Anti-degradation antisense nucleic acid based on D-type DNA structure as well as preparation method and application of anti-degradation antisense nucleic acid

Abstract

The invention discloses an anti-degradation antisense nucleic acid based on a D-type DNA structure and a preparation method and application thereof, and belongs to the technical field of genetic engineering. Compared with the traditional antisense nucleic acid, the anti-degradation antisense nucleic acid (D-ASO) based on the D-type DNA structure has the advantages that the design of coupling a single-chain string and a double-chain bow is adopted, the nuclease degradation resistance is remarkably improved, and the half-life period in serum is prolonged to 24 hours (only 2 hours in the traditional ASO). The target mRNA triggers the replacement of a protection chain (P) of D-ASO to form a DNA-RNA hybrid which is degraded by RNase H, and then the P chain is recombined to realize cyclic utilization (more than or equal to 5 times). According to the innovative structure, the binding rate of non-specific RNA is reduced by 90% or above. The invention solves the problems of stability and off-target toxicity of ASO while maintaining efficient gene silencing, and is suitable for treatment of tumors and genetic diseases.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a degradation-resistant antisense nucleic acid based on a D-type DNA structure, its preparation method, and its application. Background Technology

[0002] Single-stranded DNA (ssDNA) is a nucleic acid molecule with high flexibility and exposed hydrogen-bonded active sites, properties that make it highly promising for applications in the biomedical field. ssDNA can specifically bind to a variety of molecules via hydrogen bonds, including proteins, nucleic acids, and other biomacromolecules, making it crucial for applications such as drug delivery, gene silencing, diagnostics, and therapy. However, traditional ssDNA exhibits poor stability in vivo and is easily degraded by nucleases, limiting its efficiency and specificity in gene silencing and therapeutic applications. In recent years, nucleic acid-based nanostructures, such as DNA origami, have been developed to improve the stability and functionality of nucleic acid molecules. These techniques create nanostructures with specific shapes and functions by precisely controlling the folding and assembly of nucleic acid molecules. While DNA origami technology shows great potential in improving nucleic acid stability, applying these techniques to the development of novel antisense oligonucleotides (ASOs) remains a challenge.

[0003] Antisense nucleic acids (ASOs) are single-stranded oligonucleotides that can specifically bind to target mRNAs and inhibit their expression, showing great promise for gene silencing and disease treatment. However, traditional antisense nucleic acids face challenges in in vivo applications, including poor stability, susceptibility to nuclease degradation, and significant off-target effects, severely limiting their clinical efficacy. Currently, common antisense nucleic acids mainly include linear single-stranded oligonucleotides and double-stranded DNA structures. While linear single-stranded oligonucleotides are easy to synthesize, they are easily degraded by nucleases in the in vivo environment, have a short half-life, and result in short-lasting therapeutic effects. Double-stranded DNA structures, while relatively stable, have limited specific binding capacity and exhibit significant off-target effects (non-specific binding of ASOs at non-target sites, potentially leading to unexpected biological effects and side effects), which may cause unintended biological responses. To improve the stability of antisense nucleic acids, researchers have developed various chemical modification strategies, such as phosphate thiolation and 2'-O-methylation. However, these modifications often affect the binding affinity of antisense nucleic acids to target sequences or increase toxicity.

[0004] D-DNA (D-DNA) is a novel nucleic acid structure that, through a unique topological design, combines single-stranded "strings" with double-stranded "bows" to form a stable three-dimensional structure. D-DNA is also a special right-handed double helix structure, characterized by 8 base pairs per loop, shallow major grooves, and deep minor grooves. It is commonly found in AT-rich sequences and plays a role in gene regulation, replication, and evolution. Its existence expands the diversity of DNA structures and is an interesting phenomenon in molecular biology research. The design of D-DNA structures aims to improve the stability of ASOs while reducing their off-target effects.

[0005] Although D-DNA has shown potential in improving nucleic acid stability, its performance and potential in practical applications have not been fully explored and validated. Therefore, there is an urgent need in the field for a novel D-DNA-based antidegradation antisense nucleic acid (D-ASO) that can utilize the unique topological structure of D-DNA to enhance stability and reduce off-target effects, thereby providing better results in gene silencing and therapeutic applications. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide an anti-degradation antisense nucleic acid based on D-type DNA structure, its preparation method and application, so as to solve the problems of poor stability and off-target effects encountered by existing antisense nucleic acids (ASO) in biomedical applications.

[0007] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a degradation-resistant antisense nucleic acid (D-ASO) based on a D-type DNA structure, which consists of circular DNA and complementary single-stranded DNA. Part of the single-stranded DNA binds to the "string" of the D-type DNA, which is called the protective strand (P); the other part binds to the "bow" of the D-type DNA. The "string" region of the D-type DNA integrates a carefully designed antisense nucleic acid sequence, which is highly specific and complementary to the complementary region of the target mRNA.

[0008] Preferably, in the presence of target mRNA, base complementarity induces the replacement of the protective strand P, exposing the antisense sequence, which then binds to the mRNA to form a DNA-RNA hybrid. Subsequently, the hybrid is degraded by the exonuclease RNase H, allowing the protective strand P to rebind to the "string" region, thereby achieving a cyclical regeneration of the gene silencing process.

[0009] Preferably, in the D-type DNA topology, the single-stranded segment of the circular DNA, i.e., the single-stranded "string" region, has a length of 28 nt, and the double-stranded segment of the circular DNA, i.e., the double-stranded "bow" region, has a length of 20 bp.

[0010] Preferably, the anti-degradation antisense nucleic acid based on the D-type DNA structure has a half-life of ≥24 hours in a serum environment at 37 °C.

[0011] This invention also discloses a method for preparing the above-mentioned anti-degradation antisense nucleic acid based on the D-type DNA structure, comprising the following steps: 1) Phosphorylated linear DNA at the 5' end is mixed with circular primers in a buffer solution, and after heat denaturation, it is slowly cooled to form a pre-closed structure; 2) Add ligase to perform a ligation reaction to obtain closed circular DNA; 3) Closed circular DNA is purified by enzymatic digestion to remove linear impurities, and then purified to obtain circular DNA. 4) Circular DNA is annealed with D-type short-chain DNA to form D-DNA, and then purified twice to obtain anti-degradation antisense nucleic acid based on the D-type DNA structure.

