DNA tetrahedron capable of carrying oligonucleotides and nucleic acid analogues as well as preparation method and application of DNA tetrahedron

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a DNA tetrahedron capable of carrying oligonucleotides and nucleic acid analogues as well as a preparation method and application of the DNA tetrahedron. The method comprises the following steps: cutting off one single-chain DNA in classical tetrahedral framework nucleic acid to form three short-chain DNAs, and self-assembling six single-chain DNAs again to form a DNA tetrahedron to obtain ORT; according to the invention, the mismatching and self-matching phenomena of tFNA are improved, the yield of tFNA is improved, and the tFNA has good structural stability, high bioavailability and excellent biocompatibility; the ORT prepared by the invention can be used as a drug carrier for loading nucleic acid and nucleic acid analogues, protein drugs or small molecule drugs, and the application of the ORT in the aspects of drug delivery, gene therapy, biosensing and the like is promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a DNA tetrahedron capable of transporting oligonucleotides and nucleic acid analogs, and a preparation method and use thereof. Background Art

[0002] Driven by DNA nanotechnology, DNA has become an attractive material for assembling nanostructures. DNA-based nanomaterials have been widely developed and applied in multiple medical fields such as biosensing, bioimaging, disease diagnosis and treatment, and tissue engineering, becoming an important part of nanomedicine. DNA nanomaterials have many advantages, mainly including the following aspects: (1) DNA is the carrier and transmitter of various biological genetic information and has good biocompatibility. (2) The unit composing DNA is four different deoxynucleotide monomers, with simple components and easy synthesis. (3) DNA has diversity and flexible programmability. By designing sequence-specific bases and strict base complementary pairing principles, DNA can self-assemble into highly controllable and high-precision nanostructures. With the rise of technologies such as DNA origami, DNA tiles, and DNA polyhedra, from two-dimensional structures to three-dimensional structures, DNA almost has infinite geometric structure possibilities. This diverse structure endows DNA nanomaterials with better stability and huge carrier potential. (4) DNA has excellent modifiability. DNA nanostructures can load small molecule cargoes or be conjugated with macromolecular polymers, and are multifunctional nanorobots with controllable release. These properties of DNA are the basis for constructing DNA self-assembled intelligent nanomaterials and play important roles in drug delivery, stimulus response, etc.

[0003] Among them, tetrahedral framework nucleic acid (tFNA) is one of the most studied and widely used DNA nanomaterials. tFNA is a DNA tetrahedron self-assembled by four single-stranded DNAs (ssDNAs), which has the advantages of high editability, good biocompatibility, excellent drug loading capacity, easy modification, and stable structure. According to previous research reports, tFNA itself has functions such as promoting tissue regeneration, anti-inflammation, immune regulation, and wound repair, and can also act as a biological carrier to achieve the delivery of various drugs. Currently, tFNA mainly delivers oligonucleotide sequences (aptamers, miRNAs, siRNAs) by modifying sticky ends at the vertices to enhance the cellular uptake of oligonucleotides and resist enzymatic degradation. tFNA can bind small molecule compounds, polypeptides, and functional groups including chemotherapeutic drugs, antibiotics, and traditional Chinese medicine monomers through grooves, electrostatic adsorption, or vertex chemical ligation to improve the bioavailability of drugs. In addition, with the continuous in-depth research on tFNA, it is found that tFNA has good permeability and can be applied in various ways such as intravenous injection, skin application, eye drops, nasal inhalation, and intra-articular injection. It can be said that tFNA has broad application prospects in the biomedical field due to its unique structure and multiple functions.

[0004] However, the development of tFNA still faces some challenges. First, tFNA is a delicate nanostructure composed of complementary pairing of ssDNAs (S1, S2, S3, S4). However, S4 has the phenomenon of self-mismatch, resulting in a decrease in the yield of synthesized tFNA. Second, the synthesized tFNA is prone to aggregation under storage conditions to form nanopolymers, which is not conducive to bioavailability. Therefore, it is of great significance to design tFNA with higher yield and better stability. Summary of the Invention

[0005] Aiming at the problems of the prior art, the present invention provides a DNA tetrahedron capable of transporting oligonucleotides and nucleic acid analogs, and its preparation method and use.

[0006] A DNA tetrahedron capable of transporting oligonucleotides and nucleic acid analogs, wherein the DNA tetrahedron is assembled by base complementary pairing of three long-chain DNAs S1, S2, S3 and at least three short-chain DNAs formed by truncating S4.

[0007] Preferably, the number of short-chain DNAs formed by truncating S4 is 3.

[0008] Preferably, the nucleotide sequences of the short-chain DNAs are as shown in SEQ ID NO.4-SEQ ID NO.6.

[0009] Preferably, the truncation method is to delete the unpaired bases that form the vertices of the tetrahedral framework nucleic acid in S4.

[0010] Preferably, the unpaired base is adenine.

[0011] Preferably, the nucleotide sequences of S1, S2, and S3 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively.

[0012] The present invention also provides a method for preparing the above DNA tetrahedron, which is prepared according to one of the following steps:

[0013] Step a: Mix S1, S2, S3, and at least 3 short-chain DNAs, and incubate to obtain the product.

[0014] Or, step b: Mix S1, S2, and S3, incubate, and then add at least 3 short-chain DNAs and mix and incubate to obtain the product.

[0015] The present invention also provides the use of the above DNA tetrahedron as a vector.

[0016] Preferably, the vector can load at least one of nucleic acids and nucleic acid analogs, protein drugs, small molecule drugs, and functional groups.

[0017] Preferably, the nucleic acids and nucleic acid analogs are selected from at least one of siRNA, miRNA, saRNA, ASO, aptamer, DNA, LNA, PNA, and CpG;

[0018] And / or, the protein drugs are selected from small molecule polypeptides;

[0019] And / or, the small molecule drugs are selected from at least one of chemotherapeutic drugs, natural product drugs, and antibiotics.

