA units and the RNA clips.

FIG. 4 design and electron microscopy imaging of enlarged triangular, square and pentagonal DNA nanostructures. 21D: DNA inserted on both sides of the nanostructure (21 nt); 93D, 124D and 155D are DNA (containing 93, 124 and 155 nt, respectively) with three, four and five matching sequences of 21D, respectively. (a) New triangular, square and pentagonal nanostructures were designed by ring expansion. Each side of the triangle, square and pentagon is 31 bp. (b) And (c) native PAGE analysis and electron microscopy images of DNA nanostructures.

In summary, RNA clips can be successfully folded to form DNA triangles, quadrilaterals and pentagons, and RNA clips can also be used to fold DNA scaffold strands into the same quadrilateral shape using one, two or four folding units. In contrast, the corresponding DNA was not possible, indicating that the structural single-stranded RNA can provide sufficient structural diversity, stability and high assembly efficiency to form stable DNA nanostructures. Under this strategy of the invention, RNA clips can be folded to form amplified DNA nanostructures and confirmed in EM imaging, which provides a platform for assembly of complex DNA nanostructures using simple DNA nanostructures as building blocks. In order to stabilize RNA-guided DNA nanostructures, 2-methylated RNA strands can improve folding efficiency and chemical stability, and are ideal alternatives. The experimental results of the invention prove a new strategy: short RNA clips with structural diversity can guide DNA scaffold strands into a variety of shapes through stable duplexes and secondary RNA clip structures.

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method for constructing a DNA nanostructure, which comprises folding a scaffold DNA strand using RNA and modified RNA and sequences thereof to form the DNA nanostructure.

2. The method of claim 1, wherein the modified RNA is 2 '-Z-RNA, and Z is selected from the group consisting of methoxy, ethoxy, amino, fluoro, chloro, bromo, locked nucleic acid, and other 2' -substitution that does not affect folding.

3. The method of claim 1, further comprising: using the outer strand as an RNA clip, and folding the DNA scaffold strand containing 3-5 folding units into a triangle, a quadrangle and a pentagon respectively; wherein, the DNA nano-structure formed by folding one RNA clip comprises two oligonucleotide components with high assembly efficiency.

4. The method of claim 1, further comprising: and folding the DNA scaffold chain by using RNA with a special structure as a clamp to obtain the DNA nano structure.

5. The method of claim 1, further comprising: and (3) changing the sequence types and the number of the RNA clip binding sites of the DNA support chains to obtain DNA nano-structures with different shapes.

6. The method of claim 1, further comprising: and folding the DNA scaffold by using the 2' -OMe modified RNA as a clamp to obtain the DNA nanostructure.

7. The method of claim 1, further comprising: by introducing a piece of DNA into the RNA-folded DNA nanostructure, a larger DNA nanostructure is obtained.

8. The method of claim 1, further comprising: folding DNA by using a DNA-RNA hybrid chain obtained by replacing RNA with partial DNA and using the sequence as a clamp to obtain a DNA nano structure; and/or DNA nanostructures obtained by stably folding DNA scaffold strands by extended DNA clip strands.

9. A novel DNA nanostructure constructed by the method for constructing a DNA nanostructure according to any one of claims 1 to 8, wherein the novel DNA nanostructure has a general formula:

10. use of the novel DNA nanostructure of claim 9 in molecular instrumentation and nanomedicine.