A method for constructing DNA topology

Abstract

The application provides a method for constructing DNA topology, and relates to the field of DNA nanotechnology.The method comprises the following steps: performing directional connection after 5' phosphorylation modification of the end of a DNA single strand, and performing DNA self-assembly from bottom to top to form a braided DNA grid nanometer array structure with programmable node number; the DNA grid nanometer array structure is connected into a closed ring DNA single strand through enzymatic reaction to form a covalent bond, accurate positioning of node position is realized, and a DNA topology is constructed; and a basic structural unit of the DNA grid nanometer array structure is a DNA four-arm cross structure formed by four DNA single strands through base pairing.The application can accurately assemble the topology structure with a specific number of nodes based on the braided DNA grid nanometer array to construct the DNA topology, and a topology single strand DNA structure with a highly complex structure is synthesized.

Description

Technical Field

[0001] This invention belongs to the field of DNA nanotechnology, and specifically relates to a method for constructing DNA topological structures. Background Technology

[0002] Topology is not only an important branch of mathematics but also a crucial research area in physics and life sciences. In nature, topological structures are widely present in biological macromolecules such as DNA and proteins, playing a key role in their biological functions. For example, during protein folding, polypeptide chains form topological conformations with specific crossovers; and during the replication and transcription of genomic DNA, DNA chains also form temporary topological structures, which are ultimately precisely untied by DNA topoisomerases. The artificial synthesis of topological molecular structures plays a vital role in studying biological issues such as the activity of molecular isomerases. However, achieving controllable complexity topological compounds at the molecular scale through artificial design remains a highly challenging problem.

[0003] Existing techniques for synthesizing DNA topological structures suffer from the following drawbacks: 1) Cumbersome assembly process: Constructing complex topological structures typically requires a step-by-step process, first assembling basic topological units and then integrating them through inter-unit hybridization. This process is cumbersome and makes it difficult to construct high-order complex topological structures. 2) Simple topological structures with limited complexity: Existing methods are mostly limited to constructing simple or classical topological structures with a limited number of nodes, presenting significant bottlenecks in achieving more complex high-order chain loops. 3) Poor tolerance for folding path errors: In single-strand self-folding paths, intrastrand self-entanglement easily generates redundant sub-nodes, and the correct folding rate drops sharply with the increase in the number of crossovers. Once a folding error occurs, it is difficult to reverse, leading to the accumulation of defective structures and affecting product uniformity and yield.

[0004] To address the aforementioned problems, this invention provides a method for constructing DNA topological structures to overcome key issues in existing technologies, such as cumbersome step-by-step assembly, limited structural complexity, and low fault tolerance in the folding process. Summary of the Invention

[0005] The main objective of this invention is to provide a method for constructing DNA topological structures to overcome the shortcomings of existing technologies.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: As the first objective of this invention, it provides a method for constructing a DNA topological structure, comprising: phosphorylating the 5' ends of DNA single strands and then directionally connecting them; and performing bottom-up DNA self-assembly to form a woven DNA lattice nanoarray structure with a programmable number of nodes; wherein the DNA lattice nanoarray is then connected by enzymatic reaction to form closed circular DNA single strands through covalent bonds, achieving precise positioning of the node positions, thereby constructing a DNA topological structure; wherein the basic structural unit of the DNA lattice nanoarray structure is a DNA four-way junction formed by four DNA single strands through base pairing.

[0007] DNA self-assembly technology, due to its high programmability, holds promise for synthesizing highly complex DNA topologies. DNA self-assembly is a bottom-up assembly technique based on the principle of complementary base pairing, using DNA strands as raw materials. It can programmably synthesize complex structures with nanoscale precision, controllable dimensions, and spatial addressability. This invention applies DNA self-assembly technology to the construction of DNA topologies. Through bottom-up DNA self-assembly, a DNA lattice nanoarray with a programmable number of nodes is formed. With the aid of enzymatic reactions, the construction of DNA topologies with precisely programmable node positions and highly customizable structural complexity is ultimately achieved. This overcomes key problems in existing technologies, such as cumbersome step-by-step assembly, limited structural complexity, and low fault tolerance in the folding process. Theoretically, this construction method can realize the construction of functional materials of arbitrary complexity, demonstrating great potential in preparing DNA topologies with customizable topological nodes and complexity.

[0008] In a preferred embodiment, the DNA lattice nanoarray is synthesized from auxiliary single strands and topological single strands.

