Neuron targeting nano-carrier system based on DNA origami

Abstract

The invention relates to a DNA origami-based intracellular delivery platform for precisely targeting motor neurons, a DNA origami structure (BioDoori) comprises M13mp18 single-stranded DNA, biotin chains (biotin-DNA), fluorescent chains (fluorophore-DNA) and unmodified staple chains, each side of the DNA origami structure is loaded with 1-30 fluorescent chains and 1-10 biotin chains, and the biotin chains are loaded with 1-30 fluorescent groups and 1-10 fluorescent groups. The nucleotide sequence of the staple chain is as shown in SEQ ID NO: 1-208. According to the nano origami structure disclosed by the invention, accurate and efficient specific targeting aiming at motor neuron cells is realized, and lysosome escape efficacy of a DNA origami carrier is improved in a breakthrough manner.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a neuronal targeted intracellular delivery system based on DNA origami technology and its preparation method. Background Technology

[0002] Intracellular delivery is a challenging aspect of disease treatment, its importance stemming from the unique physiological properties of cells—multiple barriers, including cell membranes and intracellular lysosomal degradation, must be overcome to allow drugs to precisely reach their intracellular targets. Intracellular delivery to neurons is particularly difficult due to their structural complexity. A fundamental challenge in treating botulinum toxin poisoning lies in the difficulty of delivering antibody drugs into the cytoplasm of motor neurons. While engineered toxins have been reported as drug delivery carriers to deliver antibody drugs and block the toxicity of intracellular botulinum toxin, high doses pose safety risks. Therefore, there is an urgent need to develop neuronal-targeted intracellular delivery carriers with excellent biosafety and high efficiency in delivering antibody drugs.

[0003] To address the need for intracellular delivery, various nanomaterial carriers have been reported, such as liposomes, exosomes, PLGA nanoparticles, and gold nanoparticles. However, these carriers have problems such as difficulty in precisely regulating cell targeting, and are insufficient to accurately match the specific delivery needs of neurons.

[0004] DNA origami nanostructures represent an emerging delivery platform with advantages such as high biocompatibility, nanometer-precision addressability, and programmability. In recent years, drug delivery systems based on DNA origami have seen rapid development. However, DNA origami nanostructures lack the ability to target motor neurons, and their lysosomal escape efficiency is low. Therefore, developing a precise, highly lysosomal-escape-efficient intracellular delivery system based on DNA origami neurons is a key problem that this patent aims to solve. Summary of the Invention

[0005] The purpose of this invention is to provide a DNA origami nanostructure (BioDori), which comprises M13mp18 single-stranded DNA, a biotin chain (biotin-DNA), a fluorescent chain (fluorescent group-DNA), and unmodified staple chains. Each side of the DNA origami structure is loaded with 1-30 fluorescent chains and 1-10 biotin chains.

[0006] The nucleotide sequence of the staple chain is shown in SEQ ID NO: 1-208.

[0007] In a preferred embodiment of the present invention, the nucleotide sequence of the staple chain is arranged as shown in SEQ ID NO: 1-208.

[0008] In a preferred embodiment of the present invention, each side of the DNA origami structure is loaded with 1-10 fluorescent chains, preferably 1-5 fluorescent chains.

[0009] In a preferred embodiment of the present invention, the fluorescent chain is a fluorescent group modified on a staple chain.

[0010] In a preferred embodiment of the present invention, the nucleotide sequence of the fluorescent chain is any one or a combination of A30, A61, B30, B61, C30, and C61 in the staple chain, and the 5' end of the DNA sequence is modified with a fluorescent group.

[0011] In a preferred embodiment of the present invention, the fluorescent group is selected from any one or a combination of Cy3, Cy5, and Cy5.5.

[0012] In a preferred embodiment of the present invention, the biotin chain is biotin modified on a staple chain.

[0013] In a preferred embodiment of the present invention, the nucleotide sequence of the biotin chain is that of the staple chain.

[0014] Any one or a combination of A04, A12, A20, A26, A41, A49, A56, B04, B12, B20, B26, B41, B49, B56, C04, C12, C20, C26, C41, C49, C56, first modify TTTT at the 5' end of the DNA sequence, and then modify biotin at the 5' end.

[0015] In a preferred embodiment of the present invention, the nucleotide sequence of the biotin chain is A04, A12, A41, B04, B12, B41, CO4, C12 and C41 in the staple chain, and TTTT is first modified at the 5' end of the DNA sequence, and then biotin is modified at the 5' end.

[0016] In a preferred embodiment of the present invention, the nucleotide sequence of the biotin chain is A04, A12, A20, A41, A49, B04, B12, B20, B41, B49, CO4, C12, C20, C41, and C49 in the staple chain. TTTT is first modified at the 5' end of the DNA sequence, and then biotin is modified at the 5' end.

[0017] In a preferred embodiment of the present invention, the nucleotide sequence of the biotin chain is A04, A12, A20, A26, A41, A49, A56, B04, B12, B20, B26, B41, B49, B56, CO4, C12, C20, C26, C41, C49, and C56 in the staple chain. TTTT is first modified at the 5' end of the DNA sequence, and then biotin is modified at the 5' end.

[0018] In a preferred embodiment of the present invention, the nucleotide sequence of the biotin chain is shown in SEQ ID NO: 209-229.

[0019] In a preferred embodiment of the present invention, the biotin is selected from any one or a combination of NHS-PEG2-Biotin, NHS-PEG4-Biotin, NHS-PEG8-Biotin, and NHS-PEG12-Biotin.

[0020] In a preferred embodiment of the present invention, the nucleotide sequence of the unrevised staple chain in the DNA origami nanostructure is as shown in SEQ ID NO: 1-208, wherein the staple chain used to prepare the fluorescent chain and biotin chain is removed.

[0021] In a preferred embodiment of the present invention, the site spacing between the two biotin chains is 5-30 nm, preferably 5-20 nm, and more preferably 15-20 nm.

[0022] In a preferred embodiment of the present invention, the DNA origami nanostructure is any one of a triangle, a quadrilateral, or a rectangle.

[0023] In a preferred embodiment of the present invention, the length of each side of the DNA origami nanostructure is 60-180 nm, preferably 80-160 nm, and more preferably 100-120 nm.

[0024] In a preferred embodiment of the present invention, the DNA origami nanostructure is loaded with 1-5 Cy5-DNA strands on each side, preferably 1, 2, 3, 4, or 5 strands on each side.

[0025] In a preferred embodiment of the present invention, the DNA origami nanostructure is loaded with 3-7 biotin chains on each side, preferably 3, 4, 5, 6, or 7 biotin chains on each side.

[0026] Another objective of this invention is to provide a method for preparing a DNA origami nanostructure (BioDori), comprising the following steps: adding M13mp18 single-stranded DNA, biotin chain, fluorescent chain, and unmodified staple chain to a buffer system, mixing at room temperature according to a final concentration ratio of 1:5-20:5-20:5-20 for M13mp18 single-stranded DNA, biotin chain, fluorescent chain, and unmodified staple chain in the buffer system, and synthesizing the product at a programmed temperature of 95°C, and ultrafiltration through a 100kDa ultrafiltration tube to obtain the DNA origami body (BioDori).

[0027] In a preferred embodiment of the present invention, the final concentration ratio of the M13mp18 single-stranded DNA, biotin chain, fluorescent chain, and unmodified staple chain is 1:10-15:10-15:10-15.

[0028] In a preferred embodiment of the present invention, the nucleotide sequence of the unmodified staple chain in the DNA origami nanostructure is as shown in SEQ ID NO: 1-208, with the staple chain used to prepare the fluorescent chain and biotin chain removed.

[0029] In a preferred embodiment of the present invention, the fluorescent chain is a fluorescent group modified on a staple chain.

[0030] In a preferred embodiment of the present invention, the nucleotide sequence of the fluorescent chain is any one or a combination of A30, A61, B30, B61, C30, and C61 in the staple chain, and the 5' end of the DNA sequence is modified with a fluorescent group.

[0031] In a preferred embodiment of the present invention, the fluorescent group is selected from any one or a combination of Cy3, Cy5, and Cy5.5.

[0032] In a preferred embodiment of the present invention, the biotin chain is biotin modified on a staple chain.

[0033] In a preferred embodiment of the present invention, the nucleotide sequence of the biotin chain is that of the staple chain.

[0034] Any one or a combination of A04, A12, A20, A26, A41, A49, A56, B04, B12, B20, B26, B41, B49, B56, C04, C12, C20, C26, C41, C49, C56, first modify TTTT at the 5' end of the DNA sequence, and then modify biotin at the 5' end.

[0035] The nucleotide sequence of the biotin chain is shown in SEQ ID NO: 209-229.

[0036] In a preferred embodiment of the present invention, the biotin is selected from any one or a combination of NHS-PEG2-Biotin, NHS-PEG4-Biotin, NHS-PEG8-Biotin, and NHS-PEG12-Biotin.

[0037] In a preferred embodiment of the present invention, the programmed temperature control condition is that the temperature drops from 95°C to 25°C in 2.5 hours.

[0038] In a preferred embodiment of the present invention, the buffer solution system is 1×TAE-Mg 2+ Buffer system.

[0039] In a preferred embodiment of the present invention, the 1×TAE-Mg 2+The buffer system contains 40 mM Tris base, 20 mM acetic acid, 2 mM EDTA, 12.5 mM magnesium acetate, pH=8.0, and the remainder is water.

[0040] In a preferred embodiment of the present invention, the ultrafiltration conditions are 1×TAE-Mg. 2+ In a buffer system, ultrafiltration at 3000-10000g for 5-10 minutes.

