CN111939265B - DNA nano ladder of polyvalent aptamer and preparation method and application thereof - Google Patents

Abstract

The invention provides a multivalent aptamer DNA nano-ladder and a preparation method and application thereof. The invention uses natural DNA molecules as raw materials, and on the basis of DNA origami theory, uses the special properties of rolling circle amplification products to construct DNA nano-ladders, and introduces a large number of aptamers to construct a simple design and strong drug-carrying capacity. , A drug delivery system with target specificity and biocompatibility, and through aptamer or drug replacement, the carrier can also be applied to various types of target cells.

Description

A kind of multivalent aptamer DNA nano-ladder and its preparation method and application

technical field

The invention belongs to the technical field of biomedicine, and relates to a multivalent aptamer DNA nano-ladder and a preparation method and application thereof.

Background technique

The incidence and mortality of cancer in my country are increasing year by year, and it has become one of the four major chronic diseases in my country. At present, chemotherapy drugs are widely used in cancer treatment, but chemotherapy drugs can induce cytotoxicity in both tumor cells and healthy cells; and due to the phenomenon of multidrug resistance, the special "protein pump" in some tumor cells will cause chemotherapy drugs For example, doxorubicin hydrochloride (Dox) is pumped out of the cell and becomes resistant to the drug. Therefore, the development of a targeted drug carrier with a simple design that can specifically recognize tumor cells is a challenge in the field of biomedical technology.

DNA nanostructures have become a new type of drug carrier in recent years. The preparation of DNA nanostructures mainly relies on the self-assembly of DNA. Through hydrogen bonding, stacking, electrostatic and hydrophobic interaction, it drives the complementary pairing of DNA strands. Design of DNA strand sequences to obtain DNA nanostructures with specific structures and functions. DNA origami is a new self-assembly method. The design idea is to fold a long single-stranded DNA (ssDNA) back and forth as a scaffolding chain, and use multiple staple strands (short DNA strands) to fix the shape to obtain a specific structure. . In order to further simplify DNA origami, rolling circle amplification (RCA) products are used as scaffold chains, and the periodicity and large molecular weight of rolling circle amplification (RCA) products are used to combine several staple strands to construct a prefabricated chain. Designing the shape further reduces the number of staple chains used and simplifies the sequence design.

Multivalent targeted binding has attracted much attention in the biomedical field in recent years due to its high affinity and high specificity. Aptamers are developed by phylogenetic evolution of exponentially enriched ligands (SELEX), and are essentially an oligonucleotide sequence that can form stable secondary or tertiary structures and targets (such as proteins) through internal forces. ) specific binding. By immobilizing aptamers on the surface of DNA nanostructures or nanoparticles, the obtained multivalent ligands can simultaneously bind to receptors on target tumor cells to achieve high binding affinity and selectivity, and multivalent receptors on cell membranes can promote Endocytosis is more efficient than monovalent DNA nanostructures.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a multivalent aptamer DNA nano-ladder, which is a drug delivery carrier with simple design, strong drug-carrying capacity and target-specificity, which can specifically target tumor cells and induce selective cytotoxicity. , thereby greatly reducing the adverse reactions of antitumor drugs (eg, doxorubicin hydrochloride (Dox)) to normal cells, and reducing the side effects of chemotherapy.

In order to achieve the above object, on the one hand, the present invention provides a multivalent aptamer DNA nano-ladder, characterized in that, the multivalent aptamer DNA nano-ladder includes m repeating units in series, and the repeating unit includes two repeating units. A long single-stranded DNA molecule and 2n short single-stranded DNA molecules, wherein m is an integer from 80 to 320, n is an integer greater than or equal to 3 and less than or equal to 7,

Wherein, in the repeating unit, the sequences of the two long single-stranded DNA molecules are different; at least one short single-stranded DNA molecule among the 2n short single-stranded DNA molecules is connected to an aptamer, and each short single-stranded DNA molecule has Two hybridization regions, one of the two hybridization regions hybridizes with a part of the long single-stranded DNA molecule, and the other hybridization region hybridizes with a part of the other short single-stranded DNA molecule to form a double helix structure, thereby the two hybridization regions. A long single-stranded DNA molecule and 2n short single-stranded DNA molecules self-assemble into a DNA nano-ladder.

In the present invention, the "multivalent" refers to the multivalent binding of multiple ligands and multiple receptors, and the number of ligand-receptor binding pairs is the valence state. A large number of aptamers contained in the DNA nanoladder can be Achieve multivalent binding to receptors on cell membranes.