[0009] In a preferred embodiment, the topological strand is modified with 5' phosphorylation, then an auxiliary strand is added, and the assembly is annealed to form the DNA lattice nanoarray structure with phosphorylation sites. Then, T4 ligase is added to link the 5' phosphate group and 3' hydroxyl group in the DNA lattice nanoarray structure to form circular DNA, thus obtaining the DNA topological structure.

[0010] It should be noted that in the DNA grid nanoarray structure, each DNA grid structure has two different distribution patterns of DNA double helix structures in the DNA topology structure, depending on the distance between the intersections: when the distance between the intersections is an integer multiple of the number of helixes, the DNA double helixes remain on the same side; when the distance between the intersections is half a multiple of the number of helixes, the DNA double helixes are arranged in an alternating woven pattern.

[0011] As a preferred embodiment, the construction method specifically includes the following steps: S1. Design DNA single strands, including topological single strands and auxiliary single strands; S2, phosphorylation of single-stranded topological chains; Phosphorylase was added to the single-stranded topological chain and incubated once to phosphorylate its 5' end. S3. Construct a DNA nanogrid array; The phosphorylated topological single strand was mixed with the auxiliary single strand and annealed in a buffer solution to assemble a DNA grid nanoarray with phosphorylation sites. S4. Constructing DNA topology T4 ligase was added to the mixed solution of DNA grid nanoarrays with phosphorylation sites prepared in S3 for a second incubation, so that the 5' phosphate group of the single strand of the topological strand and the 3' hydroxyl group of the single strand of the auxiliary strand in the structure of the DNA grid nanoarray were linked to form a circular DNA, thus obtaining the DNA topological structure.

[0012] In a preferred embodiment, in S1, the topological chain single-chain sequence is SEQ ID NO.1~15.

[0013] Preferably, the auxiliary chain single-strand sequence is SEQ ID NO.16~25.

[0014] In a preferred embodiment, in S2, the incubation conditions are 37°C for 1 hour.

[0015] In a preferred embodiment, in S3, the molar ratio of the topological chain single chain to the auxiliary chain single chain is 1:1.

[0016] In a preferred embodiment, in S3, the buffer solution contains Mg 2+ TE buffer solution.

[0017] Preferably, Mg 2+ The concentration is 20~40mM.

[0018] Preferably, the annealing conditions include reducing the temperature from 85°C to 25°C and annealing for 24-36 hours.

[0019] In a preferred embodiment, in S4, the amount of T4 ligase added is 1 μL (4 × 10⁻⁶). 5 (units / mL).

[0020] Preferably, the secondary incubation conditions are 16°C for 19 hours.

[0021] In a preferred embodiment, S4 further includes: after generating the circular DNA, adding exonuclease III and exonuclease I, incubating again to degrade the linear DNA strands complementary to the circular DNA in the mixed solution and the remaining single-stranded DNA in the system, and finally adding a denaturing agent to inactivate exonuclease III and exonuclease I.

[0022] Preferably, the concentration of exonuclease III is 1 × 10⁻⁶. 5 The unit / mL addition volume is 0.5μL.

[0023] Preferably, the concentration of exonuclease I is 2 × 10⁻⁶. 4 The unit / mL addition volume is 1μL.

[0024] Preferably, the denaturant is 1% SDS, and the addition amount is 20 μL.

[0025] As a second objective of the invention, the present invention also provides a DNA topological structure prepared using the construction method described above.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention is based on the construction of DNA topology structures using a braided DNA grid nanoarray, which can precisely customize and assemble topology structures with a specific number of nodes, and theoretically can synthesize highly complex topological single-stranded DNA structures.

[0027] 2. This invention utilizes DNA self-assembly technology, employing a one-pot annealing and enzymatic treatment method, effectively avoiding the problems of cumbersome step-by-step assembly operations and low yields. Synthesizing complex topological single-stranded DNA does not increase experimental procedures. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a schematic diagram of a DNA grid structure provided by the present invention.

[0030] Figure 2 This is a simulated schematic diagram of the DNA lattice structure provided by the present invention used to generate single-stranded DNA topology.

[0031] Figure 3 The 3 provided in Embodiment 1 of the present invention 3. Agarose gel electrophoresis image of DNA grid nanoarray.

[0032] Figure 4 The three assemblies provided in Embodiment 1 of this invention, assembled under different annealing conditions, are 3 Comparison of atomic force microscopy characterization of DNA grid nanoarrays.