[0020] The present invention truncates one of the single-stranded DNAs in the classical tetrahedral framework nucleic acid to form 3 short-chain DNAs, and reassembles the six single-stranded DNAs into a DNA tetrahedron to obtain ORT; it improves the mismatch and self-matching phenomena of tFNA, increases the yield of tFNA, and has good structural stability, high bioavailability, and excellent biocompatibility; the ORT prepared by the present invention can be used as a drug carrier to load different types and lengths of oligonucleotides, promoting its applications in drug delivery, gene therapy, biosensing, etc.

[0021] Obviously, based on the above content of the present invention, according to the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions, or changes can also be made.

[0022] The following is a further detailed description of the above content of the present invention in the form of specific embodiments. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram and identification for the synthesis of tFNA and ORT. (A) Schematic diagram of the preparation process of tFNA and ORT. (i) Four single-stranded DNAs (ssDNAs) are assembled into tFNA in one step through a high-temperature annealing process. (ii) The S4 strand is truncated into S4-1, S4-2, and S4-3. S1, S2, S3, S4-1, S4-2, and S4-3 are mixed and ORT is synthesized in one step according to the same synthesis process as tFNA. (iii) First, a tetrahedral framework composed of S1, S2, and S3 is synthesized through a high-temperature annealing process, denoted as S123. Subsequently, S4-1, S4-2, and S4-3 are complexed with S123 at room temperature to prepare ORT. (B) The successfully synthesized ORT by the one-pot method and the two-step method was characterized by polyacrylamide gel electrophoresis (PAGE) and agarose gel electrophoresis (AGE). Lane 1, S1+S2+S3 (S123). Lanes 2-6 are the ssDNAs with S4 broken (S4-1, S4-2, S4-3) incubated with S123 at room temperature for 5, 10, 15, 20, and 30 minutes, respectively. Lane 7, ORT prepared by the one-pot method. Lane 8, tFNA. Size markers: 100 bp and 200 bp. (C) PAGE results of different nanomaterials. Lane 1, S4. Lane 2, S123. Lane 3, S123+S4-1. Lane 4, S123+S4-1+S4-2. Lane 5, ORT (one-pot method). Lane 6, tFNA (the polymer of tFNA is marked with a dashed box). Size markers: 40 bp, 100 bp, and 200 bp. (D) Size and ζ potential of ORT and tFNA verified by DLS. (E) TEM images of ORT and tFNA (scale bar: 40 nm). (E) AFM images of ORT and tFNA (scale bar: 40 nm).

[0024] Figure 2For the stability determination of ORT and tFNA. (A) Schematic diagrams of different stability determination methods. (B) Stability determination of ORT and tFNA incubated with different concentrations of DNase (37 °C, 1 h), lane 1, 0 U / mL. Lane 2, 0.1 U / mL. Lane 3, 0.5 U / mL. Lane 4, 1 U / mL. Lane 5, 2 U / mL. Lane 6, 4 U / mL. Lane 7, 8 U / mL. Lane 8, 16 U / mL. (C) Stability determination of ORT and tFNA incubated with DNase for different times (1 U / mL, 37 °C). Lane 1, 0 h, lane 2, 2 h, lane 3, 4 h, lane 4, 6 h, lane 5, 8 h, lane 6, 10 h, lane 7, 12 h, lane 8, 24 h. (D) Stability analysis of ORT and tFNA at 4 °C. Lane 1, 0 days. Lane 2, 1 day. Lane 3, 2 days. Lane 4, 3 days. Lane 5, 4 days. Lane 6, 5 days. Lane 7, 6 days. Lane 8, 7 days. (E) Stability determination of ORT and tFNA incubated with FBS (10%, 37 °C). Lane 1, 0 h. Lane 2, 2 h. Lane 3, 4 h. Lane 4, 6 h. Lane 5, 8 h. Lane 6, 10 h. Lane 7, 12 h. Lane 8, 24 h.

[0025] Figure 3 For the cellular uptake of Cy5-S1, FITC-S4, Cy5&FITC-tFNA and Cy5&FITC-ORT by different cells. (A) Schematic diagrams of the cellular uptake of various nanomaterials by several classical cell types. (B) Cellular uptake of S1, S4, tFNA and ORT by RAW 264.7 cells at 6 h, 12 h, 18 h and 24 h. Channel 1: merged, channel 2: Cy5 (red), channel 3: FITC (green), channel 4: dapi - cell nucleus (blue), channel 5: bright field (gray). The scale bar sizes of channel 1 and 2 / 3 / 4 / 5 are 20 μm and 40 μm respectively. (C) Cellular uptake of S1, S4, tFNA and ORT in bone marrow stromal cells (BMSCs) at 3 h, 6 h and 12 h. Channel 1: merged, channel 2: Cy5 (red), channel 3: FITC (green), channel 4: dapi - cell nucleus (blue), channel 5: tritc - cytoskeleton (gray). The scale bar sizes of channel 1 and 2 / 3 / 4 / 5 are 20 μm and 40 μm respectively. (D) Cellular uptake of S1, S4, tFNA and ORT by 4T1 cells at 6 h and 12 h. Channel 1: merged, channel 2: Cy5 (red), channel 3: FITC (green), channel 4: dapi - cell nucleus (blue), channel 5: tritc - cytoskeleton (gray). The scale bar sizes of channel 1 and 2 / 3 / 4 / 5 are 20 μm and 40 μm respectively.

[0026] Figure 4 For the comparison of the in vitro biological functions of tFNA and ORT. (A) Schematic diagram of the wound healing experiment of bone marrow stromal cells. (B) Wound healing experiments of bone marrow mesenchymal stem cells treated with tFNA or ORT at 24 h and 48 h. Scale bar: 500 μm. The unhealed area is marked with a yellow line. (C) Semi-quantification of wound closure at 24 h. Data are expressed as mean ± SD (n = 3). (D) Semi-quantification of wound closure at 48 h. (E) Schematic diagram of the effect of tFNA and ORT on macrophage polarization. (F) ROS staining images of each group under a fluorescence microscope. Channel 1: Merge, Channel 2: ROS (green), Channel 3: DAPI - cell nucleus (blue). Scale bar: 200 μm. (G) Semi-quantification of the ROS fluorescence intensity at 24 h. Semi-quantification of the mRNA expression levels of various inflammation-related factors (IL-6, IL-1β, TNF-α, iNOS, IL-10). Data are expressed as mean ± SD (n = 3): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The label "ns" indicates no statistical difference.