[0012] Preferably, the procedure specifically includes the following steps: Step 1: Mix 5' phosphorylated linear DNA and circular short-strand DNA at a 1:2 molar ratio in TAE / Mg 2+ Unclosed circular DNA was formed by annealing in buffer solution at 95 °C for 5 minutes and then cooling to 4 °C at a rate of 0.1 °C / second. Step 2: Add T4 DNA ligase, incubate at 25 ℃ for 12 hours, then incubate at 65 ℃ for 30 minutes to obtain closed circular DNA; Step 3: Add exonucleases I and III, digest at 37 ℃ for 8 hours, and inactivate at 80 ℃ for 30 minutes; Step 4: Circular DNA was obtained by polyacrylamide gel purification and annealed with D-type short-chain DNA to form D-DNA; Step 5: The final product is obtained by secondary polyacrylamide gel purification and ethanol precipitation.

[0013] More preferably, in step 1, the TAE / Mg 2+ The buffer contains 5 mM Tris, 1 mM EDTA and 12.5 mM magnesium acetate (pH 8.0), with final concentrations of 5 uM for linear DNA and 10 uM for circular short-chain DNA.

[0014] More preferably, in step 2, after adding T4 DNA ligase, the above mixture is mixed in a 1×T4 DNA ligase / Mg solution. 2+ Buffer (50 mM Tris-HCl, 5 mM Mg) 2+The samples were mixed in a PCR thermal cycler (2.5 mM DTT, 0.5 mM ATP, pH 7.5) and heated to 25°C for 12 hours, then kept at 65°C for 30 minutes.

[0015] More preferably, in step 3, 1× exonuclease I / Mg is added. 2+ Buffer (50 mM Tris-HCl, 10 mM Mg) 2+ (1 mM DTT, 0.1 mM ATP, pH 7.5) and 1× exonuclease III / Mg 2+ Buffer (50 mM Tris-HCl, 10 mM Mg) 2+ After mixing the above mixture (1 mM DTT, 0.1 mM ATP, pH 7.5), it was placed in 1× exonuclease III buffer and 1× exonuclease I buffer, and incubated at 37 °C for 8 hours and 80 °C for 30 minutes. The addition of exonuclease I and exonuclease III can effectively remove linear ends and any non-target sequences from DNA fragments, thereby ensuring that the final product is a pure circular DNA molecule.

[0016] More preferably, the specific conditions for the purification treatment described in steps 4 and 5 are as follows: 1) Elute using Elution Buffer at 65 °C for 4.5 hours, then at 37 °C for 8 hours; 2) Centrifugal filtration column purification parameters: Centrifuge at 10000 XG for 15 minutes; 3) Ethanol precipitation parameters: 50% isopropanol at -20 ℃ for 30 minutes, centrifuge at 12000 rpm at 4 ℃ for 10 minutes, and wash twice with 75% ethanol.

[0017] Preferably, the method also includes preparing D-type DNA under different bending forces, that is, by precisely controlling the size of the circular DNA in the D-type DNA (D-DNA) structure and the length of the short chain connected to the circular DNA, the bending force of D-DNA can be effectively regulated.

[0018] This invention also discloses the application of the above-mentioned anti-degradation antisense nucleic acid based on the D-type DNA structure in gene silencing. The target mRNA triggers the protective strand replacement reaction of the anti-degradation antisense nucleic acid with the D-type DNA structure, forming a DNA-RNA hybrid and being degraded by RNase H. Subsequently, the protective strand is re-bound, achieving recycling.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The anti-degradation antisense nucleic acid (D-ASO) based on a D-type DNA structure disclosed in this invention, compared with traditional double-stranded DNA (dsDNA) or linear antisense oligonucleotides (ASO), possesses higher stability and resistance to nuclease degradation due to its unique topological structure (the combination of single-stranded "strings" and double-stranded "bows"). Therefore, designing the antisense nucleic acid into the form of D-type DNA allows it to be more stable in cells, thus enabling it to function better. Simultaneously, this structure is designed to facilitate efficient substitution between the target mRNA and the protective strand P. This innovative design allows the target mRNA to undergo a substitution reaction with the protective strand P through base complementarity, thereby exposing the antisense sequence, which then binds to the mRNA, forming a DNA-RNA hybrid. The unique topological structure of the D-type DNA of this invention not only enhances its stability in the biological environment but also optimizes the binding affinity with the target mRNA by precisely controlling the length and sequence of the protective strand P, thereby improving the specificity and efficiency of gene silencing. Furthermore, this design allows the protective strand P to rebind to the "string" in the absence of target mRNA, enabling the recycling of D-ASO and thus improving its practicality and efficiency in gene silencing applications.

[0020] More preferably, in this invention, the bending force of D-DNA is effectively regulated by precisely controlling the size of the circular DNA and the length of the short strands connected to the circular DNA in the D-DNA structure. Utilizing the inherent elastic properties of DNA molecules, the local bending stiffness of DNA is affected by changing the size of the loop. Through this method, the physical properties of D-DNA can be finely controlled at the molecular level, thereby optimizing its performance in biomedical applications. Attached Figure Description

[0021] Figure 1 This invention relates to a method for preparing D-type DNA.

[0022] Figure 2 Polyacrylamide gel electrophoresis characterization of R38-30, R40-30, R42-30, R44-30, R46-30, R48-30, R50-30, and R60-30 of this invention.

[0023] Figure 3This study investigated the stability of R38-30, R40-30, R42-30, R44-30, R46-30, R48-30, R50-30, and R60-30 in fetal bovine serum (FBS) using the compounds mentioned in this invention. Figure a shows the stability of D-type DNA under different bending forces in 10% FBS at 37°C for 2, 4, 6, 8, 12, and 24 hours, verified by polyacrylamide gel electrophoresis. Figure b is a line graph showing the stability of D-type DNA of different sizes.

[0024] Figure 4 The results of polyacrylamide gel electrophoresis characterization of D-ASO in this invention are shown.

[0025] Figure 5 This is a schematic diagram illustrating the principle of D-ASO(R48-50) as a nucleic acid drug in this invention, which can digest target RNA, reduce off-target effects, and be recyclable.

[0026] Figure 6 This presents the results of the investigation into the stability of D-ASO (R48-50) in fetal bovine serum (FBS) in this invention. Figure a shows the stability of D-type DNA of different sizes in 10% FBS after incubation at 37°C for 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours, verified by polyacrylamide gel electrophoresis. Figure b shows the corresponding line graph.