[0033] Figure 5 The DNA topology provided in Example 1 of this invention is characterized by agarose gel electrophoresis. Detailed Implementation

[0034] Together with Figure 1 Reading the following detailed description will provide a more complete understanding of the invention. Detailed embodiments of the invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely exemplary examples of specific implementations of the invention, which may be embodied in various forms. Therefore, the specific functional details disclosed herein should not be construed as limiting, but rather as the basis for the claims and as intended to teach those skilled in the art to employ the representative basis of the invention in different ways in any suitable detailed embodiment.

[0035] This invention provides a method for constructing DNA topological structures, comprising: using DNA four-way junctions as basic units, and covalently linking the 5' end phosphorylated with the adjacent 3' hydroxyl group, assembling DNA from bottom to top into a DNA lattice nanoarray with a programmable number of nodes, and using enzymatic reactions to achieve precise positioning of the node positions, thereby constructing a DNA topological structure.

[0036] The method for constructing the DNA topology includes the following specific steps: (1) DNA grid nanoarray DNA quadruple crosses are a type of branched nucleic acid structure composed of four DNA strands that pair up through bases. DNA grid structures use DNA quadruple crosses as basic structural elements.

[0037] See Figure 1 Each set of four DNA quadruplets cross-connects to form a DNA grid structure, and several DNA grid structures constitute a DNA grid nanoarray.

[0038] Depending on the distance between the intersections, the DNA double helix can exhibit two different distributions; for example, Figure 1 As shown in Figure A, when the distance between the crossover points is an integer multiple of the number of helices, such as 21 nt (one complete integer helix), the DNA double helix containing either the yellow or blue strand remains on the same side; as... Figure 1As shown in Figure B, when the distance between the crossover points is half the number of helices, such as 26 nt (2.5 half-helical cycles), the DNA double helixes exhibit a woven, alternating arrangement.

[0039] This invention employs a woven grid structure design to achieve a DNA grid structure with a topological structure.

[0040] Based on the design principle of woven lattice structure, the present invention designs a topological DNA lattice nanostructure with a distance of (n+0.5)×10.5 bases between two nodes, where n is a positive integer (n=1, 2, 3, 4, etc.); and weaves a DNA lattice nanoarray with a certain number of nodes (3×3, 5×5, 7×7, etc.) from bottom to top based on the cross-linking of DNA four arms.

[0041] Based on the woven grid structure, the ends of the structure are connected to form a loop structure, thus forming a DNA topology.

[0042] (2) Generate single-stranded DNA topology The DNA single strands that form the topological DNA lattice structure are divided into two groups: auxiliary strands and topological strands. Topological strands are DNA single strands that form closed circular structures. See [link to documentation]. Figure 2 The black strand in the middle; the auxiliary strand is a single strand of DNA that helps the topological strand form the topological structure, such as... Figure 2 The blue chain in the middle.

[0043] To synthesize single-stranded DNA topological structures, the topological strands are phosphorylated at 5' before assembling the DNA lattice structure. After assembly, the structure is treated with T4 ligase to link the topological strands covalently, forming closed circular single-stranded DNA. Finally, the auxiliary strands are digested by DNA exonuclease to obtain the single-stranded DNA topological structure.

[0044] Specifically, DNA topological structures were constructed using enzymatic reactions: after proving the feasibility of the DNA lattice nanoarray design, First, the topological strands are modified with 5' phosphorylation. Phosphorylase is added to a mixed solution of topological strands and single strands and incubated at 37°C for 1 h to phosphorylate the 5' end. Then, topological strands are added and annealed together to assemble a DNA grid nanoarray with phosphorylation sites. T4 ligase is added to the annealed solution and incubated at 16°C for 19 h to link the 5' phosphate group and 3' hydroxyl group in the structure to form a circular DNA. Finally, exonucleases III and I are added and incubated at 37°C for 0.5 h to degrade the linear DNA strand complementary to the circular DNA and the remaining single-stranded DNA in the system, respectively. Then, 1% SDS denaturant is added to inactivate the two exonucleases, thus generating a single-stranded DNA topological loop with customized nodes.

[0045] The technical solution of the present invention will be illustrated below through specific embodiments.