[0027] Figure 5 To detect the biodistribution of tFNA and ORT in vivo after eye drops and knee joint injection of drugs. (A) Schematic diagram of the eye drop penetration experiment. (B) Fluorescence images of the expression of Cy5 / FITC-tFNA or Cy5 / FITC-ORT in eye tissues after 3 treatments. Channel 1: Merge, Channel 2: FITC (green), Channel 3: Cy5 (red), Channel 4: DAPI - cell nucleus (blue). Scale bar: 100 μm. (C) Schematic diagram of the in vivo drug metabolism experiment of intra-articular injection of tFNA and ORT. (D) Fluorescence image results of SD rats treated with intra-articular injection of Cy5 / FITC-tFNA (right) or Cy5 / FITC-ORT (left) at 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 6 h, 12 h (n = 3).

[0028] Figure 6 . For the biodistribution of tFNA and ORT in vivo after transdermal administration and tail vein injection. (A) Schematic diagram of the process of transdermal administration experiment. (B) Fluorescence images of the skin treated with Cy5 / FITC-tFNA or Cy5 / FITC-ORT for 24 hours. Channel 1: Merge, Channel 2: FITC (green), Channel 3: Cy5 (red), Channel 4: dapi - cell nucleus (blue). Scale bar: 200 μm. (C) Schematic diagram of the determination of the biodistribution of tFNA or ORT by intravenous injection. (D) Representative fluorescence images of the main organs in vitro of mice treated with Cy5 / FITC-tFNA or Cy5 / FITC-ORT at 30 min, 1 h, 2 h, and 4 h after tail vein injection (n = 3).

[0029] Figure 7. Preparation of various modified nanomaterials based on ORT. (A) Schematic diagram of the method for modifying ORT-based nanomaterials. Through base pairing, ORT can carry different types of functional strands: DNA, RNA, and ASO. By changing the sequence of the framework nucleic acid S123 composed of S1, S2, and S3, ORT can load functional strands of different lengths. (B) Two synthesis methods of ORT@siRNA were verified by AGE. Lane 1, siRNA. Lane 2, modified S123 paired with siRNA, named S123-siRNA. Lanes 3-6 are siRNA incubated with S123-siRNA at room temperature for 5 minutes, 10 minutes, 20 minutes, and 30 minutes, respectively. Lane 7, one-pot preparation of ORT@siRNA. Lane 8, tFNA. (C) Successful preparation of ORT@siRNA was detected by PAGE. Lane 1, siRNA. Lane 2, S123-siRNA. Lane 3, S123-siRNA + siRNA, ratio 1:0.5. Lane 4, S123-siRNA + siRNA, ratio 1:1. Lane 5, S123-siRNA + siRNA, ratio 1:2. Lane 6, tFNA. Lane 7, S123-siRNA + siRNA, ratio 1:3, incubated for 5 minutes. Lane 8, S123-siRNA + siRNA, ratio 1:3, incubated for 10 minutes. (D) Different synthesis methods of ORT@miR-22 were characterized by AGE. Lane 1, miR-22. Lane 2, modified S123 paired with siRNA, named S123-miR-22. Lanes 3-6 are miR-22 incubated with S123-miR-22 at room temperature for 5 minutes, 10 minutes, 20 minutes, and 30 minutes, respectively. Lane 7, one-pot preparation of ORT@miR-22. Lane 8, tFNA. (E) PAGE results of ORT@miR-22. Lane 1, miR-22. Lane 2, S123-miR-22. Lane 3, S123-miR-22 + miR-22, ratio 1:0.5. Lane 4, S123-miR-22 + miR-22, ratio 1:1. Lane 5, S123-miR-22 + miR-22, ratio 1:2. Lane 6, tFNA. Lane 7, S123-miR-22 + miR-22, ratio 1:3, incubated for 5 minutes. Lane 8, S123-miR-22 + miR-22, ratio 1:3, incubated for 10 minutes. (F) Synthesis of ORT@ASO was verified by PAGE. Lane 1, ASO. Lane 2, modified S123 paired with ASO, named S123-ASO. Lane 3, S123-ASO + ASO, ratio 1:1. Lane 4, S123-ASO + ASO, ratio 1:2. Lane 5, ORT@ASO (S123-ASO: ASO = 1:3).Lane 6, tFNA. (G) PAGE results of ORT@D26. Lane 1, D26-1. Lane 2, S123. Lane 3, S123 + D26-1. Lane 4, S123 + D26-1 + D26-2. Lane 5, ORT@D26. Lane 6, tFNA. Detailed implementation mode

[0030] In the following examples and experimental examples, reagents and raw materials not specifically described are commercially available products.

[0031] Example 1 ORT and its synthesis method

[0032] The tetrahedral framework nucleic acid is assembled by 4 single-stranded DNAs (S1, S2, S3, S4) through base complementary pairing. In the present invention, the unpaired adenine bases (adenyl, A) that constitute the vertices of the DNA tetrahedron in the S4 strand are deleted, and S4 is truncated into three segments: S4-1, S4-2, and S4-3; the six single-stranded DNAs (S1, S2, S3, S4-1, S4-2, S4-3) are reassembled into a DNA tetrahedron, which is ORT. The nucleotide sequence of the present invention is shown in Table 1.

[0033] One-pot synthesis of ORT, add equimolar concentrations of S1, S2, S3, S4-1, S4-2, and S4-3 to TM buffer (10 mM Tris-HCl and 50 mM MgCl2, and adjust the pH value of the solution to 8.0), mix well and place in a PCR instrument, quickly raise the temperature to 95 °C and stabilize for 10 min, then cool to 4 °C and stabilize for 20 min to synthesize ORT.

