DNA topological tetrahedral structure as well as preparation method and application thereof

Abstract

The invention relates to a DNA topological tetrahedral structure as well as a preparation method and application thereof. The DNA topological tetrahedral structure has the following characteristics: 1) four sulfo-modified thymine bases are extended at the 3'end of a DNA single strand corresponding to an expandable edge in the DNA topological tetrahedral structure, so that the DNA single strand can resist hydrolysis from 3'to 5 'of exonuclease III; and 2) modifying a phosphate group at the 5'end of a DNA single strand corresponding to a non-expanded side in the DNA topological tetrahedral structure, hybridizing the phosphate group with a complementary strand and connecting the phosphate group with the complementary strand end to end, so that the DNA single strand can be connected into a ring by T4DNA ligase. Compared with the prior art, the topological tetrahedron prepared by the method disclosed by the invention has more stable expandable performance, can be quickly and efficiently prepared, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a DNA topological tetrahedron structure and a preparation method and application thereof. Background Art

[0002] DNA is programmable and can assemble and construct complex and special nanostructures. The DNA double helix high-level structure with topological properties is indispensable in the process of life, so it is a biological macromolecule with important research significance and application value. DNA topological materials have been extensively studied by taking advantage of its exquisite topological properties. The topological framework of DNA enables people to design nanostructures suitable for specific applications, such as trefoil knots, DNA-based cyclohexane-like, dynamic tricyclic olefins and molecular robots of nanocarriers. Through hybridization and chain displacement reactions, large DNA origami structures can also show rapid topological conformational changes, and adjusting the twist of the DNA helix can also affect the stability of the DNA structure.

[0003] Among various DNA nanostructures, tetrahedral DNA nanostructure (TDN) is a notable example of topological materials. Its simple structure and efficient assembly enable it to be applied in various fields. The size of DNA tetrahedron can be as low as a few nanometers and can pass through the glomerular basement membrane and the blood-brain barrier. The rigid structure and good biocompatibility of tetrahedron make it an ideal choice for drug delivery and imaging probes in cells and bacteria. DNA tetrahedrons can be dynamically assembled in cells to regulate cell behavior and have been applied to intracellular imaging and spatiotemporal programmable gene therapy. In addition, DNA tetrahedral structures have been used in highly sensitive electrochemical biosensors for rapid detection of RNA, viruses, specific molecules, enzyme activity, etc.

[0004] In general, DNA tetrahedral nanostructures contain four ssDNA chains, which can be linear chains with gaps or complete circular chains. Depending on the number of circular ssDNA and the location of the cuts, TDN can have different topological conformations. However, a major challenge facing DNA topological structures is the cumbersome preparation and low purification efficiency, which limits the exploration of their potential applications. DNA enzymes have been shown to be effective for purification, but further specificity is needed. Some nucleotide and nucleic acid backbone modifications have been shown to enhance the stability of DNA structures while maintaining their functional properties.

[0005] Traditional assembly of topological structures such as double-stranded DNA rotaxanes or catenanes is usually achieved through a stepwise synthesis method, which involves hybridization of the two ends of the single strand with the template strand to form a partially nicked dsDNA structure, which is then cyclized into a ring by T4 DNA ligase. Subsequently, ssDNA is combined with the ring strand to assemble various topological structures step by step in sequence. Although this method is intuitive and reliable, the process of assembling more complex topological structures is cumbersome and inefficient because each step of the assembly produces byproducts, which are often observed during gel extraction and recovery. Summary of the invention

[0006] Based on the above problems of complicated and inefficient process in preparing DNA topological structure by stepwise synthesis method in the prior art, the present invention provides a DNA topological tetrahedral structure and a preparation method and application thereof.

[0007] The method of the present invention is a method of combining multiple biochemical methods. The method solves the problem of low purification efficiency of DNA topological tetrahedrons and can prepare more stable DNA topological tetrahedrons that retain scalability in just two steps.