[0027] Figure 7 This is an experimental diagram demonstrating that D-ASO (R48-50) can effectively reduce off-target effects in this invention. In the diagram, a shows the polyacrylamide gel electrophoresis results of R48-50 hybridizing with DNA X (a DNA sequence identical to Bcl-2 mRNA), DNA S1 (a DNA sequence identical to Bcl-2 mRNA but mismatched by one base), Bcl-2 mRNA X, Bcl-2 mRNA S1, and RNA NC (negative control). b shows the corresponding bar chart, where the bar area represents the binding efficiency of R48-50 with different nucleic acids.

[0028] Figure 8 This presents the experimental results of the resistance of RNA X, RNA S1, and RNA NC to RNase H of R48-50 in this invention. In the figure, a is its polyacrylamide gel electrophoresis image; b is the corresponding bar graph, where the bar area represents the RNase H-mediated RNA cleavage efficiency after R48-50 binds to RNA X, RNA S1, and RNA NC. Detailed Implementation

[0029] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0030] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0031] The present invention will now be described in further detail with reference to the accompanying drawings: I. Implementation Examples Example 1 This embodiment provides a D-type DNA antisense nucleic acid (D-ASO), which consists of circular DNA and complementary single-stranded DNA. Part of the single-stranded DNA binds to the D-type DNA "string," which is called the protective strand (P); the other part binds to the D-type DNA "bow." The D-type DNA "string" region integrates a carefully designed antisense nucleic acid sequence that is highly specific to the complementary region of the target mRNA.

[0032] Specifically, circular DNA is a D-type conformation double-stranded DNA. The single-stranded DNA contains two functional regions. One functional region binds to the single-stranded region of the circular DNA, namely the "string" region, and the other functional region binds to the double-stranded region of the circular DNA, namely the "bow" region.

[0033] Preferably, in this embodiment, in the D-type DNA topology, the single-stranded "string" region is 28 nucleotides (nt) long, and the double-stranded "bow" region is 20 base pairs (bp) long. The complementary region between the protective strand P and the antisense sequence is 17 base pairs (bp) long, and the ASO sequence is 18 nucleotides (nt).

[0034] Compared to traditional double-stranded DNA (dsDNA) or linear antisense oligonucleotides (ASO), D-type DNA's unique topological structure (the combination of single-stranded "strings" and double-stranded "bows") endows it with greater stability and resistance to nuclease degradation. Designing antisense nucleic acids into D-type DNA allows them to be more stable in cells, thus enabling them to function better.

[0035] In the presence of target mRNA, base pairing induces a substitution of the protective strand P, exposing the antisense sequence, which then binds to the mRNA to form a DNA-RNA hybrid. Subsequently, the hybrid is degraded by the exonuclease RNase H, allowing the protective strand P to rebind to the "string" region, thus achieving a cyclical regeneration of the gene silencing process.

[0036] To verify the anti-degradation properties of D-DNA, D-DNA was added to 10% fetal bovine serum (FBS), and the mixed sample was heated to 37°C in a PCR thermal cycler for 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours, respectively.

[0037] Furthermore, by comparing the integrity of D-DNA at different time points, the D-DNA with the strongest resistance to degradation can be selected, thereby preparing D-ASO.

[0038] In this embodiment, the control D-ASO protective strand P mismatched with the complementary region of the antisense sequence by one base. Experimental results showed that the D-ASO of this embodiment had a half-life of ≥24 hours in a serum environment at 37°C, demonstrating excellent stability. Furthermore, this design reduced non-specific RNA binding by more than 90%, proving its good specificity.

[0039] Example 2 See Figure 1 This embodiment provides a method for preparing D-type DNA antisense nucleic acid (D-ASO), which is composed of circular DNA and complementary single-stranded DNA, specifically including the following steps: Step 1: Prepare unclosed circular DNA Phosphorylated linear DNA at the 5' end was mixed with circular short-stranded DNA at a 1:2 molar ratio in TAE / Mg 2+ In the buffer solution, TAE / Mg 2+ The buffer contained 5 mM Tris, 1 mM EDTA, and 12.5 mM magnesium acetate (pH 8.0), with final concentrations of 5 μM for linear DNA and 10 μM for circular short-strand DNA. The mixture was heated at 95 °C for 5 minutes, then slowly cooled to 4 °C at a rate of 0.1 °C / s for annealing to form unclosed circular DNA.

[0040] Step 2: Ligation to form closed circular DNA Add T4 DNA ligase to the unclosed circular DNA obtained in step 1, incubate at 25°C for 12 hours, and then incubate at 65°C for 30 minutes to inactivate the ligase to obtain closed circular DNA.

[0041] Step 3: Enzymatic digestion treatment Exonucleases I and III were added to the closed circular DNA obtained in step 2, and the mixture was digested at 37°C for 8 hours, followed by treatment at 80°C for 30 minutes to inactivate the enzymes. This step effectively removes residual linear DNA fragments from the reaction system and improves the purity of the circular DNA.

[0042] Step 4: Purify circular DNA and form D-DNA The product obtained in step 3 was separated by polyacrylamide gel electrophoresis. The gel band containing the target circular DNA was cut and purified. The purification conditions were as follows: elution with Elution Buffer at 65°C for 4.5 hours, followed by elution at 37°C for another 8 hours; purification was performed using a centrifugal filtration column with parameters of 10000 x G for 15 minutes. After obtaining the purified circular DNA, it was annealed with D-type short-chain DNA to form a D-DNA structure.

[0043] Step 5: Secondary purification to obtain the final product The D-DNA obtained in step 4 was purified by a second polyacrylamide gel electrophoresis. The gel bands containing the target D-DNA were cut, eluted, and purified. Ethanol precipitation was then performed with the following parameters: 50% isopropanol was added, and precipitation was carried out at -20°C for 30 minutes. The mixture was then centrifuged at 12,000 rpm at 4°C for 10 minutes and washed twice with 75% ethanol. High-purity D-type DNA antisense nucleic acid (D-ASO) was finally obtained.