In the repeating unit of the multivalent aptamer DNA nano-ladder of the present invention, preferably, the lengths of the two long single-stranded DNA molecules can be the same. Preferably, the length of the long single-stranded DNA molecule may be 13-240 bp, more preferably 63 bp. In one embodiment, the two long single-stranded DNA molecules are shown as (GGTGCTTAGTCGAAAGAAAGAAAGGAAGAAGTTTCAAGGAAAGGAAACAAGAAGGCGAAGACA, SEQ ID NO: 13) and (AGGGCTTGGCATAGACGAGTTGACAGAGACGGAATCCGACCATTGTGCGCTATCTTCATCTTA, SEQ ID NO: 14).

In the DNA nano-ladder composed of m repeating units in series, the long single-stranded DNA finally formed has periodicity and large molecular weight. The length of the long single-stranded DNA is mainly affected by the reaction time of rolling circle amplification (RCA) and the concentration of dNTPs. Preferably, the two long single-stranded DNAs may be the same length. More preferably, the two long-chain DNA molecular chains A and B have about 127 tandem 63-base repeat sequences; the molecular weight of the long-chain DNA molecular chain A is about 19708 g/mol, and the molecular weight of the long-chain DNA molecular chain B is about 19448 g/mol. mol.

In the repeating unit of the polyvalent aptamer DNA nano-ladder of the present invention, the short single-stranded DNA molecule is an oligonucleotide (referring to the general term for short-chain nucleotides with less than 50 bases) sequence. The more GC base pairs in each strand, the higher the drug loading. In a specific embodiment, the short single-stranded DNA molecules are all 42 bp in length. In one embodiment, the 2n short single-stranded DNA molecules (wherein, n=3) are such as SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO:12. In the present invention, the length of each short single-stranded DNA molecule needs to be the same, and the short single-stranded DNA molecule has two hybridization regions which are respectively hybridized with the long single-stranded DNA molecule and another (adjacent) short single-stranded DNA molecule. Rectangular regions are finally built into ladder-like DNA nanostructures (DNA nanoladders). In the present invention, the sequence of each short single-stranded DNA molecule is divided into two hybrid regions, the sequence of one hybrid region must be complementary to the long single-stranded DNA molecule, and the sequence of one hybrid region must be complementary to another (adjacent) short single-stranded DNA. Molecular complementarity, the sequence forming the hybrid region can choose any complementary base, but the sequence design of short single-stranded DNA molecules should try to avoid the formation of special DNA secondary structures such as hairpin structure and G-quadruplex.

In the repeating unit of the multivalent aptamer DNA nano-ladder of the present invention, preferably, m can be an integer of 100-150, more preferably, m is 127; n can be an integer of 3-5, more preferably, n is 3 .

In a specific embodiment, in the multivalent aptamer DNA nano-ladder of the present invention, the 5' end of the aptamer is labeled with a fluorescent group. In one embodiment, the fluorescent group may be carboxyfluorescein (FAM), or Cy5.

In one embodiment, in the multivalent aptamer DNA nano-ladder, the theoretical width of the DNA nano-ladder is about 5.4 nm to 13.9 nm, preferably about 11 (4+21×0.34=11.1) nm; theoretical The length is about 1700 nm to 6800 nm, preferably about 2700 (8000×0.34=2720) nm. The width of the DNA nanoladder can be controlled by the design sequence of short single-stranded DNA molecules, and the length can be controlled by the rolling circle amplification (RCA) reaction.

In the present invention, the width of the DNA nano-ladder can be controlled by directly changing the length of the hybridization region of two short single-stranded DNA molecules.

In the multivalent aptamer DNA nano-ladder of the present invention, the selection of aptamers is not particularly limited. As long as specific targeting of tumor cells can be achieved. Aptamers are essentially a DNA or RNA oligonucleotide sequence, which can form stable secondary structures such as hairpin structures, pseudoknots, G-quadruplexes, etc. through base complementary pairing, forming stable complexes with receptors substances, this binding has a high degree of bond and degree and selectivity. The aptamer can be a tumor cell aptamer, and the aptamer can specifically bind to the receptor protein on the cell membrane, for example, the acute lymphoblastic leukemia T lymphocyte (CCRF-CEM) aptamer Sgc8, the aptamer Can specifically bind to protein tyrosine kinase 7 (PTK7), which is overexpressed on CCRF-CEM cells; or human breast cancer cell (MCF-7) aptamer S2.2, which can bind to MUC1 protein For specific binding, MUC1 is overexpressed on MCF-7 cells; or the aptamer AS1411, which can specifically bind to the broad-spectrum tumor marker nucleolar protein. A large number of aptamers are connected on one side of the DNA nano-ladder to form a multivalent aptamer structure.