[0008] Based on the method of the present invention, the problems of complicated and low efficiency in the preparation of the original DNA topological structure can be solved, and the method of the present invention also verifies the good stability of the DNA topological tetrahedral structure.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] The present invention provides a stable and expandable DNA topological tetrahedron structure, wherein the DNA topological tetrahedron structure has the following characteristics:

[0011] 1) The 3' end of the DNA single strand corresponding to the expandable edge in the DNA topological tetrahedral structure is extended by 4 thiolated thymine bases, so that the DNA single strand can resist the hydrolysis from 3' to 5' by the nuclease Exonuclease III (Exo III), thereby improving the stability of the DNA topological tetrahedral structure and retaining the expandability of DNA;

[0012] 2) The 5' end of the DNA single strand corresponding to the non-extended edge in the DNA topological tetrahedron structure is modified with a phosphate group, which hybridizes with the complementary strand and connects end to end, so that the DNA single strand can be connected into a ring by T4 DNA ligase, thereby improving the stability of the DNA tetrahedron.

[0013] In one embodiment of the present invention, the DNA topological tetrahedral structure has 1, 2, 3 or 4 rings, and the DNA topological tetrahedral structure with 1, 2, 3 or 4 rings is named TDN-C, TDN-2C, TDN-3C and TDN-4C, respectively.

[0014] In one embodiment of the present invention, the sequences of the four single-stranded DNAs used to prepare the common tetrahedral TDN are shown in SEQ ID NO: 1 to SEQ ID NO: 4, and the DNA strands do not need any modification.

[0015] In one embodiment of the present invention, the sequences of the four DNA single strands used to prepare TDN-C are shown in SEQ ID NO: 5 to SEQ ID NO: 8, wherein the 3' ends of three DNA single strands are extended with four thio-modified thymine bases, and the 5' end of one DNA single strand is modified with a phosphate group.

[0016] In one embodiment of the present invention, the sequences of the four DNA single strands used to prepare TDN-2C are shown in SEQ ID NO: 6 to SEQ ID NO: 9, wherein the 3' ends of two DNA single strands are extended with four thio-modified thymine bases, and the 5' ends of two DNA single strands are modified with phosphate groups.

[0017] In one embodiment of the present invention, the sequences of the four DNA single strands used to prepare TDN-3C are shown in SEQ ID NO: 7 to SEQ ID NO: 10, wherein the 3' end of one DNA single strand is extended with four thio-modified thymine bases, and the 5' ends of three DNA single strands are modified with phosphate groups.

[0018] In one embodiment of the present invention, the sequences of the four DNA single strands used to prepare TDN-C are shown in SEQ ID NO: 8 to SEQ ID NO: 11, wherein the 5' ends of the four DNA single strands are modified with phosphate groups.

[0019] The present invention further provides a method for preparing the stable and expandable DNA topological tetrahedral structure, comprising the following steps:

[0020] 1) The 5' end of the single-stranded ssDNA with a DNA topological tetrahedral structure is modified with a phosphate group, four single-stranded ssDNAs are mixed in equal stoichiometric ratios, and annealed in a PCR instrument to construct a DNA tetrahedron;

[0021] 2) connecting the DNA tetrahedron obtained in step 1) with T4 DNA ligase to form a ring chain to obtain a DNA tetrahedron containing a ring structure;

[0022] 3) The DNA tetrahedron containing the circular structure obtained in step 2) is incubated with Exo III nuclease, and Exo III nuclease hydrolyzes the uncircularized ssDNA, that is, hydrolyzes the uncircularized circular chain, and after purification, a stable and expandable DNA topological tetrahedron structure is obtained.

[0023] In one embodiment of the present invention, the different DNA topological tetrahedral structures have different numbers of circularized DNA by controlling the number of DNA with modified phosphate groups involved in assembling the tetrahedron.

[0024] In one embodiment of the present invention, in step 1), when the DNA topological tetrahedral structure is a DNA topological tetrahedral structure TDN-4C having four rings, the 5' end of each ssDNA is modified with a phosphate group.