[0041] Another object of the present invention is to provide a delivery system (BioDoriH) targeting motor neuron cells, comprising a DNA origami nanostructure (BioDori) and a targeting ligand immobilized on the biotin chain; wherein the targeting ligand, after being biotinylated, binds to the biotin chain via the action of streptavidin.

[0042] In a preferred embodiment of the present invention, the targeting ligand is GenBank:CS401852.1.

[0043] Another object of the present invention is to provide a method for preparing a delivery system for targeted motor neuron cells (BioDoriH), comprising the following steps:

[0044] (1) The targeting ligand and biotin reagent were mixed at a molar ratio of 1:50-100, reacted at room temperature for 30-60 min, and then purified by ultrafiltration at 10kDa-20kDa to obtain the biotinylated targeting ligand (Biotin-Hc / A).

[0045] (2) DNA origami nanostructure (BioDori) was mixed with streptavidin (SA) at a molar ratio of 1:5-10n and reacted at room temperature for 30-60 min to obtain SA-bound biotinylated DNA origami (SA-BioDori).

[0046] (3) The SA-binding biotinylated DNA origami (SA-BioDori) from step (2) is mixed with the biotinylated targeting ligand (Biotin-Hc / A) from step (1) at a molar ratio of 1:10-20n and reacted at room temperature for 30-60 min to obtain the Biotin-Hc / A-linked SA-BioDori (BioDoriH).

[0047] In a preferred embodiment of the present invention, in step (1), the biotin reagent is any one of NHS-PEG2-Biotin, NHS-PEG4-Biotin, NHS-PEG8-Biotin, or NHS-PEG12-Biotin.

[0048] In the preferred embodiment of the present invention, in step (1), the targeting ligand is GenBank:CS401852.1.

[0049] In the preferred embodiment of the present invention, in steps (1) and (2), n is the total number of biotin chains on the DNA origami structure.

[0050] In a preferred embodiment of the present invention, in step (1), the method for preparing the targeting ligand is as follows:

[0051] (1) Search for the Hc / A gene (sequence number: CS401852.1) of the receptor-binding domain of botulinum toxin type A on the NCBI website, link the 6×His tag to the N end of the gene sequence, clone the designed gene sequence into the pET28a (+) vector, and obtain the plasmid.

[0052] (2) The prepared plasmid was transfected into Escherichia coli BL21(DE3) to construct the strain. The strain was cultured in LB liquid medium containing kanamycin. After low temperature induction, the strain was centrifuged at 4℃ and 8000 g for 10 min, the supernatant was discarded and the precipitate was collected. The bacterial cells were collected by centrifugation and lysed by sonication to obtain the lysate.

[0053] (3) The lysis buffer was passed through a selective nickel column protein purification column to obtain the targeted ligand.

[0054] Another objective of this invention is to provide a semi-i-motif modified DNA origami nanostructure (MDori), wherein the i-motif modified DNA origami nanostructure comprises M13mp18 single-stranded DNA, a biotin chain (biotin-DNA), a fluorescent chain (fluorescent group-DNA), a semi-i-motif modified chain (semi-i-motif-DNA), and an unmodified staple chain, wherein each side of the DNA origami body is loaded with 1-30 fluorescent chains, 1-10 biotin chains, and 1-10 semi-i-motif modified chains;

[0055] The nucleotide sequence of the staple chain is shown in SEQ ID NO: 1-208.

[0056] In a preferred embodiment of the present invention, the nucleotide sequence of the staple chain is arranged as shown in SEQ ID NO: 1-208.

[0057] In a preferred embodiment of the present invention, each side of the DNA origami structure is loaded with 1-10 fluorescent chains, preferably 1-5 fluorescent chains.

[0058] In a preferred embodiment of the present invention, the fluorescent chain is a fluorescent group modified on a staple chain.

[0059] In a preferred embodiment of the present invention, the nucleotide sequence of the fluorescent chain is any one or a combination of A30, A61, B30, B61, C30, and C61 in the staple chain, and the 5' end of the DNA sequence is modified with a fluorescent group.

[0060] In a preferred embodiment of the present invention, the fluorescent group is selected from any one or a combination of Cy3, Cy5, and Cy5.5.

[0061] In a preferred embodiment of the present invention, the biotin chain is biotin modified on a staple chain.

[0062] In a preferred embodiment of the present invention, the nucleotide sequence of the biotin chain is any one or a combination of A04, A12, A20, A26, A41, A49, A56, B04, B12, B20, B26, B41, B49, B56, CO4, C12, C20, C26, C41, C49, and C56 in the staple chain, wherein TTTT is first modified at the 5' end of the DNA sequence, and then biotin is modified at the 5' end.

[0063] The nucleotide sequence of the biotin chain is shown in SEQ ID NO: 209-229.

[0064] In a preferred embodiment of the present invention, the semi-i-motif modification chain is a semi-i-motif modification on the staple chain.

[0065] In a preferred embodiment of the present invention, the nucleotide sequence of the semi-i-motif modified strand (semi-i-motif-DNA) is any one or a combination of A32, B32, and C32 in the staple strand, with the 3' end of the DNA sequence modified with AAAAAAAAAAAACCCCTAACCCC; or any one or a combination of A65, B65, and C65, with the 5' end of the DNA sequence modified with CCCCTAACCCCAAAAAAAAAAAA.

[0066] In a preferred embodiment of the present invention, the nucleotide sequence of the semi-i-motif modified strand (semi-i-motif-DNA) is shown in SEQ ID NO: 230-235.

[0067] In a preferred embodiment of the present invention, each side of the DNA origami structure is loaded with 1-5 semi-i-motif modification strands, preferably 2-3 semi-i-motif modification strands.

[0068] In a preferred embodiment of the present invention, at least one of the semi-i-motif modification chains is located near the vertex of the origami structure, preferably two semi-i-motif modification chains on each side are located near the vertex.

[0069] In a preferred embodiment of the present invention, the nucleotide sequence of the unrevised staple chain in the DNA origami nanostructure is as shown in SEQ ID NO: 1-208, wherein the staple chain used to prepare the fluorescent chain and biotin chain is removed.

[0070] In a preferred embodiment of the present invention, the sites between the two Cy5-DNA molecules are spaced 50-100 nm apart, preferably 60-80 nm apart.

[0071] In a preferred embodiment of the present invention, the sites between the two Biotin-DNAs are spaced 5-30 nm apart, preferably 5-20 nm apart, and more preferably 15-20 nm apart.

[0072] In a preferred embodiment of the present invention, the DNA origami nanostructure is any one of a triangle, a quadrilateral, or a rectangle.

[0073] In a preferred embodiment of the present invention, the sites between the two semi-i-motif-DNAs are spaced 60-100 nm apart.

[0074] In a preferred embodiment of the present invention, the length of each side of the DNA origami nanostructure is 60-80 nm, preferably 80-160 nm, and more preferably 100-120 nm.

[0075] In a preferred embodiment of the present invention, the DNA origami nanostructure is loaded with 1-5 Cy5-DNA strands on each side, preferably 1, 2, 3, 4, or 5 strands on each side.

[0076] In a preferred embodiment of the present invention, the DNA origami nanostructure is loaded with 3-7 biotin chains on each side, preferably 3, 4, 5, 6, or 7 biotin chains on each side.

[0077] In a preferred embodiment of the present invention, the DNA origami nanostructure is loaded with 1-5 semi-i-motif-DNA strands on each side, preferably 1, 2, 3, 4, or 5 strands on each side.

[0078] Another objective of this invention is to provide a method for preparing a semi-i-motif modified DNA origami nanostructure (MDori), comprising the following steps: adding M13mp18 single-stranded DNA, biotin chain (biotin-DNA), fluorescent chain (fluorescent group-DNA), semi-i-motif-DNA, and unmodified staple chain to a buffer system; mixing the M13mp18 single-stranded DNA, biotin chain (biotin-DNA), fluorescent chain (fluorescent group-DNA), semi-i-motif-DNA, and unmodified staple chain in the buffer system at a final concentration ratio of 1:5-20:5-20:5-20:5-20 at room temperature; synthesizing the product under programmed temperature control; and ultrafiltration through a 100kDa-200kDa ultrafiltration tube to obtain the DNA origami nanostructure body (MDori).

[0079] In a preferred embodiment of the present invention, the molar ratio of the M13mp18 single-stranded DNA, biotin chain (biotin-DNA), fluorescent chain (fluorescent group-DNA), semi-i-motif-DNA, and unmodified staple chain is 1:10-15:10-15:10-15:10-15.

[0080] In a preferred embodiment of the present invention, the programmed temperature control condition is that the temperature drops from 95°C to 25°C in 2.5 hours.

[0081] In a preferred embodiment of the present invention, the buffer solution system is 1×TAE-Mg 2+ Buffer system.

[0082] In a preferred embodiment of the present invention, the 1×TAE-Mg 2+ The buffer system contains 40 mM Tris base, 20 mM acetic acid, 2 mM EDTA, 12.5 mM magnesium acetate, pH=8.0, and the remainder is water.

[0083] In a preferred embodiment of the present invention, the ultrafiltration conditions are 1×TAE-Mg. 2+ In a buffer system, ultrafiltration at 3000-10000g for 5-10 minutes.

[0084] Another object of the present invention is to provide a semi-i-motif modified delivery system for targeting motor neuron cells (MDoriH), comprising a semi-i-motif modified biotinylated DNA origami structure (MDori) of the present invention and a targeting ligand fixed on the structure by a biotinylated chain using SA; wherein the targeting ligand binds to the SA after being biotinylated.