[0034] Two-step synthesis of ORT, first add equimolar concentrations of S1, S2, and S3 to TM buffer (10 mM Tris-HCl and 50 mM MgCl2, and adjust the pH value of the solution to 8.0), mix well and place in a PCR instrument, quickly raise the temperature to 95 °C and stabilize for 10 min, then cool to 4 °C and stabilize for 20 min to prepare the tetrahedral framework S123. Then, add the same concentrations of S4-1, S4-2, and S4-3 to S123 and incubate at room temperature for 5-10 min to complete the synthesis of ORT. The nucleotide sequence is shown in Table 1.

[0035] This comparative example provides the control samples used in the experiment:

[0036] Comparative Example 1 tFNA

[0037] Dissolve four designed single-stranded DNAs (S1, S2, S3, S4) in TM buffer solution (10 mM Tris-HCl and 50 mM MgCl2, and adjust the pH value of the solution to 8.0) at the same concentration. Then vortex, mix well, and centrifuge the mixture, and place it in a PCR instrument. Rapidly increase the temperature to 95 °C and stabilize for 10 min, then cool to 4 °C and stabilize for 20 min to synthesize tFNA. The nucleotide sequences are shown in Table 1.

[0038] Table 1. Nucleotide sequence table

[0039]

[0040]

[0041] The technical solution of the present invention will be further described through experiments below.

[0042] Experimental Example 1 Characterization of ORT

[0043] I. Experimental method

[0044] 1. Agarose gel electrophoresis (AGE)

[0045] a. Prepare 2% agarose gel: Weigh 1 g of agarose powder, dissolve it in 150 mL of 1x TAE solution, add 10 μl of GelRed, boil it in a microwave oven at high power for 3 - 4 minutes, cool it slightly, and pour it into a BIO-RAD electrophoresis gel casting tank, insert a gel comb, and wait for it to cool and solidify into a gel.

[0046] b. Loading and electrophoresis: Connect the BIO-RAD electrophoresis tank, transfer the prepared agarose gel to the electrophoresis tank, and add 1x TAE solution to level with the gel surface. Mix 1 μl of 6× loading buffer with 5 μl of the sample and marker evenly, and then add them to the corresponding electrophoresis tanks respectively. Under the conditions of ice bath and constant voltage of 120 V, electrophoresis for 25 - 30 minutes.

[0047] c. Exposure: Take out the gel and expose it with a gel imager for imaging.

[0048] Lane 1, S1 + S2 + S3 (S123). Lanes 2 - 6 are the three segments of ss DNA (S4-1, S4-2, S4-3) obtained by breaking S4 incubated with S123 at room temperature for 5, 10, 15, 20, and 30 minutes respectively. Lane 7, ORT prepared by one-pot method. Lane 8, tFNA.

[0049] 2) Polyacrylamide gel electrophoresis (PAGE)

[0050] a. Preparation of polyacrylamide gel: Mix a 40 wt% acrylamide solution, 10×TAE, a 10 wt% ammonium sulfate solution, distilled water, N,N,N',N'-tetramethylethylenediamine, etc. in a volume ratio of 1 - 2:1:1:1:1 to prepare the polyacrylamide gel.

[0051] b. Loading and electrophoresis: Mix 1 μL of 6×loading buffer with 5 μL of the sample and the marker respectively, and then add them to the corresponding electrophoresis tanks. Under the conditions of ice bath and constant voltage of 80 V, perform electrophoresis for 80 minutes.

[0052] c. GelRed staining and exposure: Place the polyacrylamide gel in a mixture of GelRed and distilled water mixed in a ratio of 1:50, avoid light, and shake on a shaker for 15 - 25 minutes. Then perform exposure.

[0053] Lane 1, S4. Lane 2, S123. Lane 3, S123 + S4 - 1. Lane 4, S123 + S4 - 1 + S4 - 2. Lane 5, ORT (one-pot method). Lane 6, tFNA.

[0054] 3) Dynamic light scattering

[0055] Take appropriate amounts of tFNA and ORT sample solutions and place them in a dynamic light scattering detector for detection.

[0056] 4) Transmission electron microscopy

[0057] Take appropriate amounts of tFNA and ORT sample solutions on a metal sheet, irradiate them under infrared for 5 - 10 minutes to dry, and then perform machine detection.

[0058] 5) Atomic force microscopy

[0059] Take appropriate amounts of tFNA and ORT sample solutions, ultrafilter them and place them on a metal sheet, and detect the morphology with an atomic force probe.

[0060] II. Experimental results

[0061] As Figure 1 shown in

[0062] A, the synthesis schematic diagrams of tFNA and ORT are shown. (i) Four ss DNAs are assembled into tFNA in one step through a high-temperature annealing process. (ii) The S4 strand is truncated into S4 - 1, S4 - 2, and S4 - 3. Mix S1, S2, S3, S4 - 1, S4 - 2, and S4 - 3, and synthesize ORT in one step according to the same synthesis process as tFNA. (iii) First, synthesize a tetrahedral framework composed of S1, S2, and S3 by high-temperature annealing, denoted as S123. Subsequently, incubate S4 - 1, S4 - 2, S4 - 3 with S123 at room temperature to prepare ORT. Figure 1Shown in B are the results of two synthetic methods for AGE verification of ORT, indicating the successful synthesis of ORT by the one-pot method and the two-step method; as Figure 1 Shown in C are the PAGE results of different nanomaterials in the steps of synthesizing ORT by the one-pot method, indicating the successful synthesis of ORT. As Figure 1 Shown in D, the particle sizes of tFNA and ORT are 10.7 nm and 9.8 nm respectively. The ζ potentials of tFNA and ORT are -4.53 mV and -4.43 mV respectively. As Figure 1 Shown in E, tFNA and ORT present uniformly sized nanoparticles under the transmission electron microscope, with particle sizes of approximately 10 - 20 nm. As Figure 1 Shown in F, tFNA and ORT show a tetrahedral structure similar to a triangle under the atomic force microscope, with a particle size of approximately 20 nm.