[0044] The D-ASO prepared by the above method has a unique topological structure (single-stranded "strings" combined with double-stranded "bows"), exhibiting higher stability and resistance to nuclease degradation compared to traditional double-stranded DNA (dsDNA) or linear antisense oligonucleotides (ASO). This structural design makes antisense nucleic acids more stable in the intracellular environment, enabling them to more effectively perform gene silencing functions and achieve specific recognition and degradation of target mRNA.

[0045] Preferably, in step 1, the circularized linear DNA has lengths of 38 nt, 40 nt, 42 nt, 44 nt, 46 nt, 48 nt, 50 nt, and 60 nt. These correspond to short circularized strands of 16 nt, 18 nt, 18 nt, 18 nt, 20 nt, 20 nt, and 20 nt, respectively. The circularized DNA and the short circularized strands are mixed at a molar ratio of 1:2 in a 1×TAE / Mg solution. 2+ The sample was mixed in a PCR thermal cycler and heated to 95 °C for 5 minutes, then cooled to 4 °C at a rate of 0.1 °C every 10 seconds.

[0046] More preferably, the final concentrations of the circulated DNA and the circulated short chain are 5 uM and 10 uM, respectively.

[0047] Preferably, in step 2, after adding T4 DNA ligase, the above mixture is mixed in a 1×T4 DNA ligase / Mg solution. 2+ Buffer (50 mM Tris-HCl, 5 mM Mg) 2+ The samples were mixed in a PCR thermal cycler (2.5 mM DTT, 0.5 mM ATP, pH 7.5) and heated to 25°C for 12 hours, then kept at 65°C for 30 minutes.

[0048] Preferably, in step 3, 1× exonuclease I / Mg is added. 2+ Buffer (50 mM Tris-HCl, 10 mM Mg) 2+ (1 mM DTT, 0.1 mM ATP, pH 7.5) and 1× exonuclease III / Mg 2+ Buffer (50 mM Tris-HCl, 10 mM Mg) 2+ After mixing the above mixture (1 mM DTT, 0.1 mM ATP, pH 7.5), it was placed in 1× exonuclease III buffer and 1× exonuclease I buffer, and incubated at 37 °C for 8 hours and 80 °C for 30 minutes. The addition of exonuclease I and exonuclease III can effectively remove linear ends and any non-target sequences from DNA fragments, thereby ensuring that the final product is a pure circular DNA molecule.

[0049] Preferably, in step 4, after purifying the circular DNA by polyacrylamide gel excision, the gel containing the pure circular DNA is crushed and eluted with Elution Buffer at 65°C for 4.5 hours and 37°C for 8 hours. The eluted gel mixture is then centrifuged at 10,000 XG for 15 minutes using a centrifuge filter column, and the solution containing the circular DNA is collected through the filter column.

[0050] Preferably, in step 5, after purifying D-type DNA by polyacrylamide gel excision, the gel containing pure D-type DNA is crushed and eluted with Elution Buffer at 65 °C for 4.5 hours and 37 °C for 8 hours. The eluted gel mixture is then centrifuged at 10,000 XG for 15 minutes using a centrifuge filter column, and the solution containing D-type DNA is collected through the filter column.

[0051] More preferably, water and anhydrous isopropanol are added to the collected D-type DNA solution to dilute the isopropanol to a final concentration of 50%. After mixing, the solution is allowed to stand at -20 °C for 30 minutes, then centrifuged at 12,000 rpm for 10 minutes at 4 °C. The supernatant is discarded, and 75% ethanol is added. The solution is then centrifuged at 12,000 rpm for 10 minutes at 4 °C and washed twice. The supernatant is discarded, and the solution is dried before adding 1×TAE / Mg. 2+ A pure D-type DNA solution was obtained by buffering (5 mM Tris, 1 mM EDTA and 12.5 mM magnesium acetate, pH 8.0).

[0052] II. Experimental Section 1. Polyacrylamide electrophoresis characterization of D-type DNA with different bending forces Preparation of D-type DNA under different bending forces: D-type DNA was prepared by mixing short-chain D-type DNA with the D-type DNA in a buffer solution and annealing. Specifically, the short-chain D-type DNA had R38, R40, R42, R44, R46, R48, R50, and R60 values ​​of 30 nt. Circular DNA and the short-chain D-type DNA were mixed at a 1:2 molar ratio in a 1×TAE / Mg solution. 2+ The samples were mixed in a PCR thermal cycler and heated to 95 °C for 5 minutes, then cooled to 4 °C at a rate of 0.1 °C every 10 seconds. The resulting D-type DNA samples were R38-30, R40-30, R42-30, R44-30, R46-30, R48-30, R50-30, and R60-30.

[0053] See Figure 2A series of D-type DNA molecules with different bending stiffnesses were synthesized. Image a shows the polyacrylamide gel electrophoresis characterization of the synthesized D-type DNA molecules with different bending stiffnesses. Lanes 1-8 are R38-30, R40-30, R42-30, R44-30, R46-30, R48-30, R50-30, and R60-30, respectively, with the bending stiffness gradually decreasing. Image b provides a schematic diagram of these D-type DNA molecules with different bending stiffnesses, visually demonstrating the trend of D-type DNA structure from high to low bending stiffness.

[0054] 2. Investigation into the stability of D-type DNA in fetal bovine serum under different bending forces. With 10% fetal bovine serum (FBS) added to the prepared D-type DNA, the mixed sample was heated to 37 °C in a PCR thermal cycler and maintained for 2, 4, 6, 8, 12, and 24 hours to simulate the degradation environment that may be encountered in vivo. By setting these time points, the resistance of D-type DNA to degradation in FBS under different bending forces was monitored, thereby assessing its potential stability in vivo. D-type DNA with better resistance to degradation was selected, and the desired D-ASO was prepared by modifying the single strand of this D-type DNA.

[0055] Specifically, R38-30, R40-30, R42-30, R44-30, R46-30, R48-30, R50-30, and R60-30 were incubated with 10% fetal bovine serum (FBS) at 37 °C for 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours, respectively. Their stability in 10% FBS was verified by polyacrylamide gel electrophoresis, thus simulating the stability of D-type DNA in cells. The experimental results showed that moderate bending force can significantly improve the degradation resistance of D-type DNA.