[0085] In a preferred embodiment of the present invention, the targeting ligand is GenBank:CS401852.1.

[0086] Another objective of this invention is to provide a method for preparing a semi-i-motif modified delivery system for targeting motor neuron cells (MDoriH), wherein the DNA origami structure comprises M13mp18 single-stranded DNA, a biotin chain (biotin-DNA), a fluorescent chain (fluorescent group-DNA), and a staple chain, wherein each side of the DNA origami structure is loaded with 1-30 fluorescent chains and 1-10 biotin chains;

[0087] Includes the following steps:

[0088] (1) The targeting ligand and biotin reagent were mixed at a molar ratio of 1:50-100, reacted at room temperature for 30-60 min, and then purified by ultrafiltration at 10 kDa to obtain the biotinylated targeting ligand.

[0089] (2) The biotinylated DNA origami structure modified by semi-i-motif (MDori) was mixed with streptavidin (SA) at a molar ratio of 1:5-10n and reacted at room temperature for 30-60 min to obtain the SA-modified DNA origami drug delivery system (SA-MDori), where n is the total number of biotin chains on the DNA origami structure.

[0090] (3) The SA-modified DNA origami drug delivery system (SA-MDori) from step (2) is mixed with the biotinylated targeting ligand from step (1) at a molar ratio of 1:10-20n and reacted at room temperature for 30-60 min to obtain the semi-i-motif modified delivery system for targeting motor neuron cells (MDoriH).

[0091] In a preferred embodiment of the present invention, in step (1), the biotin reagent is any one of NHS-PEG2-Biotin, NHS-PEG4-Biotin, NHS-PEG8-Biotin or NHS-PEG12-Biotin.

[0092] In the preferred embodiment of the present invention, in step (1), the targeting ligand is GenBank:CS401852.1.

[0093] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0094] 1. This invention employs DNA origami technology, which, with its high programmability, can precisely construct pre-defined nanocarrier structures (DNA origami nanostructures, DON). It can not only enhance targeting by modifying the carrier surface with neuron-specific ligands, but also flexibly control the site and quantity of targeting ligands by leveraging its unique addressability advantage, thereby more accurately identifying neurons. This effectively compensates for the shortcomings of traditional nanocarriers in terms of targeting ability, providing a new pathway for the precise delivery of intracellular neuronal therapy.

[0095] 2. This invention focuses on the challenge of precisely targeting neurons and has successfully developed a targeting system based on a DNA origami platform for precise intracellular targeting of motor neurons. This invention successfully constructs a DNA origami delivery vector that combines targeting and escape capabilities, achieving highly specific recognition and efficient intracellular delivery of diseased neurons. It enables precise localization, effectively improves the lysosomal escape efficiency of the vector, allows for precise control of drug loading, and possesses excellent biocompatibility and biosafety. Attached Figure Description

[0096] Figure 1 BioDori design diagrams, a is BioDori-3, b is BioDori-5, c is BioDori-7;

[0097] Figure 2 AFM characterization diagram of BioDoriH-3 prepared stepwise;

[0098] Figure 3 AFM characterization diagram of BioDoriH-5 prepared stepwise;

[0099] Figure 4 AFM characterization diagram of BioDoriH-7 prepared stepwise;

[0100] Figure 5 AFM characterization of MDori at different pH values;

[0101] Figure 6 A study on the uptake of BioDoriH by neuronal cells using flow cytometry.

[0102] Figure 7 Laser confocal microscopy analysis of the lysosomal acid-neutralizing properties of MDoriH;

[0103] Figure 8 Laser confocal microscopy analysis of MDoriH's lysosomal escape performance. Detailed Implementation

[0104] The present invention will be described below with reference to the embodiments. However, the present invention is not limited to the embodiments.

[0105] Example 1: Preparation and characterization of biotinylated DNA origami nanostructures (BioDori)

[0106] 1. Preparation of biotinylated DNA origami nanostructures (BioDori)

[0107] (1) The triangular DNA origami was synthesized from 208 staple chains and 1 long single strand. The 208 staple chains are shown in Table 1. Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize 208 DNA chains.