[0063] Experimental Example 2 Stability Test of ORT

[0064] I. Stability Test in DNA Enzymes with Different Concentrations

[0065] (I) Experimental Method

[0066] Take Cy5 and FITC double-labeled ORT and tFNA and mix them with DNA enzymes at different concentrations (0, 0.1, 0.5, 1, 2, 4, 8, 16 U / ml), incubate them at 37 °C for 1 hour, and take all samples for AGE.

[0067] (II) Experimental Results

[0068] As Figure 2 Shown in A is a schematic diagram of the stability test of ORT and tFNA. As Figure 2 Shown in B, ORT and tFNA start to undergo a small amount of degradation in 2 U / ml DNA enzyme. When the DNA concentration is increased to 16 U / ml, both nanomaterials are almost completely degraded. As the concentration of DNA enzyme increases, the "tailing" phenomenon of tFNA becomes gradually obvious. However, the degree of "tailing" of ORT is significantly less than that of tFNA, indicating that ORT has better enzyme stability.

[0069] II. Stability Test at Different Incubation Times of DNA Enzyme

[0070] (I) Experimental Method

[0071] Take Cy5 and FITC double-labeled ORT and tFNA and mix them with 1 U / ml DNA enzyme, place them at 37 °C for different times (0, 2, 4, 6, 8, 10, 12, and 24 h), and take all samples for AGE.

[0072] (II) Experimental Results

[0073] AsFigure 2 As shown in Figure C, ORT and tFNA can stably exist for about 10 hours, and a large amount of single-strand dissociation occurs at 24 hours. At 12 hours, the fluorescence of tFNA significantly weakens, while strong fluorescence can be detected for ORT, indicating that ORT can better resist enzymatic degradation.

[0074] III. Stability Test under Storage Conditions

[0075] (I) Experimental Method

[0076] Take Cy5- and FITC-dually labeled ORT and tFNA and place them at 4°C for different times (0, 1, 2, 3, 4, 5, 6, and 7 days), and subject all samples to AGE.

[0077] (II) Experimental Results

[0078] As Figure 2 shown in Figure D, when ORT and tFNA are placed under storage conditions at 4°C, both can stably exist for about 7 days, and the retention rate of ORT is significantly higher than that of tFNA, indicating that ORT is more stable when stored at 4°C.

[0079] IV. Stability Test in 10% Fetal Bovine Serum (FBS)

[0080] (I) Experimental Method

[0081] Take Cy5- and FITC-dually labeled ORT and tFNA and fix them with 10% FBS at 37°C for different times (0, 2, 4, 6, 8, 10, 12, and 24 h), and subject all samples to AGE.

[0082] (II) Experimental Results

[0083] As Figure 2 shown in Figure E, ORT and tFNA can be retained in 10% fetal bovine serum (FBS) at 37°C for about 6 hours, and more nanostructure disintegration occurs at 8 hours. The overall fluorescence intensity of ORT is significantly higher than that of tFNA, indicating that ORT has a more stable structure in 10% fetal bovine serum.

[0084] The above results show that compared with tFNA, the ORT prepared in the present invention has better structural stability.

[0085] Experimental Example 3 Cellular Uptake Test of ORT in Different Cells

[0086] I. Experimental Method

[0087] Fluorescence tracing technique

[0088] a. To study the endocytosis of ORT and tFNA in different cells, Cy5 and FITC were used to co-label ORT and tFNA, and Cy5-S1 and FITC-S4 were selected as the control groups.

[0089] b. Different cell suspensions (RAW 264.7 cells, BMSCs, and 4T1 cells) were seeded in confocal dishes and pre-incubated in the incubator for 24 hours (37 °C, 5% CO2).

[0090] c. Cy5-S1, FITC-S4, Cy5&FITC-tFNA, and Cy5&FITC-ORT with a concentration of 250 nM were added and cultured in the incubator. Each type of cell was treated with the drugs for different times according to the time grouping (37 °C, 5% CO2). RAW 264.7 cells: 6, 12, 18, 24 hours. BMSCs: 3, 6, 12 hours. 4T1 cells: 6, 12 hours.

[0091] d. The culture medium was aspirated and the cells were washed three times with PBS for 5 minutes each time; then fixed with 4 wt% paraformaldehyde for 25 minutes, the paraformaldehyde was aspirated, and the cells were washed three times with PBS for 5 minutes each time; then treated with DAPI for 10 minutes in the dark, the DAPI was aspirated, and the cells were washed three times with PBS for 5 minutes each time; then sealed with 10 wt% glycerol, kept in the dark at 4 °C, and detected by machine.

[0092] II. Experimental Results

[0093] As Figure 3 shown in Figure 3 A, the schematic diagram of the cellular uptake of different nanomaterials in several classical cell types is shown. As Figure 3 shown in Figure 3 B, the cellular uptake of ORT and tFNA by RAW 264.7 cells reached the peak at 18 hours. At 24 hours, the fluorescence intensities of ORT and tFNA detected were very weak, which was consistent with the previously reported half-life of tFNA. The cellular internalization of ORT and tFNA was much higher than that of S1 and S4, indicating that the tetrahedral structure of ORT and tFNA could increase the efficiency of free ssDNA entering the cells. As

[0094] Experimental Example 4 Biological Function Test of ORT

[0095] I. Experimental Methods

[0096] (1) Scratch assay

[0097] a. Seed BMSCs in six-well culture dishes and culture them in an incubator (37 °C, 5% CO2) until the dishes are covered.

[0098] b. Scratch with a sterile pipette tip to form a cross-shaped monolayer cell wound.

[0099] c. Culture HUVEC with 250 nmol / L tFNA or ORT respectively. At the time points of 24 hours and 48 hours, take photos of the scratch wound healing and count them using ImageJ.

[0100] (2) ROS scavenging assay

[0101] a. Seed RAW264.7 cells in six-well plates and culture until the cells adhere to the surface.

[0102] b. Randomly divide the cells into a control group, a lipopolysaccharide (LPS) group, an LPS + tFNA group, and an LPS + ORT group. The latter three groups are pretreated with 1 μg / mL LPS for 2 hours, and then 250 nmol / L TM buffer, ORT, and tFNA are added to the corresponding wells and treated for another 24 hours.