In one embodiment, the aptamer can be the acute lymphoblastic leukemia T lymphocyte (CCRF-CEM) aptamer Sgc8, which can specifically bind to protein tyrosine kinase 7 (PTK7), which is in It is overexpressed on CCRF-CEM cells, and a large number of aptamers are connected to one side of the DNA nano-ladder to form a multivalent aptamer structure that binds to CCRF-CEM cells to achieve specific detection.

In one embodiment, the aptamer can also be a human breast cancer cell (MCF-7) aptamer S2.2 or a broad-spectrum aptamer AS1411.

On the other hand, the present invention provides a preparation method of the above-mentioned polyvalent aptamer DNA nano-ladder, which comprises the following steps:

(1) synthesizing two long single-stranded DNAs each having a nucleotide sequence of m tandem long single-stranded DNA molecules by a rolling circle amplification (RCA) reaction;

(2) Based on DNA origami, the two long single-stranded DNAs synthesized in step (1) are used as scaffold chains and combined with m×2n short single-stranded DNA molecules to construct the DNA nano-ladder.

The above-mentioned preparation method, preferably, in step (1), the circular DNA as shown in SEQ ID NO:1 and SEQ ID NO:2 is used as a template respectively, and in step (1), as shown in SEQ ID NO:3 and SEQ ID NO:4 In the presence of the indicated DNA primers, Phi29 DNA polymerase buffer and dNTPs, add Phi29 DNA polymerase (10U/μL) to initiate the reaction, incubate at 30°C for 10 min, then raise the temperature to 65°C to inactivate Phi29 DNA polymerase, and obtain the SEQ ID NO: The long single-stranded DNA shown in ID NO:5 and SEQ ID NO:6.

In the above preparation method, preferably, in step (2), the long single-stranded DNA shown in SEQ ID NO: 5 and SEQ ID NO: 6 and the long single-stranded DNA shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 6 and SEQ ID NO: 7 are taken successively. NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 short single-stranded DNA molecules, (wherein, preferably, the concentration ratio of the long single-stranded DNA to the short single-stranded DNA molecule is 1:10～200, more preferably 1:10, 1:20, 1:50, 1:100, 1:150 or 1:200, the optimal concentration ratio is 1:100), added to the annealing buffer containing The annealing buffer was TAE buffer containing 12mM Mg ²⁺ , mixed well, centrifuged at appropriate low speed, heat-treated at 95°C for 5 min, naturally annealed to 25°C overnight, to obtain DNA nanoladders.

In one embodiment, the present invention provides a method for constructing the above-mentioned multivalent aptamer DNA nano-ladder, comprising the following steps:

1) Preparation of long single-stranded DNA:

Perform rolling circle amplification (RCA) reaction with circular DNA templates A and B shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and in each reaction system, add 2 μL of SEQ ID NO: 3 in turn. and primers shown in SEQ ID NO: 4 (10 μM), 2 μL circular DNA template (10 μM), 2 μL 10×Phi29 DNA polymerase buffer and 2 μL dNTPs (7 mM), supplemented with ddH ₂ O to a total volume of 20 μL, and added 0.8 μL Phi29 DNA polymerase (10U/μL) initiated the reaction, incubated at 30°C for 10 min, and then raised to 65°C to inactivate Phi29 DNA polymerase, and the rolling circle amplification shown in SEQ ID NO: 5 and SEQ ID NO: 6 was obtained. (RCA) product chain A, chain B;

2) Preparation of DNA nanoladders:

In step 1), rolling circle amplification (RCA) product chain A and chain B are each 1 μL, such as SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and 1 μL (concentration 100 μM) of each short chain 1-6 shown in SEQ ID NO: 12 was added to a centrifuge tube filled with annealing buffer, the annealing buffer was TAE buffer containing 12mM Mg ²⁺ , supplemented with ddH ₂ O to 100 μL, mixed evenly, centrifuged at appropriate low speed, heat-treated at 95°C for 5 min, naturally annealed to 25°C overnight, to obtain DNA nano-ladders.

In another aspect, the present invention provides a multivalent aptamer DNA nano-ladder-anti-tumor drug complex, in which the above-mentioned DNA nano-ladder serves as a drug carrier to load anti-tumor drugs.