[0108] Table 1 208 staple chains

[0109] Serial number Name Sequence (5' to 3') SEQ ID NO.1 A01 CGGGGTTTCCTCAAGAGAAGGATTTTGAATTA SEQ ID NO.2 A02 AGCGTCATGTCTCTGAATTTACCGACTACCTT SEQ ID NO.3 A03 TTCATAATCCCCTTATTAGCGTTTTTCTTACC SEQ ID NO.4 A04 ATGGTTTATGTCACAATCAATAGATATTAAAC SEQ ID NO.5 A05 TTTGATGATTAAGAGGCTGAGACTTGCTCAGTACCAGGCG SEQ ID NO.6 A06 CCGGAACCCAGAATGGAAAGCGCAACATGGCT SEQ ID NO.7 A07 AAAGACAACATTTTCGGTCATAGCCAAAATCA SEQ ID NO.8 A08 GACGGGAGAATTAACTCGGAATAAGTTTATTTCCAGCGCC SEQ ID NO.9 A09 GATAAGTGCCGTCGAGCTGAAACATGAAAGTATACAGGAG SEQ ID NO.10 A10 TGTACTGGAAATCCTCATTAAAGCAGAGCCAC SEQ ID NO.11 A11 CACCGGAAAGCGCGTTTTCATCGGAAGGGCGA SEQ ID NO.12 A12 CATTCAACAAACGCAAAGACACCAGAACACCCTGAACAAA SEQ ID NO.13 A13 TTTAACGGTTCGGAACCTATTATTAGGGTTGATATAAGTA SEQ ID NO.14 A14 CTCAGAGCATATTCACAAACAAATTAATAAGT SEQ ID NO.15 A15 GGAGGGAATTTAGCGTCAGACTGTCCGCCTCC SEQ ID NO.16 A16 GTCAGAGGGTAATTGATGGCAACATATAAAAGCGATTGAG SEQ ID NO.17 A17 TAGCCCGGAATAGGTGAATGCCCCCTGCCTATGGTCAGTG SEQ ID NO.18 A18 CCTTGAGTCAGACGATTGGCCTTGCGCCACCC SEQ ID NO.19 A19 TCAGAACCCAGAATCAAGTTTGCCGGTAAATA SEQ ID NO.20 A20 TTGACGGAAATACATACATAAAGGGCGCTAATATCAGAGA SEQ ID NO.21 A21 CAGAGCCAGGAGGTTGAGGCAGGTAACAGTGCCCG SEQ ID NO.22 A22 ATTAAAGGCCGTAATCAGTAGCGAGCCACCCT SEQ ID NO.23 A23 RELATIONSHIPSAGATGTTAGAACCTAAATATTC SEQ ID NO.24 A24 GCCGCCAGCATTGACACCACCCTC SEQ ID NO.25 A25 AGAGCCGCACCATCGATAGCAGCATGATTAT SEQ ID NO.26 A26 CACCGTCACCTTATTACGCAGTATTGAGTTAAGCCCAATA SEQ ID NO.27 A27 AGCCATTAACGTCACCAATGAACACCAACCA SEQ ID NO.28 A28 ATAAGAAACATGGCATGATTAGCTCCGACTTG SEQ ID NO.29 A29 CCATAGCAAGGCCGGGGGAATTA SEQ ID NO.30 A30 GAGCCAGCGAATACCCAAAAGAACATGAAATAGCAATAGC SEQ ID NO.31 A31 TATCTTACCGAAGCCCAAACGCATATATAACGAAAATCACCAG SEQ ID NO.32 A32 EXPANDACCAGGTTTTTAAAGAAAGTAAGTAGCCG SEQ ID NO.33 A33 CCTTTTCATTCAACAATTTCATAGTTAG SEQ ID NO.34 A34 TTTAACCTATCATAGGTCTGAGAGTTCCAGTA SEQ ID NO.35 A35 AGTATAAAATATGCGTTATACAAAGCCATCTT SEQ ID NO.36 A36 CAAGTACCTCATTCCAAGAACGGGAAATTCAT SEQ ID NO.37 A37 CONTINUATIONAAAAACAGGGAGACONTINUE SEQ ID NO.38 A38 AAAAAAAAATTAATTAATGGAAAATTACKATTAGGAAT SEQ ID NO.39 A39 TTATCAAACCGGCTTAGGTTGGGTAAGCCTGT SEQ ID NO.40 A40 TTAGTATCGCCAACGCTCAACAGGTCGGCTGTC SEQ ID NO.41 A41 TTTCCTTAGCACTCATCGAGAACAATAGCAGCCTTTACAG SEQ ID NO.42 A42 AGAGTCAAAAATCAATATATGTGATGAAACAAACATCAAG SEQ ID NO.43 A43 ACTAGAAATATATAACTATATGTACGCTGAGA SEQ ID NO.44 A44 TCAATAATAGGGCTTAATTGAGAATCATAATT SEQ ID NO.45 A45 AACGTCAAAAATGAAAAGCAAGCCGTTTTTATGAAACCAA SEQ ID NO.46 A46 GAGCAAAAGAAGATGAGTGAATAACCTTGCTTATAGCTTA SEQ ID NO.47 A47 GATTAAGAAATGCTGATGCAAATCAGAATAAA SEQ ID NO.48 A48 CACCGGAATCGCCATATTTAACAAAATTTACG SEQ ID NO.49 A49 AGCATGTATTTCATCGTAGGAATCAAACGATTTTTTGTTT SEQ ID NO.50 A50 ACATAGCGCTGTAAATCGTCGCTATTCATTTCAATTACCT SEQ ID NO.51 A51 GTTAAATACAATCGCAAGACAAAGCCTTGAAA SEQ ID NO.52 A52 CCCATCCTCGCCAACATGTAATTTAATAAGGC SEQ ID NO.53 A53 TCCCAATCCAAATAAGATTACCGCGCCCAATAAATAATAT SEQ ID NO.54 A54 TCCCTTAGAATAACGCGAGAAAACTTTTACCGACC SEQ ID NO.55 A55 GTGTGATAAGGCAGAGGCATTTTCAGTCCTGA SEQ ID NO.56 A56 ACAAGAAAGCAAGCAAATCAGATAACAGCCATATTATTTA SEQ ID NO.57 A57 GTTTGAAATTCAAATATATTTTAG SEQ ID NO.58 A58 AATAGATAGAGCCAGTAATAAGAGATTTAATG SEQ ID NO.59 A59 GCCAGTTACAAAATAATAGAAGGCTTATCCGGTTATCAAC SEQ ID NO.60 A60 TTCTGACCTAAAATATAAAGTACCGACTGCAGAAC SEQ ID NO.61 A61 GCGCCTGTTATTCTAAGAACGCGATTCCAGAGCCTAATTT SEQ ID NO.62 A62 TCAGCTAAAAAAGGTAAAGTAATT SEQ ID NO.63 A63 ACGCTAACGAGCGTCTGGCGTTTTAGCGAACCCAACATGT SEQ ID NO.64 A64 ACGACAATAAATCCCGACTTGCGGGAGATCCTGAATCTTACCA SEQ ID NO.65 A65 TGCTATTTTGCACCCAGCTACAATTTTGTTTTGAAGCCTTAAA SEQ ID NO.66 B01 TCATATGTGTAATCGTAAAACTAGTCATTTTC SEQ ID NO.67 B02 GTGAGAAAATGTGTAGGTAAAGATACAACTTT SEQ ID NO.68 B03 GGCATCAAATTTGGGGCGCGAGCTAGTTAAAG SEQ ID NO.69 B04 TTCGAGCTAAGACTTCAAATATCGGGAACGAG SEQ ID NO.70 B05 ACAGTCAAAGAGAATCGATGAACGACCCCGGTTGATAATC SEQ ID NO.71 B06 ATAGTAGTATGCAATGCCTGAGTAGGCCGGAG SEQ ID NO.72 B07 AACCAGACGTTTAGCTATATTTTCTTCTACTA SEQ ID NO.73 B08 GAATACCACATTCAACTTAAGAGGAAGCCCGATCAAAGCG SEQ ID NO.74 B09 AGAAAAGCCCCAAAAAGAGTCTGGAGCAAACAATCACCAT SEQ ID NO.75 B10 CAATATGACCCTCATATATTTTAAAGCATTAA SEQ ID NO.76 B11 CATCCAATAAATGGTCAATAACCTCGGAAGCA SEQ ID NO.77 B12 AACTCCAAGATTGCATCAAAAAGATAATGCAGATACATAA SEQ ID NO.78 B13 CGTTCTAGTCAGGTCATTGCCTGACAGGAAGATTGTATAA SEQ ID NO.79 B14 CAGGCAAGATAAAAATTTTTAGAATATTCAAC SEQ ID NO.80 B15 GATTAGAGATTAGATACATTTCGCAAATCATA SEQ ID NO.81 B16 CGCCAAAAGGAATTACAGTCAGAAGCAAAGCGCAGGTCAG SEQ ID NO.82 B17 GCAAATATTTAAATTGAGATCTACAAAGGCTACTGATAAA SEQ ID NO.83 B18 TTAATGCCTTATTTCAACGCAAGGGCAAAGAA SEQ ID NO.84 B19 TTAGCAAATAGATTTAGTTTGACCAGTACCTT SEQ ID NO.85 B20 TAATTGCTTTACCCTGACTATTTGAGGCATAGTAAGAGC SEQ ID NO.86 B21 ATAAAGCCTTTGCGGGAGAAGCCCTGGAGAGGGTAG SEQ ID NO.87 B22 TAAGAGGTCAATTCTGCGAACGAGATTAAGCA SEQ ID NO.88 B23 AACACTATCATAACCCATCAAAATCAGGTCTCCTTTTGA SEQ ID NO.89 B24 ATGACCCTGTAATACTTCAGAGCA SEQ ID NO.90 B25 TAAAGCTATATAACAGTTGATTCCCATTTTTG SEQ ID NO.91 B26 CGGATGGCACGAGAATGACCATAATCGTTTACCAGACGAC SEQ ID NO.92 B27 TAATTGCTTGGAAGTTTCATTCCAAAATCGGTTGTA SEQ ID NO.93 B28 GATAAAACCAAAATATTAAACAGTTCAGAAATTAGCT SEQ ID NO.94 B29 ACTAAAGTACGGTGTCGAATATAA SEQ ID NO.95 B30 TGCTGTAGATCCCCCTCAAATGCTGCGAGAGGCTTTTGCA SEQ ID NO.96 B31 AAAGAAGTTTTGCCAGCATAAATATTCATTGACTCAACATGTT SEQ ID NO.97 B32 AATACTGCGGAATCGTAGGGGGTAATAGTAAAATGTTTAGACT SEQ ID NO.98 B33 AGGGATAGCTCAGAGCCACCACCCCATGTCAA SEQ ID NO.99 B34 CAACAGTTTATGGGATTTTGCTAATCAAAAGG SEQ ID NO.100 B35 GCCGCTTTGCTGAGGCTTGCAGGGGAAAAGGT SEQ ID NO.101 B36 GCGCAGACTCCATGTTACTTAGCCCGTTTTAA SEQ ID NO.102 B37 ACAGGTAGAAAGATTCATCAGTTGAGATTTAG SEQ ID NO.103 B38 CCTCAGAACCGCCACCCAAGCCCAATAGGAACGTAAATGA SEQ ID NO.104 B39 ATTTTCTGTCAGCGGAGTGAGAATACCGATAT SEQ ID NO.105 B40 ATTCGGTCTGCGGGATCGTCACCCGAAATCCG SEQ ID NO.106 B41 CGACTGCGGTCAATCATAAGGGAACGGACATATATT SEQ ID NO.107 B42 AGACGTTACCATGTACCGTAACACCCCTCAGAACCGCCAC SEQ ID NO.108 B43 CACGTAGAAGTCTTTCC SEQ ID NO.109 B44 ATTGTGTCTCAGCAGGAAAGACACCATCGCC SEQ ID NO.110 B45 TTATAAAACGAACTAACCGAACTGACCAAACTCCTGATAA SEQ ID NO.111 B46 AGGTTTAGTACCGCCATGAGTTTCGTCACCAGGATCTAAA SEQ ID NO.112 B47 GTTTTGTCAGGAATTGCGAATAATCCGACAAT SEQ ID NO.113 B48 ANSWERGCATCGGAACGAGGGTGAGATTTG SEQ ID NO.114 B49 TATCATCGTTGAAAGAGGACAGATGGAAAAATCTACG SEQ ID NO.115 B50 AGCGTAACTACAAACTACAACGCCTATCACCGTACTCAGG SEQ ID NO.116 B51 TAGTTGCGAATTTTTTCACGTTGATCATAGTT SEQ ID NO.117 B52 GTACAACGAGCAACGGCTACAGAGGATACCGA SEQ ID NO.118 B53 ACCAGTCAGGACGTTGGAACGGTGTACAGACCGAAACAAA SEQ ID NO.119 B54 ACAGACAGCCCAAATCTCCAAAAAAAAATTTCTTA SEQ ID NO.120 B55 AACAGCTTGCTTTGGGACTAAGGGGTATA SEQ ID NO.121 B56 CCAAGCGCAGGCGCATAGGCTGGCAGAACTGGCTCATTAT SEQ ID NO.122 B57 CGAGGTGAGGCTCCAAAAGGAGCC SEQ ID NO.123 B58 ACCCCAGACTTTTTCATGAGGAACTTGCTTT SEQ ID NO.124 B59 ACCTTATGCGATTTTATGACCTTCATCAAGAGCATCTTTG SEQ ID NO.125 B60 CGGTTTATCAGGTTTCCATTAAACGGGAATACACT SEQ ID NO.126 B61 AAAACACTTAATCTTGACAAGAACTTAATCATTGTGAATT SEQ ID NO.127 B62 GGCAAAAGTAAAATACGTAATGCC SEQ ID NO.128 B63 TGGTTTAATTTCAACTCGGATATTCATTACCCACGAAAAA SEQ ID NO.129 B64 ACCAACCTAAAAAATCAACGTAACAAATAAATTGGGCTTGAGA SEQ ID NO.130 B65 CCTGACGAGAAACACCAGAACGAGTAGGCTGCTCATTCAGTGA SEQ ID NO.131 C01 TCGGGAGATATCAGTAACAGTACAAATAATT SEQ ID NO.132 C02 CCTGATTAAAGGAGCGGAATTATCTCGGGCCTC SEQ ID NO.133 C03 GCAAATCACCTCAATCAATATCTGCAGGTCGA SEQ ID NO.134 C04 CGACCAGTACATTGGCAGATTCACCTGATTGC SEQ ID NO.135 C05 TGGCAATTTTTAACGTCAGATGAAAACAATAACGGATTCG SEQ ID NO.136 C06 AAGGAATTACAAAGAACCACAGTCAGATGA SEQ ID NO.137 C07 GGACATTCACCTCAAATATCAAACACAGTTGA SEQ ID NO.138 C08 TTGACGAGCACGTATACTGAAATGGATTATTTAATAAAAG SEQ ID NO.139 C09 CCTGATTGCTTTGAATTGCGTAGATTTTCAGGCATCAATA SEQ ID NO.140 C10 TAATCCTGATTATCATTTTGCGGAGAGGAAGG SEQ ID NO.141 C11 TTATCTAAAGCATCACCTTGCTGATGGCCAAC SEQ ID NO.142 C12 AGAGATAGTTTGACGCTCAATCGTACGTGCTTTCCTCGTT SEQ ID NO.143 C13 GATTATACACAGAAATAAGAAATACCAAGTTACAAAATC SEQ ID NO.144 C14 TAGGAGCATAAAAGTTTGAGTAACATTGTTTG SEQ ID NO.145 C15 TGACCTGACAAATGAAAAATCTAAAATATCTT SEQ ID NO.146 C16 AGAATCAGAGCGGGAGATGGAAATACCTACATAACCCTTC SEQ ID NO.147 C17 GCGCAGAGGCGAATTAATTTTTGCACGTAAATTCTGAAT SEQ ID NO.148 C18 AATGGAAGCGAACGTTATTTCTCTAACAAC SEQ ID NO.149 C19 TAATAGATCGCTGAGAGCCAGCAGAGGCGTAA SEQ ID NO.150 C20 GAATACGTAACAGGAAAAACGCTCCTAAACAGGAGGCCGA SEQ ID NO.151 C21 TCAATAGATATTAATCCTTTGCCGGTTAGAACCT SEQ ID NO.152 C22 CAATATTTGCCTGCAACAGTGCCATAGAGCCG SEQ ID NO.153 C23 TTAAAGGGATTTTAGATACCGCCAGCCATTGCGGCACAGA SEQ ID NO.154 C24 ACAATTCGACAACTCGTAATACAT SEQ ID NO.155 C25 TTGAGGATGGTCAGTATTAACACTTGAATGG SEQ ID NO.156 C26 CTATTAGTATATCCAGAACAATATCAGGAACGGTACGCCA SEQ ID NO.157 C27 CGCGAACTAAAACAGAGGTGAGGCTTAGAAGTATT SEQ ID NO.158 C28 GAATCCTGAGAAGTGTATCGGCCTTGCTGGTACTTTAATG SEQ ID NO.159 C29 ACCACCAGCAGAAGATGATAGCCC SEQ ID NO.160 C30 TAAAACATTAGAAACTCAAACTTTTTATAATCAGTGAG SEQ ID NO.161 C31 GCCACCGAGTAAAAGAACATCACTTGCCTGAGCGCCATTAAAA SEQ ID NO.162 C32 TCTTTGATTAGTAATAGTCTGTCCATCACGCAAAATTAACCGTT SEQ ID NO.163 C33 CGCGTCTGATAGGAACGCCATCAACTTTTACA SEQ ID NO.164 C34 AGGAAGATGGGGACGACGACAGTAATCATATT SEQ ID NO.165 C35 CTCTAGAGCAAGCTTGCATGCCTGGTCAGTTG SEQ ID NO.166 C36 CCTTCACCGTGAGACGGGCAACAGCAGTCACA SEQ ID NO.167 C37 CGAGAAAGGAAGGGAAGCGTACTATGGTTGCT SEQ ID NO.168 C38 GCTCATTTTTTAACCAGCCTTCCTGTAGCCAGGCATCTGC SEQ ID NO.169 C39 CAGTTTGACGCACTCCAGCCAGCTAAACGACG SEQ ID NO.170 C40 GCCAGTGCGATCCCCGGGTACCGAGTTTTTCT SEQ ID NO.171 C41 TTTCACCAGCCTGGCCCTGAGAGAAAGCCGGCGAACGTGG SEQ ID NO.172 C42 GTAACCGTCTTTCATCAACATTAAAATTTTTGTTAAATCA SEQ ID NO.173 C43 ACGTTGTATTCCGGCACCGCTTCTGGCGCATC SEQ ID NO.174 C44 CCAGGGTGGCTCGAATTCGTAATCCAGTCACG SEQ ID NO.175 C45 TAGAGCTTGACGGGGAGTTGCAGCAAGCGGTCATTGGGCG SEQ ID NO.176 C46 GTTAAAATTCGCATTAATGTGAGCGAGTAACACACGTTGG SEQ ID NO.177 C47 TGTAGATGGGTGCCGGAAACCAGGAACGCCAG SEQ ID NO.178 C48 GGTTTTCCATGGTCATAGCTGTTTGAGAGGCG SEQ ID NO.179 C49 GTTTGCGTCACGCTGGTTTGCCCCAAGGGAGCCCCCGATT SEQ ID NO.180 C50 GGATAGGTACCCGTCGGATTCTCCTAAACGTTAATATTTT SEQ ID NO.181 C51 AGTTGGGTCAAAGCGCCATTCGCCCCGTAATG SEQ ID NO.182 C52 CGCGCGGGCCTGTGTGAAATTGTTGGCGATTA SEQ ID NO.183 C53 CTAAATCGGAACCCTAAGCAGGCGAAAATCCTTCGGCCAA SEQ ID NO.184 C54 CGGCGGATTGAATTCAGGCTGCGCAACGGGGGATG SEQ ID NO.185 C55 TGCTGCAAATCCGCTCACAATTCCCAGCTGCA SEQ ID NO.186 C56 TTAATGAAGTTTGATGGTGTTCCGAGGTGCCGTAAAGCA SEQ ID NO.187 C57 TGGCGAAATGTTGGGAAGGGCGAT SEQ ID NO.188 C58 TGTCGTGCACACAACATACGAGCCACGCCAGC SEQ ID NO.189 C59 CAAGTTTTTTGGGGTCGAAATCGGCAAAATCCGGGAAACC SEQ ID NO.190 C60 TTCTTCGCTATTGGAAGCATAAAGTGTATGCCCGCT SEQ ID NO.191 C61 TTCCAGTCCTTATAAATCAAAAGAGAACCATCACCCAAAT SEQ ID NO.192 C62 GCGCTCACAAGCCTGGGGTGCCTA SEQ ID NO.193 C63 CGATGGCCCACTACGTATAGCCCGAGATAGGGATTGCGTT SEQ ID NO.194 C64 AACTCACATTATTGAGTGTTGTTCCAGAAACCGTCTATCAGG SEQ ID NO.195 C65 ACGTGGACTCCAACGTCAAAGGGCGAATTTGGAACAAGAGTCC SEQ ID NO.196 Link-A1C TTAATTAATTTTTTACCATATCAAA SEQ ID NO.197 Link-A2C TTAATTTCATCTTAGACTTTACAA SEQ ID NO.198 Link-A3C CTGTCCAGACGTATACCGAACGA SEQ ID NO.199 Link-A4C TCAAGATTAGTGTAGCAATACT SEQ ID NO.200 Link-B1A TGTAGCATTCCTTTTATAAACAGTT SEQ ID NO.201 Link-B2A TTTAATTGTATTTCCACCAGAGCC SEQ ID NO.202 Link-B3A ACTACGAAGGCTTAGCACCATTA SEQ ID NO.203 Link-B4A ATAAGGCTTGCAACAAAGTTAC SEQ ID NO.204 Link-C1B GTGGGAACAAATTTCTATTTTTGAG SEQ ID NO.205 Link-C2B CGGTGCGGGCCTTCCAAAAACATT SEQ ID NO.206 Link-C3B ATGAGTGAGCTTTTAAATATGCA SEQ ID NO.207 Link-C4B ACTATTAAAGAGGATAGCGTCC SEQ ID NO.208 Loop GCGCTTAATGCGCCGCTACAGGGC