[0103] c. Collect all samples and detect the ROS level using a ROS detection kit. Analyze the captured fluorescence images using ImageJ.

[0104] (3) Experiment for regulating macrophage polarization

[0105] a. Seed RAW264.7 cells in six-well plates and culture until the cells adhere to the surface.

[0106] b. Randomly divide the cells into a control group, an LPS group, an LPS + tFNA group, and an LPS + ORT group. The latter three groups are pretreated with 1 μg / mL LPS for 2 hours, and then 250 nmol / L TM buffer, ORT, and tFNA are added to the corresponding wells and treated for another 24 hours.

[0107] c. Isolate and purify total RNA from the cells in each group using the RNeasy Plus Mini Kit (Tiangen, Nanjing, China), and obtain cDNA by reverse transcription using the SYBR Master Mix (TaKaRa) kit.

[0108] d. Finally, detect the expression levels of target mRNAs (IL-6, IL-1β, TNF-α, iNOS, IL-10) in the ABI QuantStudio 7 system (Thermo Fisher, USA), with GAPDH as the internal reference gene.

[0109] II. Experimental Results

[0110] As Figure 4 shown in A is a schematic diagram of the scratch assay of BMSCs. As Figure 4 shown in B, at 24 hours, good wound healing was already evident in the ORT group and the tFNA group. ORT and tFNA treatments caused the scratched BMSCs monolayer wound to completely close at 48 hours, while there was still a large area without cell migration in the control group. As Figure 4 shown in C and 4D, data analysis of the wound healing rates at two time periods demonstrated that the ORT group and the tFNA group showed good wound healing ability compared with the control group, and there was no significant difference between the ORT group and the tFNA group.

[0111] As Figure 4 shown in F, after LPS treatment, macrophages secreted a large amount of ROS, while ORT and tFNA could scavenge most of the LPS-induced ROS. The fluorescence intensities of ROS in each group were statistically analyzed. As Figure 4 shown in G, the ORT group and the tFNA group could significantly reduce ROS compared with the control group; as Figure 4 shown in E, the gene expression levels of pro-inflammatory factors (IL-6, IL-1β, TNF-α, iNOS) secreted by M1 macrophages and anti-inflammatory factor (IL-10) secreted by M2 macrophages after LPS pretreatment and tFNA and ORT treatments were determined by RT-PCR technology. As Figure 4 shown in G, ORT significantly reduced the expression of pro-inflammatory factors and greatly inhibited LPS-induced M1 polarization. The above results indicate that ORT has anti-inflammatory ability similar to that of tFNA and good wound healing ability.

[0112] Experimental Example 5 Biological Distribution and Pharmacokinetics Test of ORT in Vivo

[0113] I. Experimental Methods

[0114] (1) Ocular Penetration Experiment

[0115] Five-week-old C57 mice were instilled with Cy5&FITC double-labeled ORT and tFNA into the eyes (10 μL, 1 μM) respectively, three times within 24 hours, once every eight hours (3 mice per group). After instillation, the mice were euthanized, and the eyeballs were dissected intact and made into frozen sections, and the distribution of the materials in the eyes was collected using a fluorescence microscope.

[0116] (2) Pharmacokinetics Test of ORT in the Rat Joint Cavity

[0117] Cy5&FITC double-labeled ORT and tFNA (30 μL, 1 μM) were injected into the knee joint cavities of 5-week-old SD rats (3 rats). ORT was injected into the left joint cavity of each rat, and tFNA was injected into the right. The rats were anesthetized, the hair near the knee joints was removed, and the fluorescence intensities of different drugs were detected using a small animal in vivo imaging system at different time points after injection (15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 6 h, 12 h).

[0118] (3) Skin penetration experiment

[0119] The hair on the back skin of 5-week-old C57 mice was completely removed using scissors and depilatory cream. Fluorescently labeled tFNA and ORT (1 μM) were respectively mixed with an equal volume of Aquaphor, evenly applied to the back skin of the mice, and the drugs were fixed using Tegaderm (3 mice per group). The mice were transferred to a completely dark environment for 24 hours. The mice were euthanized, the back skin was dissected, frozen sections were made, and the distribution of fluorescence was detected.

[0120] (4) Biological distribution and metabolism test of ORT in vivo

[0121] Fluorescently labeled ORT and tFNA (100 μL, 1 μM) were respectively injected into C57 mice via the tail vein (3 mice per group). The mice were sacrificed at different time points after injection (30 min, 1 h, 2 h, 4 h), the main organs were dissected, and analyzed using a small animal in vivo imaging system.

[0122] II. Experimental results

[0123] As Figure 5 shown in A, the schematic diagram of the eye drop experiment. As Figure 5 shown in B, after three administrations, strong fluorescence of the material could be detected in the eyeballs. ORT and tFNA were mainly distributed in the posterior segment of the eye, especially in the retina. As Figure 5 shown in C is the schematic diagram of the in vivo drug metabolism test of tFNA and ORT after joint injection. Figure 5 D is the retention of different materials collected by the in vivo imaging system. ORT and tFNA showed similar metabolic rates, the degradation of the materials began at 2 hours, and they remained in the joint cavity for at least 6 hours.

[0124] As Figure 6 shown in A is the schematic diagram of the transdermal experiment. As Figure 6 shown in B, the fluorescence detection results of the skin frozen sections showed that ORT and tFNA could reach the dermis layer where the hair follicles are located and even enter the subcutaneous tissue, both showing good permeability. As Figure 6 shown in C is the schematic diagram of the determination of the biological distribution of tFNA or ORT after intravenous injection.Figure 6 As shown in D, the circulation time of ORT in vivo is about 2 hours, and it is mainly metabolized and cleared by the liver and kidneys. The above results indicate that the metabolic kinetics of ORT and tFNA in vivo are similar, showing a short half-life.