In the present invention, the manner of loading the antitumor drug is not particularly limited. For example, in one embodiment, the anti-tumor drug can be inserted between the DNA double strands through physical binding to form a DNA nano-ladder-anti-tumor drug complex. In this case, the antitumor drug may be anthracycline anticancer drugs such as doxorubicin hydrochloride (Dox), daunorubicin (DNR) and epirubicin (EPR), the aromatic groups of which can be Partially intercalates between DNA base pairs and coordinates with guanine bases on the DNA strand. In one embodiment, the antitumor drug may be doxorubicin hydrochloride (Dox). Alternatively, in another example, in the present invention, small interfering RNA (siRNA), cytosine-phosphate-bird can also be anchored by extending at least one of the short single-stranded DNA molecules without aptamer to the sticky end. Gene anti-tumor drugs such as glycosides (CPG), and obtain DNA nano-ladder multiple drug-carrying system.

In another aspect, the present invention provides a method for preparing the above-mentioned polyvalent aptamer DNA nano-ladder-anti-tumor drug complex, which comprises the step of using the above-mentioned DNA nano-ladder as a drug carrier to load an anti-tumor drug.

In one embodiment, the DNA nanoladder obtained by the above preparation method is co-incubated with an antitumor drug (eg, doxorubicin hydrochloride (Dox)) to prepare a complex. In the preparation method of the above complex, preferably, the DNA nano-ladder is incubated with 200 nM anti-tumor drug (for example, doxorubicin hydrochloride (Dox)) at 30°C for 30 min to prepare the DNA nano-ladder-anti-tumor drug (for example, Dox complex) ), about 2000 antitumor drug molecules (eg, doxorubicin hydrochloride (Dox)) can be loaded on each DNA nanoladder.

In one embodiment, the present invention provides a method for preparing a DNA nano-ladder-Dox complex, which comprises the following steps: incubating 2 μL of the above-prepared DNA nano-ladder with 98 μL of 200 nM doxorubicin hydrochloride (Dox) at 30°C for 30 min, Preparation of DNA nanoladder-Dox complexes.

The multivalent aptamer DNA nano-ladder of the present invention may have sustained release properties. DNA nano-ladders exist stably in phosphate-buffered saline (PBS) or fetal bovine serum (FBS) simulating the extracellular environment; under the condition that the intracellular environment contains DNA endonuclease (DNase I), the DNA nano-ladders The ladder is broken and the embedded drug is slowly released.

Drug release of the DNA nano-ladder-anti-tumor drug (eg, Dox) complex of the present invention: To simulate the intracellular environment, 3 μL of 1 U/μL DNA was added to the DNA nano-ladder-anti-tumor drug (eg, Dox) complex. Dicer (DNaseI), the DNA double-stranded structure is destroyed, and the drug is slowly released within 20min.

On the other hand, the present invention provides the application of the above-mentioned multivalent aptamer DNA nano-ladder in preparing a drug carrier, especially an antitumor drug carrier.

In another aspect, the present invention provides the application of the above polyvalent aptamer DNA nano-ladder in the preparation of diagnostic reagents.

In a specific embodiment, the multivalent aptamer DNA nanoladder comprises Sgc8 aptamer, and the diagnostic reagent can be used to detect acute lymphoblastic leukemia T lymphocytes.

In another aspect, the present invention provides the application of the above-mentioned DNA nano-ladder-Dox complex in the preparation of a medicament for tumor treatment and/or diagnosis.

beneficial effect

Compared with the prior art, the advantages of the present invention are:

(1) The DNA nano-ladder in the present invention has the advantages of suitable size, spatial addressability, strong biocompatibility, easy functional modification, etc., and can be used to deliver tumor targeting ligands, small interfering RNA (siRNA), A variety of anticancer drugs such as cytosine-phosphate-guanosine (CPG) and cytosine-phosphate-guanosine (CPG) are excellent drug carriers. (2) Rolling circle amplification (RCA) technology in the present invention, as an efficient isothermal enzymatic amplification method, the product has periodicity and high molecular weight, and can be used as a scaffold chain for periodic assembly of DNA nano-ladders, simplifying assembly design. (3) Due to its special two-dimensional and three-dimensional structure, the aptamer in the present invention has high affinity and selectivity for binding to the target, and can specifically recognize the target protein on the cell membrane.

The invention uses natural DNA molecules as raw materials, and on the basis of DNA origami theory, uses the special properties of rolling circle amplification (RCA) products to construct DNA nano-ladders, and introduces a large number of aptamers to construct a simple design, load-bearing A drug delivery system with strong drug capacity, target specificity and biocompatibility, and through aptamer or drug replacement, the carrier can also be applied to various types of target cells.