[0110] (2) Preparation of fluorescent chain solution:

[0111] Select DNAs A30, A61, B30, B61, C30, and C61 from Table 1. Sangon Biotech (Shanghai) Co., Ltd. was commissioned to modify the 5' end of the DNA sequences with Cy5, synthesizing six Cy5-modified DNA strands. Each test tube was prepared with 100 μmol of ultrapure water; then, a biotinylate solution with a final concentration of 400 nM was prepared.

[0112] (3) Design triangular DNA origami with 3, 5, or 9 biotin chains on each side. For example... Figure 1 As shown, 10.67 bases in the triangular DNA origami structure is 3.6 nm. Therefore, each biotin chain is spaced 20 nm apart to construct BioDori-3, BioDori-5 and BioDori-7.

[0113] (4) Preparation of Biotin Chain Solution 1 (3 biotin chains loaded on each side of the triangular DNA origami, for a total of 9 chains): Select A04, A12, A41, B04, B12, B41, C04, C12, and C41 from Table 1, and entrust Sangon Biotech (Shanghai) Co., Ltd. to first modify the 5' end of the DNA sequence with TTTT, and then modify the 5' end with Biotin to synthesize 9 Biotin (biotinylated) DNA chains respectively; the sequences are shown in Table 2 SEQ ID NO.209-217; add the corresponding ultrapure water to each test tube to prepare a 100 μmol DNA chain solution; and then prepare a Biotin Chain 1 solution with a final concentration of 400 nM.

[0114] (5) Preparation of Biotin Chain Solution 2 (5 biotin chains loaded on each side of the triangular DNA origami, for a total of 15 chains): Select A04, A12, A20, A41, A49, B04, B12, B20, B41, B49, C04, C12, C20, C41, and C49 from Table 1. Entrust Sangon Biotech (Shanghai) Co., Ltd. to first modify the 5' end of the DNA sequence with TTTT, and then modify the 5' end with Biotin to synthesize 15 Biotin (biotinylated) DNA chains respectively; the sequences are shown in Table 2 SEQ ID NO.209-223; add the corresponding ultrapure water to each test tube to prepare a 100 μmol DNA chain solution; and then prepare a Biotin Chain 2 solution with a final concentration of 400 nM.