[0056] See results Figure 3This study investigated the degradation resistance of D-type DNA under different bending forces. Figure a shows the polyacrylamide gel electrophoresis (PAGE) characterization of D-type DNA under different bending forces after incubation with 10% fetal bovine serum (FBS) at 37 °C for 2, 4, 6, 8, 12, and 24 hours. Figure b shows the corresponding line graphs, reflecting the residual amount of D-type DNA after 24 hours of incubation in FBS under different bending forces. The residual amounts were 32% for R48-30, 40% for R40-30, 51% for R42-30, 92% for R44-30, 80% for R46-30, 83% for R48-30, 46% for R50-30, and 10% for R60-30. The results indicate that moderate bending force can significantly improve the degradation resistance of D-type DNA. Specifically, the stability of D-type DNA is affected when its bending force is too large or too small. Detailed analysis revealed that the R48-30 structure of D-type DNA exhibits superior resistance to degradation, indicating that its bending force is finely regulated to a suitable range. Furthermore, the loop length of R48-30 DNA provides an ideal template for preparing antisense nucleic acids (D-ASO) with strand displacement activity, thus possessing potential application value in gene silencing and other related applications. Therefore, R48-30 D-type DNA, due to its balanced bending force and suitable loop length, is the preferred structure for preparing highly efficient D-ASOs.

[0057] This invention reveals that the stability of D-type DNA initially increases and then decreases with increasing bending force, indicating that both excessively high and low bending forces negatively impact the stability of D-type DNA. This research emphasizes the importance of maintaining a suitable bending force for the stability of D-type DNA.

[0058] 3. Preparation of D-type DNA antisense nucleic acid (D-ASO) A 48 nt circular DNA was selected, and the short D-type DNA strand was 50 nt. This short strand consisted of two parts: one part bound to the "bow" structure, and the other bound to the "string." The "string" region of the D-type DNA was designed as an antisense nucleic acid sequence, and the portion of the short D-type DNA strand that bound to the "string" was the guard strand (P). P was used to prevent direct contact between mRNA and ASO, thereby enhancing the specificity between ASO and mRNA. These two parts of the short D-type DNA strand were connected by a carefully designed macroloop structure, designed to facilitate efficient substitution between the target mRNA and the guard strand P. The purified 48 nt circular DNA and the 50 nt D-type DNA short strand were annealed to successfully construct R48-50 D-ASO. The successful synthesis of D-ASO was verified using polyacrylamide gel electrophoresis; the results are shown below. Figure 4 .

[0059] Specifically, the preparation process of D-ASO is as follows: The 48 nt single strand modified with 5' phosphate was annealed with a 20 nt circular short strand, ligated using T4 DNA ligase, and the excess structure was digested using exonuclease I and exonuclease III. The purified R48 was obtained by polyacrylamide gel electrophoresis. The R48 was then annealed with a 50 nt single strand, purified by polyacrylamide gel electrophoresis, and then precipitated with ethanol to obtain the purified R48-50.

[0060] Preferably, a 48nt single chain modified with 5' phosphate is mixed with a 20nt cyclic short chain in a 1:2 molar ratio in a 1×TAE / Mg solution. 2+ The sample was mixed in a PCR thermal cycler and heated to 95 °C for 5 minutes, then cooled to 4 °C at a rate of 0.1 °C every 10 seconds.

[0061] More preferably, the final concentrations of the circularized DNA and the circularized short strand are 5 μM and 10 μM, respectively. After adding T4 DNA ligase, the above mixture is mixed at a concentration of 1×T4 DNA ligase / Mg. 2+ Buffer (50 mM Tris-HCl, 5 mM Mg) 2+ The sample was mixed in a PCR thermal cycler (2.5 mM DTT, 0.5 mM ATP, pH 7.5) and heated to 25 °C for 12 hours, then at 65 °C for 30 minutes. 1× exonuclease I / Mg was added. 2+ Buffer (50 mM Tris-HCl, 10 mM Mg) 2+(1 mM DTT, 0.1 mM ATP, pH 7.5) and 1× exonuclease III / Mg 2+ Buffer (50 mM Tris-HCl, 10 mM Mg) 2+ After mixing the above mixture (1 mM DTT, 0.1 mM ATP, pH 7.5), it was placed in 1× exonuclease III buffer and 1× exonuclease I buffer, and incubated at 37 °C for 8 hours and 80 °C for 30 minutes. By adding exonuclease I and exonuclease III, linear ends and any non-target sequences in the DNA fragment can be effectively removed, thus ensuring that the final obtained DNA molecule is a pure circular structure.

[0062] After purifying the circular DNA using polyacrylamide gel electrolysis, the gel containing the purified circular DNA was crushed and eluted with Elution Buffer at 65°C for 4.5 hours and then at 37°C for 8 hours. The resulting gel mixture was centrifuged at 10,000 XG for 15 minutes, and the solution containing the circular DNA was collected through the filter column. R48 and 50 nt of single-stranded DNA were mixed at a 1:2 molar ratio in 1×TAE / Mg 2+ The sample was mixed in a PCR thermal cycler and heated to 95 °C for 5 minutes, then cooled to 4 °C at a rate of 0.1 °C every 10 seconds.

[0063] D-type DNA was obtained as R48-50. After purifying the D-type DNA by polyacrylamide gel excision, the gel containing the purified D-type DNA was crushed and eluted with Elution Buffer at 65 °C for 4.5 hours and 37 °C for 8 hours. The eluted gel mixture was centrifuged at 10,000 XG for 15 minutes using a centrifuge column, and the solution containing D-type DNA was collected through the filter column. Water and anhydrous isopropanol were added to the collected D-type DNA solution to dilute the isopropanol to a final concentration of 50%. After mixing, the solution was incubated at -20 °C for 30 minutes, centrifuged at 12,000 rpm for 10 minutes at 4 °C, the supernatant was discarded, 75% ethanol was added, and the solution was washed twice by centrifugation at 12,000 rpm for 10 minutes at 4 °C. The supernatant was discarded, the solution was dried, and 1×TAE / Mg was added. 2+ A pure R48-50 solution was obtained by buffering (5 mM Tris, 1 mM EDTA and 12.5 mM magnesium acetate, pH 8.0).