Description of drawings

Figure 1 shows the self-assembly of DNA nanoladders for drug delivery and the schematic diagram. Among them: A schematically shows the process of RCA reaction with circular DNA as a template to obtain long single-stranded DNA molecules; B schematically shows the self-assembly of 2 long-chain ssDNA molecules and 6 short-chain ssDNA molecules into a DNA nano-ladder repeating unit , and the process of the antitumor drug Dox (indicated by the solid circle) intercalated between DNA double strands to form a DNA nanoladder-Dox complex by physical binding; C schematically shows a DNA nanoladder-Dox complex with 3 repeating units The aptamer can specifically bind to the target receptor on tumor cells, and due to the multivalent effect, the endocytosis of the cell is enhanced, and the DNA nanoladder-Dox complex is finally degraded by the intracellular nuclease to release the drug.

FIG. 2 is an agarose gel electrophoresis image of rolling circle amplification (RCA) products in Example 1. FIG.

Figure 3 shows the characterization of the morphology of the DNA nano-ladder in Example 2, wherein (a) is a transmission electron microscope image of the DNA nano-ladder; (b) is an atomic force microscope image.

FIG. 4 is a graph showing the results of drug loading and release from DNA nanoladders in Example 3 and Example 4. FIG. (a) Monitoring of Doxorubicin HCl (Dox) loading on DNA nanoladders based on doxorubicin HCl (Dox) fluorescence quenching; (b) Doxorubicin HCl (Dox) loading from DNA nanoladders-Dox in the presence of DNase I complex release.

Detailed ways

In order to make the content of the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below, but the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The scope of the right protection of the present invention is subject to the claims.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The DNA sequence used in the present invention was purchased from Sangon Biotechnology Co., Ltd. Agarose, TAE buffer solution and DNA Ladder were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Doxorubicin hydrochloride was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The experimental water was ultrapure water prepared by Milli-Q IQ7000 ultrapure water system in Millipore, France.

Example 1: Preparation method of rolling circle amplification (RCA) product

Rolling circle amplification (RCA) reactions were performed with circular DNA templates A and B, respectively, and 2 μL of primers (10 μM), 2 μL of circular DNA template (10 μM), and 2 μL of 10×Phi29 DNA polymerase buffer were added to each reaction system in turn. and 2 μL of dNTPs (7 mM), supplemented with ddH ₂ O to a total volume of 20 μL, added 0.8 μL of Phi29 DNA polymerase (10 U/μL) to initiate the reaction, mixed well, centrifuged at low speed and placed in a constant temperature shaker for 10 min at 30 °C. Then, the Phi29 DNA polymerase was inactivated at a constant temperature of 65° C. for 10 min to obtain rolling circle amplification (RCA) product chain A and chain B. Rolling circle amplification (RCA) products were characterized by agarose gel electrophoresis about 8000 nt in length.

Table 1: Oligonucleotide sequences involved in Example 1

Example 2: Preparation method of DNA nano-ladder

The rolling circle amplification (RCA) products prepared in Example 1, 1 μL each of chain A and chain B, and 1 μL each of short chains 1-6 (concentration 100 μM), were added to a solution containing 42 μL of 2×TAE-Mg ²⁺ (24 mM). In the centrifuge tube, add ddH ₂ O to 100 μL, mix well, centrifuge at low speed, heat treatment at 95° C. for 5 min, and anneal to 25° C. naturally to prepare DNA nano-ladder.

Transmission electron microscopy was performed at an accelerating voltage of 200KV. Sample preparation for transmission electron microscopy: 5 μL of DNA nano-ladder sample was dropped onto the copper grid, allowed to stand for 5 minutes, stained with 1% uranyl acetate for 1 minute, and tested after the sample was completely dried.

The self-assembled product was characterized by transmission electron microscopy, as shown in Fig. 3(a), it can be seen that the shape of the product is linear with a width of about 10 nm, which is in line with the designed theoretical width.

Atomic force microscopy selects the Tapping Mode (AFM) test. Atomic force microscopy sample preparation: 10 μL of DNA nanoladder samples were dropped onto mica sheets and deposited for 10 minutes, washed with ddH ₂ O for 10 times, and tested after the samples were completely dried.

Due to the large length-diameter ratio of the DNA nano-ladder, it has a certain flexibility, as shown in Fig. 3(b), the entanglement between the DNA nano-ladders can be seen on the atomic force microscope image.

Table 2: Oligonucleotide sequences used in Example 2

Note: The underlined part in short chain 6 represents the Sgc8 aptamer sequence. In this example, the 5' end of the aptamer sequence is labeled with a fluorescent group.

Example 3: Drug Loading Experiment

The concentration of immobilized Dox was 200nM, and the concentration of DNA nanoladder was 1nM, 0.4nM, 0.2nM, 0.1nM, 0.04nM, 0.02nM, 0.01nM, 0.005nM, 0.0025nM, respectively, mixed well, centrifuged, and incubated at 30°C for 30min.