[0115] (6) Preparation of Biotin Chain Solution 3 (7 biotin chains loaded on each side of the triangular DNA origami, for a total of 21 chains): Select A04, A12, A20, A26, A41, A49, A56, B04, B12, B20, B26, B41, B49, B56, C04, C12, C20, C26, C41, C49, and C56 from Table 1. Entrust Sangon Biotech (Shanghai) Co., Ltd. to first modify the 5' end of the DNA sequence with TTTT, and then modify the 5' end with Biotin to synthesize 21 Biotin (biotinylated) DNA chains respectively; the sequences are shown in Table 2 SEQ ID NO. 209-229; add the corresponding ultrapure water to each test tube to prepare a 100 μmol DNA chain solution; and then prepare a Biotin Chain 3 solution with a final concentration of 400 nM.

[0116] Table 2 21 biotin chains

[0117] sequence name Sequence (5' to 3') SEQ ID NO.209 Biotin chain 1 Biotin-TTTTATGGTTTATGTCACAATCAATAGATATTAAAC SEQ ID NO.210 Biotin chain 2 Biotin-TTTTCATTCAACAAACGCAAAGACACCAGAACACCCTGAACAAA SEQ ID NO.211 Biotin chain 3 Biotin-TTTTTTTCCTTAGCACTCATCGAGAACAATAGCAGCCTTTTACAG SEQ ID NO.212 Biotin chain 4 Biotin-TTTTTTCGAGCTAAGACTTCAAATATCGGGAACGAG SEQ ID NO.213 Biotin chain 5 Biotin-TTTTAACTCCAAGATTGCATCAAAAAGATAATGCAGATACATAA SEQ ID NO.214 Biotin chain 6 Biotin-TTTTCGACCTGCGGTCAATCATAAGGGAACGGAACAACATTATT SEQ ID NO.215 Biotin chain 7 Biotin-TTTTCGACCAGTACATTGGCAGATTCACCTGATTGC SEQ ID NO.216 Biotin chain 8 Biotin-TTTTAGAGATAGTTTGACGCTCAATCGTACGTGCTTTCCTCGTT SEQ ID NO.217 Biotin chain 9 Biotin-TTTTTTTCACCAGCCTGGCCCTGAGAGAAAGCCGGCGAACGTGG SEQ ID NO.218 Biotin chain 10 Biotin-TTTTTTGACGGAAATACATACATAAAGGGCGCTAATATCAGAGA SEQ ID NO.219 Biotin chain 11 Biotin-TTTTAGCATGTATTTCATCGTAGGAATCAAACGATTTTTTGTTT SEQ ID NO.220 Biotin chain 12 Biotin-TTTTTAATTGCTTTACCCTGACTATTATGAGGCATAGTAAGAGC SEQ ID NO.221 Biotin chain 13 Biotin-TTTTTATCATCGTTGAAAGAGGACAGATGGAAGAAAAAATCTACG SEQ ID NO.222 Biotin chain 14 Biotin-TTTTGAATACGTAACAGGAAAAACGCTCCTAAACAGGAGGCCGA SEQ ID NO.223 Biotin chain 15 Biotin-TTTTGTTTGCGTCACGCTGGTTTGCCCCAAGGGAGCCCCGATT SEQ ID NO.224 Biotin chain 16 Biotin-TTTTCACCGTCACCTTATTACGCAGTATTGAGTTAAGCCCAATA SEQ ID NO.225 Biotin chain 17 Biotin-TTTTACAAGAAAGCAAGCAAATCAGATAACAGCCATATTATTTA SEQ ID NO.226 Biotin chain 18 Biotin-TTTTCGGATGGCACGAGAATGACCATAATCGTTTTACCAGACGAC SEQ ID NO.227 Biotin chain 19 Biotin-TTTTCCAAGCGCAGGCGCATAGGCTGGCAGAACTGGCTATTAT SEQ ID NO.228 Biotin chain 20 Biotin-TTTTCTATTAGTATATCCAGAACAATATCAGGAACGGTACGCCA SEQ ID NO.229 Biotin chain 21 Biotin-TTTTTTAATGAAGTTTGATGGTGGTTCCGAGGTGCCGTAAAGCA

[0118] (7) Preparation of staple solution 1: As shown in Table 1, remove the 9 biotin chains from step (4) and the 6 fluorescent chains from step (2). Add the corresponding amount of ultrapure water to each of the remaining 193 staple chains to prepare a 100 μmol DNA chain solution; then prepare a staple chain 1 solution with a final concentration of 400 nM.

[0119] (8) Preparation of staple solution 2: As shown in Table 1, remove the 15 biotin chains from step (5) and the 6 fluorescent chains from step (2). Add the corresponding amount of ultrapure water to each of the remaining 187 staple chains to prepare a 100 μmol DNA chain solution; then prepare a staple chain 2 solution with a final concentration of 400 nM.

[0120] (9) Preparation of staple solution 3: As shown in Table 1, remove the 21 biotin chains from step (6) and the 6 fluorescent chains from step (2). Add the corresponding amount of ultrapure water to each of the remaining 181 staple chains to prepare a 100 μmol DNA chain solution; then prepare a staple chain 3 solution with a final concentration of 400 nM.

[0121] (10) 100 nM M13mp18 single-stranded DNA (commercially purchased from New England Biolabs): Take the purchased DNA powder, centrifuge at 4000 rpm for 1 minute, then dissolve it in ultrapure water to prepare a 100 μM M13mp18 single-stranded DNA solution.

[0122] (11) 10×TAE-Mg 2+ Buffer solution: containing 40 mM Tris base, 20 mM acetic acid, 2 mM EDTA, 12.5 mM magnesium acetate, pH=8.0, mixed well.

[0123] (12) Preparation of BioDori-3 (each side contains 3 biotin chains): Prepare a 200 μL centrifuge tube and add the above M13mp18 single-stranded DNA solution, fluorescent chain solution, biotin chain 1 solution, and staple chain 1 solution in a molar ratio of 1:10:10:10. Then, add 10×TAE-Mg to the tube. 2+ Buffer and ultrapure water (containing 10X TAE-Mg) 2+ The buffer solution and ultrapure water were mixed at a volume ratio of 1:9 to form 1× TAE-Mg. 2+ The buffer system was prepared, and the final magnesium ion concentration reached 12.5 mM. The mixture was vortexed until homogeneous, centrifuged appropriately, and then placed in a PCR instrument for annealing: first, it was maintained at 95°C for 5 minutes, and then slowly cooled from 95°C to 25°C at a rate of 0.1°C every 10 seconds. After annealing, it was centrifuged at 5000 g for 3 minutes using a 100 kDa ultrafiltration tube to remove excess short-chain DNA. This ultrafiltration operation was repeated three times to obtain purified biotinylated DNA origami nanostructures (BioDori).

[0124] Similarly, referring to the above preparation method, M13mp18 single-stranded DNA, fluorescent chain solution, biotin chain 2 solution, and staple chain 2 solution were mixed to prepare BioDori-5 (each side contains 5 biotin chains).

[0125] Similarly, referring to the above preparation method, M13mp18 single-stranded DNA, fluorescent chain solution, biotin chain 3 solution, and staple chain 3 solution were mixed to prepare BioDori-7 (each side contains 7 biotin chains).

[0126] The synthesis of BioDori was verified by visualization using atomic force microscopy (AFM). The obtained AFM plot is shown below. Figure 2 A, Figure 3 A, Figure 4 As shown in Figure A, the synthesized BioDori is an equilateral triangle with uniform size, good dispersion, and stable shape, further verifying the successful preparation of BioDori (scale bar 200 nm).

[0127] Example 2: Preparation and characterization of the DNA origami neuron-targeting nanocarrier (BioDoriH) of the present invention.

[0128] 1. Preparation of DNA Origami Neuron-Targeting Nanocarriers

[0129] (1) The biotin reagent was NHS-PEG12-Biotin, purchased from Thermo Fisher Scientific, and dissolved in acetonitrile to prepare a 10 mM biotin reagent solution. Streptavidin SA was purchased from MedChemexpress Biotechnology, Inc., and dissolved in double-distilled water to prepare a 20 μM streptavidin SA solution.

[0130] (2) The gene sequence of the targeting ligand from GenBank:CS401852.1 was cloned into the pET28a plasmid vector to obtain the Hc / A recombinant plasmid. The obtained Hc / A recombinant plasmid was transfected into Escherichia coli to obtain plasmid bacteria, which were then activated and expanded in LB medium at 37°C and 180 rpm. When the OD600 value was monitored to be approximately 0.6, protein expression was induced at low temperature, and the culture was continued for 18-24 hours. The bacteria expressing the protein were collected, washed with PBS, and then sonicated. After centrifugation, the supernatant and the lysed bacteria were separated. The supernatant was purified to obtain the targeting ligand (Hc / A).

[0131] (3) The targeting ligand Hc / A was mixed with 10 mM biotin reagent solution at a molar ratio of 1:50, and reacted at room temperature for 0.5 h. Then, 10 kDa Amicon was used. ® Using an Ultra 0.5 mL ultrafiltration tube, the biotinylated targeting ligand (Biotin-Hc / A) was obtained by ultrafiltration three times at 10000g for 15min in a double-distilled water system.