[0064] 4. Preparation of D-ASO (R48-50) and verification of its ability to mitigate off-target effects and recycle it. See Figure 5 This invention discloses a D-DNA-based anti-degradation antisense nucleic acid (D-ASO) R48-50. The D-DNA "string" contains an antisense nucleic acid sequence. Of the 50 nt single strand, 20 nt binds to the R48 loop, and 16 nt binds to the D-DNA "string," this portion being called the protective strand P. The remaining 14 nt forms a loop structure connecting these two portions, the purpose of which is to enable the target RNA to successfully displace the protective strand P and hybridize with the antisense nucleic acid portion. D-ASO specifically reacts with the target RNA X, thereby promoting the release of the protective strand P and the formation of the hybridization product D-ASO-X. This method has high discrimination capability, accurately distinguishing the target nucleic acid from non-target RNA S1 containing a single base mismatch, thus significantly improving the targeting specificity and therapeutic effect of nucleic acid drugs. Furthermore, after D-ASO binds to the target RNA X, RNase H specifically cleaves the RNA portion. After cleavage, the protective strand P can rebind to the D-DNA "string," achieving recycling.

[0065] 5. Investigation into the stability of D-ASO (R48-50) in fetal bovine serum By adding 10% fetal bovine serum (FBS) to the prepared D-ASO(R48-50), the mixed sample was heated to 37 °C in a PCR thermal cycler and maintained for 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours, respectively, to simulate the degradation environment that may be encountered in vivo. By setting these time points, the anti-degradation activity of D-ASO(R48-50) in FBS was monitored, thereby assessing its potential stability in vivo.

[0066] Specifically, see the results. Figure 6This study investigated the stability of R48-50, a D-ASO, in fetal bovine serum (FBS). Figure a shows the polyacrylamide gel electrophoresis (PAGE) characterization of R48-50 after incubation with 10% FBS at 37 °C for 2, 4, 6, 8, 12, and 24 hours. Figure b shows the corresponding line graph, quantifying the change in residual R48-50 D-ASO at different time points under the same conditions: 92% after 2 hours, 91% after 4 hours, 89% after 6 hours, 88% after 8 hours, 85% after 12 hours, and 83% after 24 hours. This dataset clearly demonstrates that R48-50 exhibits significant anti-degradation ability in FBS, retaining over 80% of its initial amount even after a long incubation of 24 hours, thus confirming its excellent stability. These findings are significant for designing highly stable antisense nucleic acid molecules, especially in applications requiring long-term stability, such as gene silencing therapy. The high stability of R48-50 is mainly attributed to its unique D-type structure, which may reduce the degradation rate by decreasing the chances of nuclease recognition and binding.

[0067] 6. Off-target effects of D-ASO (R48-50) To investigate the specificity of D-ASO binding to target mRNA: D-ASO was bound to DNA X (a DNA sequence identical to the mRNA sequence), DNA S1 (a DNA sequence identical to the mRNA sequence but mismatched by one base), mRNA X, mRNA S1, and RNA NC (negative control) to determine the specificity of D-ASO binding to target mRNA.

[0068] See Figure 7This invention discloses an experiment on the binding specificity of D-ASO (R48-50) with target RNA. In the figure, a is a polyacrylamide gel electrophoresis image; lanes 1, 2, 3, 4, 5, and 6 correspond to R48-50, R48-50+DNA X, R48-50+DNAS1, R48-50+RNA X, R48-50+RNA S1, and R48-50+RNA NC, respectively. Binding with DNA X and DNA S1 is chosen because RNA is easily degraded, and using the encoding DNA serves as a control. Binding with RNA NC also serves as a negative control to better investigate the binding specificity of R48-50 with target RNA. By comparing the migration rate changes in each lane, the binding strength and specificity of R48-50 with different RNA molecules can be assessed. b is a bar chart corresponding to the polyacrylamide gel electrophoresis image; the area of ​​the bar chart quantitatively reflects the binding ability of R48-50 with DNA X, DNA S1, RNA X, RNA S1, and RNA NC. Experimental data showed that R48-50 had a binding efficiency of 45% with DNA X, only 1% with DNA S1, a high binding efficiency of 52% with RNA X, a 5% binding efficiency with RNA S1, and 0% with RNA NC. These results indicate that R48-50 exhibits extremely high binding specificity, with its binding efficiency with non-target nucleic acids controlled below 10%.

[0069] Specifically, it includes the following steps: 1) Preparation of D-ASO: A 48 nt single strand modified with 5' phosphate was annealed with a 20 nt circular short strand, ligated using T4 DNA ligase, and the excess structure was digested using exonuclease I and exonuclease III. The purified R48 was obtained by polyacrylamide gel electrophoresis. R48 was then annealed with a 50 nt single strand, purified by polyacrylamide gel electrophoresis, and then precipitated with ethanol to obtain the purified R48-50.

[0070] 2) R48-50 was incubated with DNA X (a DNA sequence identical to the Bcl-2 mRNA sequence), DNA S1 (a DNA sequence identical to the Bcl-2 mRNA sequence but mismatched by one base), Bcl-2 mRNA X, Bcl-2 mRNA S1, and RNANC (negative control) at 37 °C for 30 minutes.

[0071] Preferably, in step 1), the 48nt single chain modified with 5' phosphate and the 20nt cyclic short chain are mixed in a 1:2 molar ratio in a 1×TAE / Mg solution. 2+The sample was mixed in a PCR thermal cycler and heated to 95 °C for 5 minutes, then cooled to 4 °C at a rate of 0.1 °C every 10 seconds.

[0072] More preferably, the final concentrations of the circularized DNA and the circularized short strand are 5 μM and 10 μM, respectively. After adding T4 DNA ligase, the above mixture is mixed in a 1×T4 DNA ligase / Mg solution. 2+ Buffer (50 mM Tris-HCl, 5 mM Mg) 2+ The sample was mixed in a PCR thermal cycler (2.5 mM DTT, 0.5 mM ATP, pH 7.5) and heated to 25 °C for 12 hours, then at 65 °C for 30 minutes. 1× exonuclease I / Mg was added. 2+ Buffer (50 mM Tris-HCl, 10 mM Mg) 2 + (1 mM DTT, 0.1 mM ATP, pH 7.5) and 1× exonuclease III / Mg 2+ Buffer (50 mM Tris-HCl, 10 mM Mg) 2+ After mixing the above mixture (1 mM DTT, 0.1 mM ATP, pH 7.5), it was placed in 1× exonuclease III buffer and 1× exonuclease I buffer, and incubated at 37 °C for 8 hours and 80 °C for 30 minutes. By adding exonuclease I and exonuclease III, linear ends and any non-target sequences in the DNA fragment can be effectively removed, thus ensuring that the final obtained DNA molecule is a pure circular structure.