[0046] Table 1. List of Single-Linked Topological Chains

[0047] Table 2. List of single-chain sequences of auxiliary chains

[0048] Example 1 This embodiment constructs a 3-based 3. The single-stranded DNA topology of the DNA nanogrid array, specifically including the following steps: I. Constructing a DNA Nanogrid Array (1) Design of DNA nanogrid array: DNA sequences were designed and generated using cadnano software, and finally, single-stranded DNA was synthesized by Genewiz Biotechnology Co., Ltd.

[0049] (2) Assembly of DNA nanogrid array: 3D DNA structures were constructed using four-armed cross-linked DNA as structural design units. 3. The braided DNA nanogrid array structure specifically includes: annealing synthesized DNA single strands in a solution of a certain concentration by mixing them at a 1:1 molar concentration ratio (temperature reduced from 85℃ to 25℃) to assemble a DNA grid nanoarray.

[0050] The assembled product was separated and recovered by agarose gel electrophoresis to separate the target band, and then characterized by agarose gel electrophoresis. See [link to documentation]. Figure 3 This is a schematic diagram of the agarose gel electrophoresis characterization results of the assembled products. Lanes (1), (2), and (3) in the diagram represent the agarose gel electrophoresis results of the assembled products. 2+ Annealing for 24 hours, 40 mM Mg 2+ Annealing for 24 hours and 20 mM Mg 2+ The assembly products obtained under the annealing 36 h assembly condition (shown in the red box) show that the product bands obtained under the three assembly conditions are very uniform and in the same position, indicating that the target product can be obtained under all three assembly conditions.

[0051] Subsequently, the target strip was cut and recovered, and then imaged and observed using an atomic force microscope, such as... Figure 4 As shown, Figure (1) and Figure (2) are for 20 mM Mg 2+ Annealing for 24 hours, 40 mM Mg 2+The morphology of the assembled product obtained after annealing for 24 h is shown in the images, which are 2 μm × 2 μm and 1 μm × 1 μm in size. The structure in the images is relatively loose and is about square. The length of the measured product is about 30 nm, which is close to the theoretical size (length and width are 25.7 nm), proving that the structure meets the expectations.

[0052] II. Constructing Single-Stranded DNA Topology This embodiment uses an enzymatic reaction to construct a DNA topological structure. The specific steps include: mixing 15 single-stranded topological strands that make up the DNA lattice nanoarray (SEQ ID NO. 1~15, see Table 1, each strand concentration 500 nM); and adding phosphorylase (1×10⁻⁶) to the mixed solution. 4 (units / mL, 1 μL), incubate at 37°C for 1 h to phosphorylate its 5' end.

[0053] Then, auxiliary single-stranded strands with sequences SEQ ID NO.16~25 (see Table 2) were added, and the mixture was annealed and assembled into a DNA lattice nanoarray. 1 μL (4 × 10⁻⁶) of the annealed solution was added. 5 Incubation with T4 ligase (units / mL) at 16°C for 19 h allowed the 5' phosphate group of the topological strands within the DNA grid nanoarray structure to be covalently linked to the 3' hydroxyl group of the adjacent strands into a single-stranded circular DNA.

[0054] Add another 0.5 μL (1 × 10⁻⁶) 5 Exonuclease III (units / mL, New England Biolabs) was 1 μL (2 × 10) 4 Units / mL, New England Biolabs) exonuclease I. After incubation at 37°C for 0.5 h, the linear auxiliary DNA strands (auxiliary strands A1-A10) complementary to the single-stranded circular DNA and the free single-stranded DNA in the system were degraded, respectively. Finally, 20 μL of denaturing agent at a final concentration of 1% (w / v) SDS was added to inactivate the enzyme in the solution, ultimately generating a single-stranded DNA topological circular with customized nodes.