[0132] (4) The DNA origami nanocarrier (BioDori-3) of Example 1 was mixed with 20 μM streptavidin SA solution at a molar ratio of 1:5n (n is the total number of biotin chains on the triangular BioDori, n=9), and reacted at room temperature for 30 min. After the reaction was completed, the free SA was removed by centrifugation at 5000 g for 3 min using a 100 kDa ultrafiltration tube to obtain the SA-conjugated DNA origami nanocarrier (SA-BioDori).

[0133] (5) The SA-conjugated DNA origami nanocarrier (SA-BioDori) from step (4) and the Biotin-Hc / A from step (3) were mixed at a molar ratio of 1:10n (n is the total number of biotin chains on the triangular BioDori, n=9), and reacted at room temperature for 30 min. After completion, the unconnected Biotin-Hc / A was removed by centrifugation at 5000 g for 3 min using a 100 kDa ultrafiltration tube, and the targeting ligand was assembled onto SA-BioDori to obtain the purified DNA origami targeting nanocarrier (BioDoriH-3).

[0134] Similarly, referring to the preparation method described above, the BioDori-5 from Example 1 was reacted, with n=15 during the reaction, to obtain BioDoriH-5.

[0135] Similarly, referring to the preparation method described above, the BioDori-7 from Example 1 was reacted, with n=21 during the reaction, to obtain BioDoriH-7.

[0136] 2. Characterization by atomic force microscopy

[0137] Biotinylated DNA origami nanocarriers (BioDori), SA-conjugated DNA origami nanocarriers (SA-BioDori), and DNA origami-targeting nanocarriers (BioDoriH) were added to freshly cut mica sheets and allowed to adhere for 2-3 min. The substrate was then washed with distilled water to remove unabsorbed sample, and the mica sheet surface was dried with high-purity nitrogen. AFM testing was performed using a BRUKER RTESP-300 probe in Tapping Mode at a Drive Amplitude of 45 mV. AFM characterization was performed, and the cross-sectional height of the obtained images was analyzed. Figure 2-4In the biotinylated DNA origami nanocarrier (BioDori), the SA-conjugated DNA origami nanodrug delivery system (SA-BioDori), and the DNA origami-targeting nanocarrier (BioDoriH), it was observed that the cross-sectional height increased with the increase of modifications on the main structure. This indicates the successful construction of the DNA origami-targeting nanocarrier (BioDoriH-n, where n is the total number of biotin chains on the triangular BioDori).

[0138] Example 3: Preparation and characterization of the i-motif-modified biotinylated DNA origami nanostructure (MDori) of the present invention.

[0139] 1. Preparation of i-motif-modified biotinylated DNA origami nanocarriers

[0140] (1) Preparation of semi-i-motif solution: Select A32, B32 and C32 from Table 1, and commission Sangon Biotech (Shanghai) Co., Ltd. to modify the 3' end of the DNA sequence with AAAAAAAAAAAACCCCTAACCCC; Select A65, B65 and C65 from Table 1, and commission Sangon Biotech (Shanghai) Co., Ltd. to modify the 5' end of the DNA sequence with CCCCTAACCCCAAAAAAAAAAAA; Synthesize 6 semi-i-motif modified DNA strands respectively, and the sequences are shown in Table 3; Add the corresponding ultrapure water to each test tube to prepare a 100 μmol DNA strand solution; Then prepare a semi-i-motif solution with a final concentration of 400 nM.

[0141] Table 3. Six DNA strands modified with semi-i-motif.

[0142] Serial Number name Sequence (5' to 3') SEQ ID NO.230 i-motif-A32 CAGAAGGAAACCGAGGTTTTTAAGAAAAGTAAGCAGATAGCCGAAAAAAAAAAAAACCCTAACCCC SEQ ID NO.231 i-motif-B32 AATACTGCGGAATCGTAGGGGGTAATAGTAAAATGTTTAGACTAAAAAAAAAAAACCCCTAACCCC SEQ ID NO.232 i-motif-C32 TCTTTGATTAGTAATAGTCTGTCCATCACGCAAATTAACCGTTAAAAAAAAAAAAAACCCTAACCCC SEQ ID NO.233 i-motif-A65 CCCCTAACCCCAAAAAAAAAAAATGCTATTTTGCACCCAGCTACAATTTTGTTTTGAAGCCTTAAA SEQ ID NO.234 i-motif-B65 CCCCTAACCCCAAAAAAAAAAAACCTGACGAGAAACACCAGAACGAGTAGGCTGCTCATTCAGTGA SEQ ID NO.235 i-motif-C65 CCCCTAACCCCAAAAAAAAAAAAACGTGGACTCCAACGTCAAAGGGCGAATTTGGAACAAGAGTCC

[0143] (2) Preparation of staple solution 4: As shown in Table 1, remove 21 biotinylated strands, 6 fluorescent strands, and 6 semi-i-motif modified DNA strands. Add the corresponding amount of ultrapure water to each of the remaining 175 staple strands to prepare a 100 μmol DNA strand solution; then prepare a staple strand 4 solution with a final concentration of 400 nM.

[0144] (3) Preparation of MDori: Prepare a 200 μL centrifuge tube and, according to a molar ratio of 1:10:10:10:10, add M13mp18 single-stranded DNA, biotin chain 3 solution, fluorescent chain solution, semi-i-motif modified DNA chain solution, and staple chain 4 solution to a 1×TAE-Mg 2+ Mix thoroughly in the system.

[0145] The mixture was vortexed until homogeneous, centrifuged appropriately, and then placed in a PCR instrument for annealing: initially maintained at 95°C for 5 minutes, then slowly cooled from 95°C to 25°C at a rate of 0.1°C every 10 seconds. After annealing, the mixture was centrifuged at 5000 g for 3 minutes using a 500 μL, 100 kDa ultrafiltration tube to remove excess short-chain DNA. This ultrafiltration process was repeated three times to obtain purified MDori.

[0146] 2. Characterization by atomic force microscopy

[0147] The prepared semi-i-motif modified DNA origami nanocarriers (MDori) were characterized by atomic force microscopy (AFM) according to the method in Example 1. The morphology of MDori in normal physiological environments (pH-7.4) and lysosomal environments (pH-5.0) was characterized using AFM. The results showed that at pH-7.4, MDori existed in a uniformly distributed triangular structure, exhibiting good dispersibility; while at pH-5.0, MDori significantly aggregated, forming micron-sized aggregates. Figure 5 This phenomenon indicates that MDori possesses acid-responsive aggregation properties and can undergo structural rearrangement in a lysosomal environment.

[0148] Example 4: Preparation of an i-motif-modified delivery platform for targeting motor neurons (MDoriH)

[0149] The preparation of the i-motif-modified delivery platform for targeting motor neurons (MDoriH-7) includes the following steps:

[0150] (1) The biotin reagent was NHS-PEG12-Biotin, purchased from Thermo Fisher Scientific, and dissolved in acetonitrile to prepare a 10 mM biotin reagent solution. Streptavidin SA was purchased from MedChemexpress Biotechnology, Inc., and dissolved in double-distilled water to prepare a 20 μM streptavidin SA solution;

[0151] (2) The gene sequence of the targeting ligand from GenBank:CS401852.1 was cloned into the pET28a plasmid vector to obtain the Hc / A recombinant plasmid. The obtained Hc / A recombinant plasmid was transfected into Escherichia coli to obtain plasmid bacteria, which were then activated and expanded in LB medium at 37°C and 180 rpm. When the OD600 value was monitored to be approximately 0.6, protein expression was induced at low temperature, and the culture was continued for 18-24 hours. The bacteria expressing the protein were collected, washed with PBS, and then sonicated. After centrifugation, the supernatant and the lysed bacteria were separated. The supernatant was purified to obtain the targeting ligand (Hc / A).

[0152] (3) The targeting ligand Hc / A was mixed with 10 mM biotin reagent solution at a molar ratio of 1:50, and reacted at room temperature for 0.5 h. Then, 10 kDa Amicon was used. ® Using an Ultra 0.5 mL ultrafiltration tube, the biotinylated targeting ligand (Biotin-Hc / A) was obtained by ultrafiltration three times at 10000g for 15min in a double-distilled water system.

[0153] (4) The i-motif modified biotinylated DNA origami nanostructure (MDori) of Example 3 was mixed with 20 μM streptavidin SA solution at a molar ratio of 1:5n (n is the total number of biotin chains on the triangular BioDori, n=21), and reacted at room temperature for 30 min. After the reaction was completed, the free SA was removed by centrifugation at 5000 g for 3 min using a 100 kDa ultrafiltration tube to obtain the SA-conjugated DNA origami drug delivery system (SA-MDori).

[0154] (5) The SA-conjugated DNA origami drug delivery system (SA-MDori) from step (4) and the biotinylated targeting ligand Biotin-Hc / A from step (3) were mixed at a molar ratio of 1:10n (n is the total number of biotin chains on the triangular BioDori, n=21) and reacted at room temperature for 30 min. After completion, the mixture was centrifuged at 5000 g for 3 min using a 100 kDa ultrafiltration tube to remove unconnected Biotin-Hc / A, and the targeting ligand was assembled onto SA-MDori to obtain the purified i-motif-modified delivery platform for targeting motor neurons (MDoriH-7).