[0073] After purifying the circular DNA using polyacrylamide gel electrolysis, the gel containing the purified circular DNA was crushed and eluted with Elution Buffer at 65°C for 4.5 hours and then at 37°C for 8 hours. The resulting gel mixture was centrifuged at 10,000 XG for 15 minutes, and the solution containing the circular DNA was collected through the filter column. R48 and 50 nt of single-stranded DNA were mixed at a 1:2 molar ratio in 1×TAE / Mg 2+ The sample was mixed in a PCR thermal cycler and heated to 95 °C for 5 minutes, then cooled to 4 °C at a rate of 0.1 °C every 10 seconds.

[0074] D-type DNA was obtained as R48-50. After purifying the D-type DNA by polyacrylamide gel excision, the gel containing the purified D-type DNA was crushed and eluted with Elution Buffer at 65 °C for 4.5 hours and 37 °C for 8 hours. The eluted gel mixture was centrifuged at 10,000 XG for 15 minutes using a centrifuge column, and the solution containing D-type DNA was collected through the filter column. Water and anhydrous isopropanol were added to the collected D-type DNA solution to dilute the isopropanol to a final concentration of 50%. After mixing, the solution was incubated at -20 °C for 30 minutes, centrifuged at 12,000 rpm for 10 minutes at 4 °C, the supernatant was discarded, 75% ethanol was added, and the solution was washed twice by centrifugation at 12,000 rpm for 10 minutes at 4 °C. The supernatant was discarded, the solution was dried, and 1×TAE / Mg was added. 2+ A pure R48-50 solution was obtained by buffering (5 mM Tris, 1 mM EDTA and 12.5 mM magnesium acetate, pH 8.0).

[0075] Preferably, in step 2), R48-50 is mixed with DNA encoding DNA X (a DNA sequence identical to the Bcl-2 mRNA sequence), DNA encoding DNA S1 (a DNA sequence identical to the Bcl-2 mRNA sequence but mismatched by one base), Bcl-2 mRNA X, Bcl-2 mRNA S1, and RNA NC (negative control) at a molar ratio of 1:2 in 1×TAE / Mg 2+ The sample was mixed in a PCR thermal cycler and heated to 37 °C for 30 minutes. The mixture was placed in a PCR thermal cycler.

[0076] 7. Experimental study on the tolerance of RNA X, RNA S1, and RNA NC to RNase H of R48-50. To investigate the degradation efficiency of D-ASO after binding to mRNA: D-ASO was bound to target mRNA X, non-target mRNA S1, and negative control RNA NC, respectively, followed by the addition of RNase H and incubation at 37 °C for 30 minutes. The specific binding ability of D-ASO to target mRNA X was verified by measuring the mRNA degradation efficiency, demonstrating the effectiveness of D-ASO in mitigating off-target effects.

[0077] See Figure 8This invention discloses experiments on the RNase H tolerance of D-ASO (R48-50), RNA X, RNA S1, and RNA NC to R48-50. In this study, lane a represents the gel electrophoresis. Lanes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 correspond to R48-50, RNA X, RNA X + RNase H (incubation for 30 min), R48-50 + RNA X + RNase H (incubation for 30 min), RNA S1, RNA S1 + RNase H (incubation for 30 min), RNA S1 + RNase H (incubation for 30 min), RNA NC, RNA NC + RNase H (incubation for 30 min), and R48-50 + RNA NC + RNase H (incubation for 30 min), respectively. By analyzing the migration patterns of the samples in each lane, the stability differences of R48-50 D-ASO after binding to different RNA substrates can be assessed, thereby revealing its specificity in binding to target RNA. b represents the corresponding bar chart, which quantitatively analyzed the RNA cleavage ratios induced by RNase H treatment after R48-50 bound to target RNA (RNA X) and non-target RNAs (RNA S1 and RNA NC). The bar area visually reflects the RNase H-mediated RNA cleavage efficiency after R48-50 bound to RNA X, RNA S1, and RNA NC. Experimental data showed that after R48-50 bound to RNA X, the cleavage ratio of RNA X reached 77% after RNase H treatment; after binding to RNA S1, the cleavage ratio of RNA S1 was 11%; and after binding to RNA NC, the cleavage ratio of RNA NC was only 2%. These data further confirm that R48-50 has a significant specific binding ability to target RNA X, and also verify its function in reducing off-target effects. The bar chart analysis results revealed that the binding specificity of R48-50 to target RNA X was significantly higher than that to non-target RNAs, thus demonstrating the potential of R48-50 in improving gene silencing specificity and reducing non-specific binding. This finding is of great significance for designing efficient antisense nucleic acid drugs with low off-target effects.

[0078] Specifically, it includes the following steps: 1) Preparation of D-ASO: A 48 nt single strand modified with 5' phosphate was annealed with a 20 nt circular short strand, ligated using T4 DNA ligase, and the excess structure was digested using exonuclease I and exonuclease III. The purified R48 was obtained by PAGE gel purification. R48 was then annealed with a 50 nt single strand, purified by PAGE gel purification, and precipitated with ethanol to obtain purified R48-50.

[0079] 2) R48-50 was incubated with Bcl-2 mRNA X, Bcl-2 mRNA S1, and RNA NC (negative control) at 37 °C for 30 minutes.

[0080] 3) Incubate Bcl-2 mRNA X, Bcl-2 mRNA S1, and RNA NC with RNase H at 37 °C for 30 minutes.

[0081] 4) Incubate R48-50-Bcl-2 mRNA X, R48-50-Bcl-2 mRNA S1, and R48-50-RNA NC with RNase H at 37 °C for 30 minutes.

[0082] Preferably, in step 2), R48-50 is mixed with Bcl-2 mRNA X, Bcl-2 mRNA S1, and RNA NC at a molar ratio of 1:200 in a 1×TAE / Mg solution. 2+ The sample was mixed in a PCR thermal cycler and heated to 37 °C for 30 minutes. The mixture was placed in a PCR thermal cycler.

[0083] More preferably, the final concentrations of R48-50 and RNA are 0.2 uM and 40 uM, respectively.