The detection method of the microplate reader is fluorescence intensity detection, the detection type is end-point scanning mode, the excitation wavelength is set to 485 nm, the emission wavelength is set to 590 nm, and the detection temperature is 30 °C.

Preparation method of DNA nanoladder-Dox complex: 200nM antitumor drug adriamycin hydrochloride (Dox) was mixed with 1nM, 0.4nM, 0.2nM, 0.1nM, 0.04nM, 0.02nM, 0.01nM, 0.005nM, 0.0025nM, respectively. DNA nanoladders were incubated at 30°C for 30min.

The antitumor drug doxorubicin hydrochloride (Dox) is inserted between the G-C bases of the DNA double-strand by physical binding to form a DNA nano-ladder-Dox complex, and its autofluorescence is quenched. As shown in Figure 4(a), DNA nanoladders with different concentrations were incubated with 200 nM doxorubicin hydrochloride (Dox), and the fluorescence intensity of Dox was measured by a microplate reader. As the concentration of DNA nanoladder increases, the degree of Dox fluorescence quenching increases. When the concentration of DNA nanoladder is 0.1 nM, the curve reaches an inflection point. The maximum drug loading of DNA nanoladder is determined by the molecular ratio of DNA nanoladder to drug. Represents about 1:2000.

Example 4: Drug release experiment

The microplate reader selects kinetic detection, the detection time is 90min, the detection time interval is 30s, the detection method is fluorescence intensity detection, the detection type is endpoint scanning mode, the excitation wavelength is set to 485nm, the emission wavelength is set to 590nm, and the detection temperature is 30℃ .

The DNA nano-ladder-Dox complex simulates drug release in the intracellular environment: 200 nM of Dox was incubated with 0.1 nM of DNA nano-ladder at 30°C for 30 min to obtain the DNA nano-ladder-Dox complex. 3 μL of 1U/μL DNA endonuclease (DNaseI) was added to the DNA nanoladder-Dox complex, and the DNA double-stranded structure was destroyed, as shown in Figure 4(b). 590nm) to measure the recovery of Dox fluorescence intensity, and the drug was slowly released within 20min.

sequence listing

<110> China University of Petroleum (East China)

Anqiu Plasticizer Factory

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aaagaaagaa aggaagaagt ttcaaggaaa ggaaacaaga aggcgaagac aggtgcttag 2340