[0155] Experimental Example 1: Study on the Highly Efficient Neuronal Targeting Performance of BioDoriH

[0156] Cortical neurons cultured for 14 days were collected. Under light-protected conditions, BioDori (prepared in Example 1), BioDori H-3 (prepared in Example 2), BioDori H-5, and BioDori H-7 were added to 12-well plates. A PBS group was set up as a negative control. Each sample was prepared in triplicate. The plates were placed in a cell culture incubator and incubated at 37°C and 5% CO2 for 12 hours. After incubation, the cell culture plates were removed from the incubator. Under light-protected conditions, the supernatant was discarded, and the cells were gently washed three times with pre-chilled PBS. An appropriate amount of 0.25% trypsin was added to each well, and the plates were incubated at 37°C for approximately 5 minutes. After adding DMEM medium to stop digestion, the cells were gently pipetted and collected as a single-cell suspension. The suspension was centrifuged (4°C, 500 g, 5 minutes), and the pellet was collected. The cells were washed three times with pre-chilled PBS and analyzed.

[0157] The results are as follows Figure 6 As shown, fluorescence intensity was positively correlated with the valence state of Hc / A within the same unit time, indicating that the uptake efficiency of BioDoriH by neuronal cells gradually increased with the increase of Hc / A valence state. The uptake rate of the BioDoriH-7 system was significantly higher than that of other groups, with a total uptake rate of approximately 95%. This indicates that a high valence state of Hc / A can significantly enhance the targeting binding ability and cellular uptake effect of BioDoriH.

[0158] Experimental Example 2: Verification of the lysosomal acid-neutralizing properties of BioDoriH and MDoriH

[0159] Preparation of Hochest 33258 staining working solution: Dissolve 10 mg of Hochest 33258 fluorescent dye in 10 mL of PBS and dilute to prepare a stock solution with a concentration of 1 mg / mL. Take an appropriate amount of the stock solution and dilute it with PBS to a concentration of 10 μg / mL for use.

[0160] Preparation of Lyso-Tracker Green fluorescent dye working solution: Dilute 1 mM Lyso-Tracker Green stock solution (commercially purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) to 2 µM using PBS to prepare Lyso-Tracker Green fluorescent dye working solution.

[0161] Neuronal cells cultured for 14 days were harvested. Under light-protected conditions, BioDoriH-7 cells prepared in Example 2 and MDoriH-7 cells prepared in Example 4 were placed in laser confocal microarrays, respectively. Each sample was prepared in triplicate and incubated at 37°C for 18 hours in a cell culture incubator.

[0162] After incubation, the drug-containing supernatant was discarded, and the cells were gently rinsed three times with PBS. The PBS was then discarded, and at room temperature, 1 mL of 4% paraformaldehyde was added to each dish to fix the cells for 10 min. The supernatant was discarded, and the cells were rinsed three times with PBS. Next, 2 mL of 10 μg / mL Hochest 33258 fluorescent dye working solution was added to each dish, and the cell nuclei were stained for 10 min at room temperature. The staining solution was discarded, and the cells were rinsed three times with PBS. 2 mL of Lyso-Tracker Green fluorescent dye working solution was added to the confocal microscopy dish, and the cells were incubated at 37°C for 30 min. The staining solution was discarded, and the cells were washed three times with PBS. The cells were then stored at 4°C in HBSS solution, protected from light. Cells were observed using a laser confocal microscope, and the images were analyzed using three-channel composite analysis in XYZ tomographic scanning and XZ longitudinal analysis modes.

[0163] The results are as follows Figure 7As shown, the fluorescence intensity of the lysosomal sensor in BioDoriH-treated cells was significantly higher than that in MDoriH-treated cells, indicating that MDoriH significantly reduced the acidity of lysosomes through proton consumption.

[0164] Experimental Example 3: Verification of the Lysosomal Escape Performance of MDoriH

[0165] Neuronal cells cultured for 14 days were collected. Under light-protected conditions, MDoriH-7 from Example 4 was added to a laser confocal microplate and incubated at 37°C for 24 h in a cell culture incubator.

[0166] After incubation, the drug-containing supernatant was discarded, and the cells were gently rinsed three times with PBS. The PBS was then discarded, and the cells were fixed for 10 min with 1 mL of 4% paraformaldehyde per dish at room temperature. The supernatant was discarded, and the cells were rinsed three times with PBS. 2 mL of Lyso-Tracker Green working solution was added to each confocal microscopy dish, and the cells were incubated at 37°C for 30 min. The staining solution was discarded, and the cells were washed three times with PBS. The cells were then stored in HBSS solution at 4°C in the dark. Cells were observed using a laser confocal microscope, and the images were analyzed using three-channel composite analysis in XYZ tomographic scanning and XZ longitudinal analysis modes.

[0167] The results showed that the colocalization of MDoriH's fluorescence signal with lysosomes was significantly reduced, and some red fluorescence had detached from the lysosomal region, indicating that MDoriH could successfully escape from the lysosome. Figure 8 MDoriH improves lysosomal escape efficiency.

[0168] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A DNA origami nanostructure (BioDori), wherein, The DNA origami structure comprises M13mp18 single-stranded DNA, a biotin chain (biotin-DNA), a fluorescent chain (fluorescent group-DNA), and an unmodified staple chain. Each side of the DNA origami structure is loaded with 1-30 fluorescent chains and 1-10 biotin chains. The nucleotide sequence of the staple chain is shown in SEQ ID NO: 1-208.

2. A method for preparing the DNA origami nanostructure (BioDori) as described in claim 1, comprising the following steps: adding M13mp18 single-stranded DNA, biotin chain, fluorescent chain, and unmodified staple chain to a buffer system, mixing at room temperature according to a final concentration ratio of 1:5-20:5-20:5-20 for M13mp18 single-stranded DNA, biotin chain, fluorescent chain, and unmodified staple chain in the buffer system, synthesizing the product at a programmed temperature of 95°C, and ultrafiltration through a 100kDa ultrafiltration tube to obtain the DNA origami main body (BioDori).

3. A delivery system for targeting motor neuron cells (BioDoriH), comprising the DNA origami nanostructure (BioDori) as described in claim 1 and a targeting ligand immobilized on the biotin chain; wherein, The targeting ligand, after being modified with biotinylation, binds to the biotin chain via the action of streptavidin.

4. A method for preparing the BioDoriH delivery system for targeted motor neuron cells as described in claim 3, comprising the following steps: (1) The targeting ligand and biotin reagent were mixed at a molar ratio of 1:50-100, reacted at room temperature for 30-60 min, and then purified by ultrafiltration at 10kDa-20kDa to obtain the biotinylated targeting ligand (Biotin-Hc / A). (2) DNA origami nanostructure (BioDori) was mixed with streptavidin (SA) at a molar ratio of 1:5-10n and reacted at room temperature for 30-60 min to obtain SA-bound biotinylated DNA origami (SA-BioDori). (3) The SA-binding biotinylated DNA origami (SA-BioDori) from step (2) is mixed with the biotinylated targeting ligand (Biotin-Hc / A) from step (1) at a molar ratio of 1:10-20n and reacted at room temperature for 30-60 min to obtain the Biotin-Hc / A-linked SA-BioDori (BioDoriH).

5. A semi-i-motif modified DNA origami nanostructure (MDori), wherein, The i-motif-modified DNA origami nanostructure comprises M13mp18 single-stranded DNA, a biotin chain (biotin-DNA), a fluorescent chain (fluorescent group-DNA), a semi-i-motif modified chain (semi-i-motif-DNA), and an unmodified staple chain. Each side of the DNA origami body is loaded with 1-30 fluorescent chains, 1-10 biotin chains, and 1-10 semi-i-motif modified chains. The nucleotide sequence of the staple chain is shown in SEQ ID NO: 1-208.

6. The semi-i-motif modified DNA origami nanostructure according to claim 5, wherein, The nucleotide sequence of the semi-i-motif modified strand (semi-i-motif-DNA) is any one or a combination of A32, B32, and C32 in the staple strand, with the 3' end of the DNA sequence modified with AAAAAAAAAAAACCCCTAACCCC; or any one or a combination of A65, B65, and C65, with the 5' end of the DNA sequence modified with CCCCTAACCCCAAAAAAAAAAAA.

7. A method for preparing the semi-i-motif modified DNA origami nanostructure (MDori) as described in claim 5, comprising the following steps: adding M13mp18 single-stranded DNA, biotin chain (biotin-DNA), fluorescent chain (fluorescent group-DNA), semi-i-motif-DNA, and unmodified staple chain to a buffer system; mixing at room temperature according to a final concentration ratio of 1:5-20:5-20:5-20:5-20 in the buffer system; synthesizing the product under programmed temperature control; and ultrafiltration through a 100kDa-200kDa ultrafiltration tube to obtain the DNA origami nanostructure body (MDori).

8. The preparation method according to claim 7, wherein, The molar ratio of the M13mp18 single-stranded DNA, biotin chain (biotin-DNA), fluorescent chain (fluorescent group-DNA), semi-i-motif-DNA, and unmodified staple chain is 1:10-15:10-15:10-15:10-15.

9. A semi-i-motif-modified delivery system for targeting motor neuron cells (MDoriH), comprising a semi-i-motif-modified biotinylated DNA origami structure (MDori) as described in any one of claims 5-6, and a targeting ligand immobilized on the structure by a biotinylate chain using SA; wherein, The targeting ligand is biotinylated and then binds to the SA.

10. A method for preparing a semi-i-motif-modified targeted motor neuron cell delivery system (MDoriH) as described in claim 9, wherein the DNA origami structure comprises M13mp18 single-stranded DNA, a biotin chain (biotin-DNA), a fluorescent chain (fluorescent group-DNA), and a staple chain, wherein each side of the DNA origami structure is loaded with 1-30 fluorescent chains and 1-10 biotin chains.

Images