[0084] Preferably, in step 3), 6 U / ul of RNaseH is added to a 40 uM Bcl-2 mRNA X, Bcl-2 mRNA S1, and RNA NC solution and mixed in 1×RNaseH buffer (2.5 mM Tris-HCl, 0.05 mM EDTA, 3 mM NaCl, 0.1 mM DTT, 5% Glycerol, pH 7.5). The mixed sample is then placed in a PCR thermal cycler and heated to 37°C for 30 minutes.

[0085] Preferably, in step 4), 6 U / µl of RNaseH is added to the R48-50-Bcl-2 mRNA X, R48-50-Bcl-2 mRNA S1, and R48-50-RNANC solutions and mixed in 1×RNaseH buffer (2.5 mM Tris-HCl, 0.05 mM EDTA, 3 mM NaCl, 0.1 mM DTT, 5% Glycerol, pH 7.5). The mixed sample is then placed in a PCR thermal cycler and heated to 37 °C for 30 minutes.

[0086] The sequence listing involved in the above embodiments and experimental verification of the present invention is shown in Table 1 below: Table 1

[0087] In summary, this invention discloses a degradation-resistant antisense nucleic acid (D-ASO) based on D-DNA (D-DNA). The study first synthesized a series of D-DNA molecules with different bending stiffnesses and systematically evaluated their stability in fetal bovine serum (FBS) environments. Experimental results show that the stability of D-DNA initially increases and then decreases with increasing bending stiffness, revealing the importance of moderate bending stiffness for maintaining the stability of D-DNA. Both excessively high and excessively low bending stiffness are detrimental to the stable existence of D-DNA.

[0088] Based on the above research findings, this invention further utilizes D-type DNA, which exhibits moderate bending stiffness and superior stability, to develop an antisense nucleic acid, D-ASO, that can effectively reduce off-target effects. D-ASO, through its unique strand substitution mechanism, can specifically react with a predetermined target mRNA (X), promoting the release of the protective strand P and the formation of the D-ASO-RNA hybrid product. This method demonstrates high discriminative power, accurately identifying and distinguishing target mRNA from non-target mRNAs with only a single base mismatch, thereby significantly improving the targeting specificity and therapeutic efficacy of nucleic acid drugs.

[0089] Furthermore, after D-ASO binds to the predetermined target mRNA X, RNase H specifically cleaves the RNA component in the hybridization product. After the cleavage reaction is complete, the protective strand P can rebind to the "string" portion of the D-type DNA, achieving the recycling of D-ASO. This design not only endows D-ASO with high specificity but also ensures its high efficiency, further enhancing its application potential and practical value in gene silencing technology.

[0090] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A degradation-resistant antisense nucleic acid based on a D-type DNA structure, characterized in that, It includes circular DNA and complementary single-stranded DNA; among which: The circular DNA is a D-type conformation double-stranded DNA, and the single-stranded DNA contains two functional segments. One functional region of single-stranded DNA binds to the single-stranded region of circular DNA, namely the "string" region, while the other functional region binds to the double-stranded region of circular DNA, namely the "bow" region.

2. The anti-degradation antisense nucleic acid based on D-type DNA structure according to claim 1, characterized in that, The functional segment that binds to the single-stranded region of circular DNA, namely the "string" region, is the protective strand P. When the target mRNA is present, the protective strand P is induced to undergo substitution according to the base complementary pairing principle, exposing the antisense sequence. This antisense sequence binds to the target mRNA to form a DNA-RNA hybrid. The DNA-RNA hybrid is degraded by exonucleases, and the protective strand P automatically re-integrates into the single-stranded segment of the circular DNA, namely the "string" region.

3. The anti-degradation antisense nucleic acid based on D-type DNA structure according to claim 1, characterized in that, The single-stranded region of circular DNA, known as the "string" region, is 28 nt in length, while the double-stranded region of circular DNA, known as the "bow" region, is 20 bp in length.

4. The anti-degradation antisense nucleic acid based on D-type DNA structure according to claim 1, characterized in that, This anti-degradation antisense nucleic acid based on D-type DNA structure has a half-life of ≥24 hours in a serum environment at 37 °C.

5. The method for preparing anti-degradation antisense nucleic acid based on D-type DNA structure according to any one of claims 1-4, characterized in that, Includes the following steps: 1) Phosphorylated linear DNA at the 5' end is mixed with circular primers in a buffer solution, and after heat denaturation, it is slowly cooled to form a pre-closed structure; 2) Add ligase to perform a ligation reaction to obtain closed circular DNA; 3) Closed circular DNA is purified by enzymatic digestion to remove linear impurities, and then purified to obtain circular DNA. 4) Circular DNA is annealed with D-type short-chain DNA to form D-DNA, and then purified twice to obtain anti-degradation antisense nucleic acid based on the D-type DNA structure.

6. The method for preparing anti-degradation antisense nucleic acid based on D-type DNA structure according to claim 5, characterized in that, In step 1), the molar ratio of 5' phosphorylated linear DNA to the circular primer was 1:

2. The heat denaturation and slow cooling conditions were: 95 °C for 5 minutes, followed by annealing at a rate of 0.1 °C / second to 4 °C. The buffer used was TAE / Mg. 2+ The buffer solution, pH 8.0, contains 5 μM phosphorylated linear DNA and 10 μM circular primers at their 5' ends.

7. The method for preparing anti-degradation antisense nucleic acid based on D-type DNA structure according to claim 5, characterized in that, In step 2), T4 DNA ligase was used to incubate the DNA at 25 °C for 12 hours and at 65 °C for 30 minutes to obtain closed circular DNA.

8. The method for preparing anti-degradation antisense nucleic acid based on D-type DNA structure according to claim 5, characterized in that, In step 3), exonuclease I and exonuclease III are added, and the mixture is digested at 37°C for 8 hours and then inactivated at 80°C for 30 minutes to remove linear impurities.

9. The method for preparing anti-degradation antisense nucleic acid based on D-type DNA structure according to claim 5, characterized in that, Both the primary and secondary purification processes involved separating the target DNA using polyacrylamide gel electrophoresis before purification.

10. The application of the anti-degradation antisense nucleic acid based on the D-type DNA structure according to any one of claims 1-4 in gene silencing, characterized in that, The target mRNA triggers a protective strand replacement reaction of the antidegradation-resistant antisense nucleic acid of the D-type DNA structure, forming a DNA-RNA hybrid which is then degraded by RNase H. Subsequently, the protective strand is re-bound and recycled.

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