tcgaaagaaa gaaaggaaga agtttcaagg aaaggaaaca agaaggcgaa gacaggtgct 2400

tagtcgaaag aaagaaagga agaagtttca aggaaaggaa acaagaaggc gaagacaggt 2460

gcttagtcga aagaaagaaa ggaagaagtt tcaaggaaag gaaacaagaa ggcgaagaca 2520

ggtgcttagt cgaaagaaag aaaggaagaa gtttcaagga aaggaaacaa gaaggcgaag 2580

acaggtgctt agtcgaaaga aagaaaggaa gaagtttcaa ggaaaggaaa caagaaggcg 2640

aagacaggtg cttagtcgaa agaaagaaag gaagaagtttt caaggaaagg aaacaagaag 2700

gcgaagacag gtgcttagtc gaaagaaaga aaggaagaag tttcaaggaa aggaaacaag 2760

aaggcgaaga caggtgctta gtcgaaagaa agaaaggaag aagtttcaag gaaaggaaac 2820

aagaaggcga agacaggtgc ttagtcgaaa gaaagaaagg aagaagtttc aaggaaagga 2880

aacaagaagg cgaagacagg tgcttagtcg aaagaaagaa aggaagaagt ttcaaggaaa 2940

ggaaacaaga aggcgaagac aggtgcttag tcgaaagaaa gaaaggaaga agtttcaagg 3000

aaaggaaaca agaaggcgaa gacaggtgct tagtcgaaag aaagaaagga agaagtttca 3060

aggaaaggaa acaagaaggc gaagacaggt gcttagtcga aagaaagaaa ggaagaagtt 3120

tcaaggaaag gaaacaagaa ggcgaagaca ggtgcttagt cgaaagaaag aaaggaagaa 3180

gtttcaagga aaggaaacaa gaaggcgaag acaggtgctt agtcgaaaga aagaaaggaa 3240

gaagtttcaa ggaaaggaaa caagaaggcg aagacaggtg cttagtcgaa agaaagaaag 3300

gaagaagttt caaggaaagg aaacaagaag gcgaagacag gtgcttagtc gaaagaaaga 3360

aaggaagaag tttcaaggaa aggaaacaag aaggcgaaga caggtgctta gtcgaaagaa 3420

agaaaggaag aagtttcaag gaaaggaaac aagaaggcga agacaggtgc ttagtcgaaa 3480

gaaagaaagg aagaagtttc aaggaaagga aacaagaagg cgaagacagg tgcttagtcg 3540

aaagaaagaa aggaagaagt ttcaaggaaa ggaaacaaga aggcgaagac aggtgcttag 3600

tcgaaagaaa gaaaggaaga agtttcaagg aaaggaaaca agaaggcgaa gacaggtgct 3660

tagtcgaaag aaagaaagga agaagtttca aggaaaggaa acaagaaggc gaagacaggt 3720

gcttagtcga aagaaagaaa ggaagaagtt tcaaggaaag gaaacaagaa ggcgaagaca 3780

ggtgcttagt cgaaagaaag aaaggaagaa gtttcaagga aaggaaacaa gaaggcgaag 3840

acaggtgctt agtcgaaaga aagaaaggaa gaagtttcaa ggaaaggaaa caagaaggcg 3900

aagacaggtg cttagtcgaa agaaagaaag gaagaagttt caaggaaagg aaacaagaag 3960

gcgaagacag gtgcttagtc gaaagaaaga aaggaagaag tttcaaggaa aggaaacaag 4020

aaggcgaaga caggtgctta gtcgaaagaa agaaaggaag aagtttcaag gaaaggaaac 4080

aagaaggcga agacaggtgc ttagtcgaaa gaaagaaagg aagaagtttc aaggaaagga 4140

aacaagaagg cgaagacagg tgcttagtcg aaagaaagaa aggaagaagt ttcaaggaaa 4200

ggaaacaaga aggcgaagac aggtgcttag tcgaaagaaa gaaaggaaga agtttcaagg 4260

aaaggaaaca agaaggcgaa gacaggtgct tagtcgaaag aaagaaagga agaagtttca 4320

aggaaaggaa acaagaaggc gaagacaggt gcttagtcga aagaaagaaa ggaagaagtt 4380

tcaaggaaag gaaacaagaa ggcgaagaca ggtgcttagt cgaaagaaag aaaggaagaa 4440

gtttcaagga aaggaaacaa gaaggcgaag acaggtgctt agtcgaaaga aagaaaggaa 4500

gaagtttcaa ggaaaggaaa caagaaggcg aagacaggtg cttagtcgaa agaaagaaag 4560

gaagaagttt caaggaaagg aaacaagaag gcgaagacag gtgcttagtc gaaagaaaga 4620

aaggaagaag tttcaaggaa aggaaacaag aaggcgaaga caggtgctta gtcgaaagaa 4680

agaaaggaag aagtttcaag gaaaggaaac aagaaggcga agacaggtgc ttagtcgaaa 4740

gaaagaaagg aagaagtttc aaggaaagga aacaagaagg cgaagacagg tgcttagtcg 4800

aaagaaagaa aggaagaagt ttcaaggaaa ggaaacaaga aggcgaagac aggtgcttag 4860

tcgaaagaaa gaaaggaaga agtttcaagg aaaggaaaca agaaggcgaa gacaggtgct 4920

tagtcgaaag aaagaaagga agaagtttca aggaaaggaa acaagaaggc gaagacaggt 4980

gcttagtcga aagaaagaaa ggaagaagtt tcaaggaaag gaaacaagaa ggcgaagaca 5040