[0125] Experimental Example 6 Preparation of Various Modified Nanomaterials Based on ORT

[0126] I. Synthesis of ORT@siRNA

[0127] (I) Experimental Method

[0128] Design siRNA sequences with a length less than 20 bp. According to the siRNA sequences, modify parts S1, S2, and S3 into sequences paired with siRNA. First, mix the modified S1, S2, S3, and siRNA (molar concentration ratio of 1:1:1:3) and add them to TM buffer, place them at 95 °C for 10 minutes, and then cool to 4 °C and stabilize for 20 min to synthesize ORT@siRNA by one-pot method.

[0129] For the two-step method, first add the modified S1, S2, S3 to TM buffer and mix well, then quickly raise the temperature to 95 °C and stabilize for 10 min, and then cool to 4 °C and stabilize for 20 min to synthesize the tetrahedral framework S123-siRNA. Add siRNA to S123-siRNA (molar concentration ratio of siRNA and S123-siRNA is 3:1), and incubate at room temperature for 10 minutes to synthesize ORT@siRNA. Use AGE to verify the two synthesis methods of ORT@siRNA. Use PAGE to verify in detail the ORT@siRNA synthesized at different siRNA ratios. The oligonucleotide sequences used are shown in Table 2.

[0130] (II) Experimental Results

[0131] As Figure 7 Figure A shows the modification idea of ORT. The first is the attempt to carry different types of oligonucleotides. Based on ORT, replace the truncated S4 with different types of functional oligonucleotides (siRNA, miRNA, ASO, DNA) to make it a DNA tetrahedron that can perform multiple biological functions. According to the base complementary pairing principle, at the same time, some bases of S1, S2, and S3 need to be designed as sequences paired with different functional strands. The side length of the tetrahedron of ORT is 21 bp. Excluding the adenine base at the vertex, the length of the oligonucleotide that ORT can accommodate is only 20 bp, but the length of most functional strands is greater than 20 bp. Therefore, the second modification method is to design an ORT nanostructure that can be used to carry oligonucleotides of different lengths. As Figure 7As shown in Figure B, two synthesis methods of ORT@siRNA were verified by AGE. Lane 1, siRNA. Lane 2, S123 modified to pair with siRNA, named S123-siRNA. Lanes 3-6 were siRNA incubated with S123-siRNA for 5 minutes, 10 minutes, 20 minutes, and 30 minutes at room temperature respectively. Lane 7, ORT@siRNA prepared by the one-pot method. Lane 8, tFNA. The results show that for RNA sequences (siRNA) with poor thermal stability, it is not feasible to synthesize DNA-RNA complexes by the one-pot method. As Figure 7 As shown in Figure C, the successful preparation of ORT@siRNA was detected by PAGE. Lane 1, siRNA. Lane 2, S123-siRNA. Lane 3, S123-siRNA + siRNA, ratio 1:0.5. Lane 4, S123-siRNA + siRNA, ratio 1:1. Lane 5, S123-siRNA + siRNA, ratio 1:2. Lane 6, tFNA. Lane 7, S123-siRNA + siRNA, ratio 1:3, incubated for 5 minutes. Lane 8, S123-siRNA + siRNA, ratio 1:3, incubated for 10 minutes. The PAGE results show that the ORT framework can fully accommodate siRNA with a length less than 20 bp. Incubated at room temperature for 5-10 minutes, all siRNA can bind to ORT, achieving an ideal ratio of framework:functional strand = 1:3. The results show that the synthesized ORT@siRNA has a slightly smaller molecular weight than tFNA, has no aggregation problem, and has a higher yield.

[0132] II. Synthesis of ORT@miRNA

[0133] (I) Experimental method

[0134] Design the miRNA (miR-22) sequence, and modify the S1, S2, and S3 parts according to the miR-22 sequence to sequences that pair with miR-22. For miR-22 with a length greater than 20 bp, design to bind the first 20 bases (base sequence 5'-3') of miR-22 to the edges of the tetrahedral framework of ORT, and the extra two bases are unpaired and free in the framework. First, mix the modified S1, S2, S3, and miR-22 (molar concentration ratio of 1:1:1:3), add them to TM buffer, quickly raise the temperature to 95 °C and stabilize for 10 min, then cool to 4 °C and stabilize for 20 min to synthesize ORT@miR-22 by one-pot method. For the two-step method, first add the modified S1, S2, S3 to TM buffer and mix well, quickly raise the temperature to 95 °C and stabilize for 10 min, then cool to 4 °C and stabilize for 20 min to synthesize the tetrahedral framework S123-miR-22. Add miR-22 to S123-miR-22 and incubate at room temperature for 10 minutes to synthesize ORT@miR-22. Use AGE to verify the two synthesis methods of ORT@miR-22. Use PAGE to verify in detail the ORT@miR-22 synthesized under different miR-22 ratios.

[0135] (II) Experimental Results

[0136] As Figure 7 shown in D, the two synthesis methods of ORT@miR-22 were verified by AGE. Lane 1, miR-22. Lane 2, the modified S123 paired with miR-22, named S123-miR-22. Lanes 3-6 are miR-22 and S123-miR-22 incubated at room temperature for 5 minutes, 10 minutes, 20 minutes, and 30 minutes respectively. Lane 7, ORT@miR-22 prepared by one-pot method. Lane 8, tFNA. The results show that for the RNA sequence (miR-22) with poor thermal stability, the one-step synthesis method is not feasible. As Figure 7As shown in Figure E, the successful preparation of ORT@miR-22 was detected by PAGE. Lane 1, miR-22. Lane 2, S123-miR-22. Lane 3, S123-miR-22 + miR-22, ratio 1:0.5. Lane 4, S123-miR-22 + miR-22, ratio 1:1. Lane 5, S123-miR-22 + miR-22, ratio 1:2. Lane 6, tFNA. Lane 7, S123-miR-22 + miR-22, ratio 1:3, incubated for 5 minutes. Lane 8, S123-miR-22 + miR-22, ratio 1:3, incubated for 10 minutes. The PAGE results verified the successful preparation of ORT@miR-22. Similar to ORT@siRNA, miR-22 bound to ORT quickly and efficiently with almost no by-products.