[0025] In one embodiment of the present invention, in step 1), when the DNA topological tetrahedral structure is a DNA topological tetrahedral structure TDN-C, TDN-2C, TDN-3C having 1, 2, or 3 rings, the 5' phosphate group of the ssDNA to be ringed in TDN-C, TDN-2C, or TDN-3C is modified, and the 3' ends of the remaining ssDNA are connected to a structure that protects DNA hydrolysis.

[0026] It should be understood that the efficiency of T4 DNA ligase in ligating circular chains is limited, and some circular chains are not circularized, thereby generating byproducts that need to be separated by purification in step 3).

[0027] In one embodiment of the present invention, in step 1), the partially annular tetrahedron is composed of four DNA single strands, and the structure that protects DNA hydrolysis is a thio-modified thymine base, which is thio-modified on the four thymine nucleotides at the 3' end of the DNA single strand, and the tolerance of DNA to the nuclease Exo III is enhanced by thio-modification.

[0028] It should be understood that, in the present invention, the structure for protecting DNA from hydrolysis is not limited to the thiolation modification used in the examples, and in fact any molecular structure that enhances nucleic acid stability is applicable to the present invention.

[0029] In one embodiment of the present invention, in step 1), the 3' end of the non-circular chain is extended with four thio-modified thymine bases.

[0030] In one embodiment of the present invention, in step 3), the commercial Exo III nuclease is used to hydrolyze the unligated circularized chain, and the circularized and 3'-terminal thiolated linear DNA chain is retained.

[0031] The DNA topological tetrahedron prepared by the preparation method of the present invention has good biological, chemical and physical stability.

[0032] Preferably, the stability includes: serum stability, pH stability and thermal stability.

[0033] The present invention further provides application of the DNA topological tetrahedron structure in preparing medicines.

[0034] The present invention adopts 5'-phosphate modified and 3'-thiophosphate modified ssDNA and T4 DNA ligase to guide intramolecular cyclization and protect the structure from being degraded by exonuclease III (Exo III). This method successfully solves the problem of low purification efficiency, and only needs two steps to prepare stable DNA tetrahedrons with 1, 2, 3 and 4 rings, which are named TDN-C, TDN-2C, TDN-3C and TDN-4C respectively. Subsequent experiments confirmed that these DNA tetrahedrons showed higher stability under various physiological and physicochemical conditions. With the enhancement of stability, these DNA tetrahedrons can withstand more stringent external environments, paving the way for wider applications.

[0035] According to the method for preparing a stable and expandable tetrahedral topological structure provided by the present invention, its working principle is: Exo III nuclease acts on double-stranded DNA, has exonuclease activity from 3'→5', can hydrolyze 3' terminal deletion or blunt end, but cannot hydrolyze 3' protruding end. It should be understood that the 3' protruding end can achieve tolerance to Exo III, but the specific situation is related to the terminal base. The use of thiolation modification can improve the tolerance, and the number of thiolation-modified bases is not limited to four or thymine bases, thereby obtaining a stable and expandable tetrahedron.

[0036] According to the present invention, the inventors prepared a variety of DNA topological tetrahedral nanostructures with a side length of 20 bp, among which TDN-C, TDN-2C and TDN-3C were studied in detail. The research found that the stability of TDN-3C was second only to TDN-4C without expansion function.

[0037] As described in the background technology section, the traditional method for synthesizing topological nucleic acids has many processes and low efficiency, and cannot be truly applied on a large scale. However, the creativity of the present invention compared to the closest existing method mainly lies in: utilizing a combined method of chemical modification and biocatalysis to quickly and efficiently prepare a DNA topological tetrahedral structure.

[0038] Compared with the prior art, the advantages and beneficial effects of the present invention are embodied in the following aspects:

[0039] The improved effect of the present invention over the existing methods is that the method for preparing DNA topological tetrahedrons provided by the present invention is more efficient and simple, and only requires two steps to complete the assembly and purification process. In practical applications, optimally, the method can be completed within 36 hours with a yield of about 70%, thus solving the problems of slow assembly and low efficiency of topological structures.