[0055] The obtained DNA topological loops were recovered and characterized by agarose gel electrophoresis. (See [reference]) Figure 5 The results are from agarose gel electrophoresis. Lanes 1 to 6 in the figure represent: Lane 1 contains 15 unphosphorylated DNA single strands; Lane 2 contains 10 helper DNA strands; Lane 3 contains 15 unphosphorylated DNA single strands + 10 DNA helper single strands annealed and assembled. 3 DNA grid nanoarrays; lane 4 consists of 15 phosphorylated DNA single strands + 10 annealed DNA single strands assembled in 3 layers. 3. DNA grid nanoarray treated with T4 ligase; lanes 5 and 4 products treated with exonuclease III; lane 6 product of lane 5 treated with exonuclease I. The results showed that lane 6 still contained product bands after exonuclease treatment, indicating the formation of a single-stranded DNA topological structure. Compared to lanes 1 and 2, lane 3 had a higher molecular weight, proving the successful assembly of the DNA grid nanoarray. Lane 4 had a higher molecular weight than lane 3, indicating that lane 4 was a phosphorylated DNA nanoarray treated with T4 ligase. The molecular weights of lanes 5 and 6 treated with exonuclease decreased compared to lane 4 but were higher than lanes 1 and 2, preliminarily demonstrating that the DNA strands complementary to the single-stranded DNA topological loops in the DNA nanoarray were degraded, and the final bands were single-stranded DNA topological loops.

[0056] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims.

Claims

1. A method for constructing DNA topological structures, characterized in that, This includes phosphorylating the 5' ends of single-stranded DNA with phosphorylation and then directionally connecting them, as well as bottom-up DNA self-assembly to form a woven DNA grid nanoarray structure with a programmable number of nodes; the DNA grid nanoarray is then connected by enzymatic reaction to form closed circular single-stranded DNA through covalent bonds, achieving precise positioning of node positions and constructing a DNA topological structure. The basic structural unit of the DNA grid nanoarray structure is a four-armed cross structure formed by four DNA single strands through base pairing.

2. The method according to claim 1, characterized in that, The DNA grid nanoarray was synthesized from auxiliary single strands and topological single strands; The single-stranded topological strand is modified with 5' phosphorylation, and then an auxiliary single-stranded strand is added. The strands are then annealed and assembled to form the DNA lattice nanoarray structure with phosphorylation sites. T4 ligase is then added to link the 5' phosphate groups and 3' hydroxyl groups within the DNA lattice nanoarray structure to form circular DNA, thus obtaining the DNA topological structure.

3. The method according to claim 1 or 2, characterized in that, The specific steps include: S1. Design DNA single strands, including topological single strands and auxiliary single strands; S2, phosphorylation of single-stranded topological chains; Phosphorylase was added to the single-stranded topological chain and incubated once to phosphorylate its 5' end. S3. Construct a DNA nanogrid array; The phosphorylated topological single strand was mixed with the auxiliary single strand and annealed in a buffer solution to assemble a DNA grid nanoarray with phosphorylation sites. S4. Constructing DNA topology T4 ligase was added to the mixed solution of DNA grid nanoarrays with phosphorylation sites prepared in S3 for a second incubation, so that the 5' phosphate group of the single strand of the topological strand and the 3' hydroxyl group of the single strand of the auxiliary strand in the structure of the DNA grid nanoarray were linked to form a circular DNA, thus obtaining the DNA topological structure.

4. The method according to claim 3, characterized in that: In S1, the topological chain single-chain sequence is SEQ ID NO.1~15; And / or, the auxiliary chain single-chain sequence SEQ ID NO.16~25.

5. The method according to claim 3, characterized in that: In S2, the incubation conditions are 37°C for 1 hour.

6. The method according to claim 3, characterized in that: In S3, the buffer solution contains Mg 2+ TE buffer solution; Mg 2+ The concentration is 20~40mM; And / or, the annealing conditions include reducing the temperature from 85°C to 25°C and annealing for 24 to 36 hours; And / or, the molar ratio of the topological chain single chain to the auxiliary chain single chain is 1:

1.

7. The method according to claim 3, characterized in that: S4, 4×10 5 The amount of T4 ligase (units / mL) added is 1 μL; And / or, the conditions for the secondary incubation are 16°C for 19 h.

8. The method according to any one of claims 3-7, characterized in that: S4 further includes: after generating the double-stranded circular DNA with the nick, adding exonuclease III and exonuclease I, incubating again to degrade the linear DNA strand complementary to the single-stranded circular DNA in the mixed solution and the remaining unassembled single-stranded DNA in the system, and finally adding a denaturing agent to inactivate exonuclease III and exonuclease I.

9. The method according to claim 8, characterized in that: The concentration of exonuclease III is 1×10⁻⁶. 5 The concentration of exonuclease I was 2 × 10⁻⁶ units / mL, and the addition volume was 0.5 μL. 4 units / mL, with an addition volume of 1μL; The denaturant is 1% SDS, and the addition amount is 20 μL.

10. A DNA topological structure prepared by the method described in any one of claims 1-9.

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