[0137] III. Synthesis of ORT@ASO

[0138] (I) Experimental method

[0139] Design an ASO sequence with a length less than 20 bp, and modify the S1, S2, and S3 parts according to the ASO sequence to be paired with the ASO. Considering the heat resistance of the ASO sequence, ORT@ASO was synthesized by a one-pot method. First, the modified S1, S2, S3, and ASO (molar concentration ratio 1:1:1:3) were mixed and added to TM buffer, the temperature was quickly raised to 95 °C and stabilized for 10 min, then cooled to 4 °C and stabilized for 20 min, and ORT@ASO was synthesized by a one-pot method, and the results were characterized by PAGE.

[0140] (II) Experimental results

[0141] As Figure 7 shown in Figure F, ORT can load short-length ASO. Lane 1, ASO. Lane 2, the modified S123 paired with ASO, named S123-ASO. Lane 3, S123-ASO + ASO, ratio 1:1. Lane 4, S123-ASO + ASO, ratio 1:2. Lane 5, ORT@ASO. Lane 6, tFNA. The above results indicate that ORT@ASO can be efficiently self-assembled and synthesized by a one-pot method, and the yield is significantly higher than that of tFNA.

[0142] IV. Synthesis of ORT@DNA

[0143] (I) Experimental method

[0144] Verify the possibility of loading oligonucleotides with longer sequences using ORT. Design DNA sequences up to 26 bp in length according to the S1, S2, and S3 sequences of ORT composition, and pair them with S1, S2, and S3 respectively, namely D26-1, D26-2, and D26-3. Fold the 20 bp extra part in the middle of D26 so that it does not pair with ORT, keeping the original size of ORT@D26. Considering the heat resistance of the DNA sequence, ORT@D26 was synthesized by the one-pot method. Mix S1, S2, S3 and D26-1, D26-2, D26-3 (molar concentration ratio of 1:1:1:1:1:1) and add them to TM buffer. Rapidly raise the temperature to 95 °C and stabilize for 10 min, then cool to 4 °C and stabilize for 20 min. Synthesize ORT@D26 by the one-pot method, and the results were characterized by PAGE.

[0145] (III) Experimental Results

[0146] As Figure 7 Figure G shows the successful synthesis of ORT@D26, and its size is similar to that of tFNA. Lane 1, D26-1. Lane 2, S123 modified to pair with ASO, named S123-D26. Lane 3, S123-D26 + D26-1. Lane 4, S123-D26 + D26-1 + D26-2. Lane 5, ORT@D26. Lane 6, tFNA. The results show that although there are a small amount of by-products in ORT@D26, there is no phenomenon similar to the inseparability of tFNA in the loading well, showing a relatively pure product.

[0147] The above results indicate that the ORT prepared by the present invention can load siRNA, miRNA, ASO, and DNA sequences up to 26 bp in length, and has a higher yield.

[0148] Table 2. Base sequences of various ORT-based nanomaterials

[0149]

[0150]

[0151] The ORT prepared by the present invention has a structure and biological function similar to those of traditional tetrahedral framework nucleic acid (tFNA). Compared with tFNA, ORT has better structural stability and yield. Moreover, ORT can load functional oligonucleotides inside the framework to replace the S4 strand, so it has more modification sites and vector potential than tFNA. ORT can be used as a vector to load nucleic acids and nucleic acid analogs, protein drugs, small molecule drugs, functional groups, etc.; the loading methods of ORT include: loading drugs inside through base complementary pairing after sequence modification, or loading through sticky ends, chemical ligation loading, groove binding loading, electroincubation loading, etc. The cargos that can be loaded by ORT are shown in Table 3.

[0152] Table 3. Cargos that can be loaded by ORT

[0153]

[0154] In summary, the present invention constructs a novel DNA tetrahedron ORT by truncating long-chain ssDNA and reducing self-pairing, which improves the mismatch and self-pairing phenomena of tFNA, increases the yield of tFNA, and has good structural stability, high bioavailability and excellent biocompatibility; ORT is expected to be used as a vector to load nucleic acids and nucleic acid analogs, protein drugs, small molecule drugs, functional groups, etc., improve the synthesis yield of tFNA, and promote its applications in drug delivery, gene therapy, biosensing and other aspects.

Claims

1. A DNA tetrahedron capable of carrying oligonucleotides and nucleic acid analogs, characterized in that: The described DNA tetrahedron is assembled by base complementary pairing of three long-chain DNAs, S1, S2, and S3, and at least three short-chain DNAs formed after truncating S4.

2. The DNA tetrahedron according to claim 1, characterized in that: The number of short-chain DNAs formed after truncating S4 is three.

3. The DNA tetrahedron according to claim 2, wherein: The nucleotide sequences of the short-chain DNAs are as shown in SEQ ID NO.4 - SEQ ID NO.

6.

4. The DNA tetrahedron according to claim 1, wherein: The truncating method is to delete the unpaired bases that form the vertices of the nucleic acid framework of the tetrahedron in S4.

5. The DNA tetrahedron according to claim 4, wherein: The unpaired base is adenine.

6. The DNA tetrahedron according to claim 1, characterized in that: The nucleotide sequences of S1, S2, and S3 are as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively.

7. The preparation method of the DNA tetrahedron according to any one of claims 1-6, characterized in that, It is prepared according to one of the following steps: Step a: Mix S1, S2, S3, and at least three short-chain DNAs, and incubate to obtain. Or, step b: Mix S1, S2, and S3, incubate, then add at least three short-chain DNAs and mix, and incubate to obtain.

8. Use of the DNA tetrahedron according to any one of claims 1 - 6 as a carrier.

9. The use according to claim 8, characterized in that: The carrier can load at least one of nucleic acids and nucleic acid analogs, protein drugs, small molecule drugs, and functional groups.

10. The use according to claim 9, wherein: The nucleic acids and nucleic acid analogs are selected from at least one of siRNA, miRNA, saRNA, ASO, aptamer, DNA, LNA, PNA, CpG; and / or, the protein drugs are selected from small molecule polypeptides; and / or, the small molecule drugs are selected from at least one of chemotherapeutic drugs, natural product drugs, and antibiotics.

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