[0040] In summary, the DNA topological tetrahedron prepared according to the method of the present invention has a fast process and high yield, ensuring the application basis of efficient preparation; 2) while preparing the topological structure, its structural stability is enhanced and its expanded function is retained. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The non-denaturing PAGE results of the assembled DNA tetrahedron in Example 1 are shown;

[0042] Figure 2 The denaturing PAGE image of topological tetrahedrons with different numbers of rings in Example 1 after being connected by T4 DNA ligase is shown;

[0043] Figure 3 Shown are the schematic diagram of the Exo III hydrolysis of topological tetrahedrons with different numbers of rings in Example 1 and the quantitative statistical results of native PAGE;

[0044] Figure 4A The quantitative statistical results of serum stability of different ring-forming DNA topology tetrahedrons in Example 2 are shown;

[0045] Figure 4B The quantitative statistical results of pH stability of different ring-forming DNA topology tetrahedrons in Example 3 are shown;

[0046] Figure 4C The statistical analysis results of the thermal stability of different ring-forming DNA topology tetrahedrons in Example 4 are shown;

[0047] Figure 5 The difference between the traditional method and the method of the present invention in preparing DNA topological tetrahedron is shown. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are carried out according to conventional methods and conditions, or are selected according to the product specifications.

[0049] In the following specific embodiments, the present invention mainly synthesizes a variety of DNA topological tetrahedrons, and verifies the stability of the DNA topological tetrahedron structure prepared by the present invention in a variety of environments. The following examples specifically illustrate the implementation effects of the present invention.

[0050] Example 1 Preparation of DNA topological tetrahedrons with different numbers of rings

[0051] The following steps are involved:

[0052] DNA modification: The 5' end of the circularized DNA is modified with a phosphate group, and four thio-modified thymine bases are added to the 3' end of the non-circularized DNA chain. No modification is required for assembling the DNA chain of the ordinary tetrahedron. The sequences used in the DNA nanostructure (SEQ ID No. 1-11) are shown in Table 1 below:

[0053] Table 1 Sequences used in DNA nanostructures

[0054]

[0055]

[0056] Synthesis of ordinary tetrahedron:

[0057] The ssDNA: S1, S2, S3, and S4 accurately quantified by VIS-UC spectrophotometer were added to the TM buffer at a stoichiometric ratio of 1:1:1:1, and the final concentration of each single strand was 1 μM. After being fully mixed, they were placed in a PCR instrument for high-temperature annealing, and the tetrahedral TDN was obtained by gel excision and recovery.

[0058] Synthesis of TDN-C tetrahedron: The ssDNA: S1-P, S2*, S3*, S4* accurately quantified by VIS-UC spectrophotometer was added to TM buffer at a stoichiometric ratio of 1:1:1:1. The final concentration of the four single strands was 1 μM. After thorough mixing, they were placed in a PCR instrument for annealing and assembly. Then, T4 DNA ligase was used for overnight connection. After hydrolysis with Exo III, the gel was cut and recovered to obtain the topological tetrahedral structure TDN-C.

[0059] The synthesis of TDN-2C, TDN-3C, and TDN-4C topological tetrahedrons is consistent with the synthesis steps of TDN-C, but they are assembled using S1-P, S2-P, S3*, S4*, S1-P, S2-P, S3-P, S4*, and S1-P, S2-P, S3-P, S4-P, respectively.

[0060] Results: Figure 1 The results of non-denaturing polyacrylamide gel electrophoresis (PAGE) imaging showed that the phosphate-modified tetrahedron was correctly synthesized. Figure 2The results of denaturing PAGE imaging showed that T4 DNA ligase can connect phosphate-modified DNA, but there are by-products, and not all phosphate-modified ssDNA can be connected into a circle.

[0061] like Figure 3 a, Schematic diagram showing the method of removing byproducts, the results of which are shown in Figure 3 b As shown in non-denaturing PAGE, Figure 3 c The preparation efficiency of the product was statistically analyzed, which proved the feasibility of this method and that other topological tetrahedrons can also be prepared in high yield.

[0062] Example 2

[0063] Comparison of serum stability of tetrahedrons with a side length of 20 bp

[0064] In order to demonstrate the effect of serum on the stability of DNA tetrahedral structure, RPMI 1640 medium containing 10% FBS was used to incubate five tetrahedrons at a final concentration of 0.1 μM at 37°C for different time periods. After the incubation, non-denaturing PAGE imaging was immediately used to observe the effect of serum on the stability of the topological tetrahedral structures of the five different ring numbers.

[0065] Results: Figure 4A The non-denaturing PAGE showed that the stability of ordinary tetrahedrons was quickly destroyed, while the ringed tetrahedrons were relatively stable and could still maintain more than 80% stability after incubation for 36 hours, especially TDN-3C and TDN-4C, whose stability was almost unaffected by serum. The results show that ring formation helps to improve the serum stability of tetrahedrons.

[0066] Example 3

[0067] Comparison of pH stability of DNA tetrahedron

[0068] In order to demonstrate the effect of pH on the stability of DNA structure, TM buffers with different pH values ​​were used to incubate with five DNA tetrahedrons with different numbers of loops at a final concentration of 0.1 μM. After the incubation, non-denaturing PAGE imaging was immediately used to observe the effect of serum on the stability of the topological tetrahedral structures with five different numbers of loops.

[0069] Results: Figure 4BThe results of non-denaturing PAGE showed that the tetrahedron stability was acceptable in the pH range of 4-11, but when the pH was below 4, the stability of the common tetrahedron decreased rapidly, TDN-4C remained almost completely stable, and the stability of other tetrahedrons decreased to a limited extent; when the pH was higher than 11, except for TDN-4C, which remained stable, the structures of other tetrahedrons were destroyed, but when the pH was higher than 12, all tetrahedron structures were destroyed. The results show that ring formation helps to improve the stability of tetrahedrons in extreme pH environments.

[0070] Example 4

[0071] Comparison of thermal stability of tetrahedron

[0072] In order to demonstrate the effect of pH on the stability of DNA structure, the thermal stability of tetrahedrons with five different numbers of rings was measured by using a temperature-controlled UV spectrophotometer, the temperature was set to heat from 20°C to 96°C, and the tetrahedron concentration was 0.1 μM. The absorbance values ​​of tetrahedrons at different temperatures were observed.

[0073] Results: Figure 4C The results of statistical analysis of the absorbance values ​​of tetrahedrons at different temperatures showed that the thermal stability of tetrahedrons gradually increased with the increase in the number of rings, especially TDN-4C. Therefore, according to the Tm results, it is proved that ring formation helps to improve the thermal stability of DNA tetrahedral structure, and the more rings there are, the more stable it is.

[0074] refer to Figure 5 The traditional method of synthesizing topological nucleic acids has many processes and low efficiency, and cannot be truly applied in large-scale applications. However, the creativity of the present invention compared with the closest existing method mainly lies in: using a combined method of chemical modification and biocatalysis to quickly and efficiently prepare a DNA topological tetrahedral structure.

[0075] The improved effect of the present invention over the existing methods is that the method for preparing DNA topological tetrahedrons provided by the present invention is more efficient and simple, and only requires two steps to complete the assembly and purification process. In practical applications, optimally, the method can be completed within 36 hours with a yield of about 70%, thus solving the problems of slow assembly and low efficiency of topological structures.

[0076] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A stable and scalable DNA topological tetrahedron structure, characterized in that: The DNA topological tetrahedral structure has the following characteristics: 1) The 3' end of the DNA single strand corresponding to the expandable edge in the DNA topological tetrahedral structure is extended by four thio-modified thymine bases, so that the DNA single strand can resist the hydrolysis from 3' to 5' by the nuclease Exonuclease III; 2) The 5' end of the DNA single strand corresponding to the non-extended edge in the DNA topological tetrahedral structure is modified with a phosphate group, which hybridizes with the complementary strand and connects end to end, so that the DNA single strand can be connected into a ring by T4 DNA ligase.

2. A stable and scalable DNA topological tetrahedron structure according to claim 1, characterized in that: The DNA topological tetrahedral structure has 1, 2, 3 or 4 rings, and the DNA topological tetrahedral structure with 1, 2, 3 or 4 rings is named TDN-C, TDN-2C, TDN-3C and TDN-4C respectively.

3. A stable and scalable DNA topological tetrahedron structure according to claim 2, characterized in that: The sequences of the four single-stranded DNAs used to prepare the common tetrahedral TDN are shown in SEQ ID NO: 1 to SEQ ID NO: 4, and the DNA strands are not modified in any way; The sequences of the four DNA single strands used to prepare TDN-C are shown in SEQ ID NO: 5 to SEQ ID NO: 8, wherein the 3' ends of three DNA single strands are extended with four thio-modified thymine bases, and the 5' end of one DNA single strand is modified with a phosphate group; The sequences of the four DNA single strands used to prepare TDN-2C are shown in SEQ ID NO: 6 to SEQ ID NO: 9, wherein the 3' ends of two DNA single strands are extended with four thio-modified thymine bases, and the 5' ends of two DNA single strands are modified with phosphate groups. The sequences of the four DNA single strands used to prepare TDN-3C are shown in SEQ ID NO: 7 to SEQ ID NO: 10, wherein the 3' end of one DNA single strand is extended with four thio-modified thymine bases, and the 5' ends of three DNA single strands are modified with phosphate groups; The sequences of the four DNA single strands used to prepare TDN-C are shown in SEQ ID NO: 8 to SEQ ID NO: 11, wherein the 5' ends of the four DNA single strands are modified with a phosphate group.

4. A stable and scalable DNA topological tetrahedron structure according to claim 1, characterized in that: The DNA topological tetrahedral structure has serum stability, pH stability and thermal stability.

5. The method for preparing a stable and expandable DNA topological tetrahedral structure according to any one of claims 1 to 4, characterized in that: The following steps are involved: 1) The 5' end of the single-stranded ssDNA with a DNA topological tetrahedral structure is modified with a phosphate group, four single-stranded ssDNAs are mixed in equal stoichiometric ratios, and annealed in a PCR instrument to construct a DNA tetrahedron; 2) connecting the DNA tetrahedron obtained in step 1) with T4 DNA ligase to form a ring chain to obtain a DNA tetrahedron containing a ring structure; 3) The DNA tetrahedron containing the circular structure obtained in step 2) is incubated with Exo III nuclease, and Exo III nuclease hydrolyzes the uncircularized ssDNA and the uncircularized circular chain, and after purification, a stable and expandable DNA topological tetrahedron structure is obtained.

6. The method for preparing a stable and scalable DNA topological tetrahedral structure according to claim 5, characterized in that: By controlling the amount of DNA with modified phosphate groups involved in assembling the tetrahedron, the different DNA topological tetrahedral structures have different amounts of looped DNA.

7. The method for preparing a stable and scalable DNA topological tetrahedral structure according to claim 5, characterized in that: In step 1), when the DNA topological tetrahedral structure is a DNA topological tetrahedral structure TDN-4C having four rings, the 5' end of each ssDNA is modified with a phosphate group; In step 1), when the DNA topological tetrahedral structure is a DNA topological tetrahedral structure TDN-C, TDN-2C, TDN-3C having 1, 2, or 3 rings, the 5' phosphate group of the ssDNA to be ringed in TDN-C, TDN-2C, or TDN-3C is modified, and the 3' ends of the remaining ssDNA are connected to a structure that protects DNA hydrolysis.

8. The method for preparing a stable and scalable DNA topological tetrahedral structure according to claim 7, characterized in that: In step 1), the partially annular tetrahedron is composed of four DNA single strands, and the structure protecting DNA hydrolysis is a thio-modified thymine base, which is thio-modified on the four thymine nucleotides at the 3' end of the DNA single strand. The thio-modification enhances the tolerance of DNA to the nuclease Exo III.

9. The method for preparing a stable and scalable DNA topological tetrahedral structure according to claim 5, characterized in that: In step 3), Exo III nuclease is used to hydrolyze the unligated circularized chain, retaining the circularized DNA chain and the linear DNA chain with 3' terminal thiolation modification.

10. Use of the stable and expandable DNA topological tetrahedral structure according to any one of claims 1 to 4 in the preparation of drugs.

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