ggtgcttagt cgaaagaaag aaaggaagaa gtttcaagga aaggaaacaa gaaggcgaag 5100

acaggtgctt agtcgaaaga aagaaaggaa gaagtttcaa ggaaaggaaa caagaaggcg 5160

aagacaggtg cttagtcgaa agaaagaaag gaagaagtttt caaggaaagg aaacaagaag 5220

gcgaagacag gtgcttagtc gaaagaaaga aaggaagaag tttcaaggaa aggaaacaag 5280

aaggcgaaga caggtgctta gtcgaaagaa agaaaggaag aagtttcaag gaaaggaaac 5340

aagaaggcga agacaggtgc ttagtcgaaa gaaagaaagg aagaagtttc aaggaaagga 5400

aacaagaagg cgaagacagg tgcttagtcg aaagaaagaa aggaagaagt ttcaaggaaa 5460

ggaaacaaga aggcgaagac aggtgcttag tcgaaagaaa gaaaggaaga agtttcaagg 5520

aaaggaaaca agaaggcgaa gacaggtgct tagtcgaaag aaagaaagga agaagtttca 5580

aggaaaggaa acaagaaggc gaagacaggt gcttagtcga aagaaagaaa ggaagaagtt 5640

tcaaggaaag gaaacaagaa ggcgaagaca ggtgcttagt cgaaagaaag aaaggaagaa 5700

gtttcaagga aaggaaacaa gaaggcgaag acaggtgctt agtcgaaaga aagaaaggaa 5760

gaagtttcaa ggaaaggaaa caagaaggcg aagacaggtg cttagtcgaa agaaagaaag 5820

gaagaagttt caaggaaagg aaacaagaag gcgaagacag gtgcttagtc gaaagaaaga 5880

aaggaagaag tttcaaggaa aggaaacaag aaggcgaaga caggtgctta gtcgaaagaa 5940

agaaaggaag aagtttcaag gaaaggaaac aagaaggcga agacaggtgc ttagtcgaaa 6000

gaaagaaagg aagaagtttc aaggaaagga aacaagaagg cgaagacagg tgcttagtcg 6060

aaagaaagaa aggaagaagt ttcaaggaaa ggaaacaaga aggcgaagac aggtgcttag 6120

tcgaaagaaa gaaaggaaga agtttcaagg aaaggaaaca agaaggcgaa gacaggtgct 6180

tagtcgaaag aaagaaagga agaagtttca aggaaaggaa acaagaaggc gaagacaggt 6240

gcttagtcga aagaaagaaa ggaagaagtt tcaaggaaag gaaacaagaa ggcgaagaca 6300

ggtgcttagt cgaaagaaag aaaggaagaa gtttcaagga aaggaaacaa gaaggcgaag 6360

acaggtgctt agtcgaaaga aagaaaggaa gaagtttcaa ggaaaggaaa caagaaggcg 6420

aagacaggtg cttagtcgaa agaaagaaag gaagaagtttt caaggaaagg aaacaagaag 6480

gcgaagacag gtgcttagtc gaaagaaaga aaggaagaag tttcaaggaa aggaaacaag 6540

aaggcgaaga caggtgctta gtcgaaagaa agaaaggaag aagtttcaag gaaaggaaac 6600

aagaaggcga agacaggtgc ttagtcgaaa gaaagaaagg aagaagtttc aaggaaagga 6660

aacaagaagg cgaagacagg tgcttagtcg aaagaaagaa aggaagaagt ttcaaggaaa 6720

ggaaacaaga aggcgaagac aggtgcttag tcgaaagaaa gaaaggaaga agtttcaagg 6780

aaaggaaaca agaaggcgaa gacaggtgct tagtcgaaag aaagaaagga agaagtttca 6840

aggaaaggaa acaagaaggc gaagacaggt gcttagtcga aagaaagaaa ggaagaagtt 6900

tcaaggaaag gaaacaagaa ggcgaagaca ggtgcttagt cgaaagaaag aaaggaagaa 6960

gtttcaagga aaggaaacaa gaaggcgaag acaggtgctt agtcgaaaga aagaaaggaa 7020

gaagtttcaa ggaaaggaaa caagaaggcg aagacaggtg cttagtcgaa agaaagaaag 7080

gaagaagttt caaggaaagg aaacaagaag gcgaagacag gtgcttagtc gaaagaaaga 7140

aaggaagaag tttcaaggaa aggaaacaag aaggcgaaga caggtgctta gtcgaaagaa 7200

agaaaggaag aagtttcaag gaaaggaaac aagaaggcga agacaggtgc ttagtcgaaa 7260

gaaagaaagg aagaagtttc aaggaaagga aacaagaagg cgaagacagg tgcttagtcg 7320

aaagaaagaa aggaagaagt ttcaaggaaa ggaaacaaga aggcgaagac aggtgcttag 7380

tcgaaagaaa gaaaggaaga agtttcaagg aaaggaaaca agaaggcgaa gacaggtgct 7440

tagtcgaaag aaagaaagga agaagtttca aggaaaggaa acaagaaggc gaagacaggt 7500

gcttagtcga aagaaagaaa ggaagaagtt tcaaggaaag gaaacaagaa ggcgaagaca 7560

ggtgcttagt cgaaagaaag aaaggaagaa gtttcaagga aaggaaacaa gaaggcgaag 7620

acaggtgctt agtcgaaaga aagaaaggaa gaagtttcaa ggaaaggaaa caagaaggcg 7680

aagacaggtg cttagtcgaa agaaagaaag gaagaagttt caaggaaagg aaacaagaag 7740

gcgaagacag gtgcttagtc gaaagaaaga aaggaagaag tttcaaggaa aggaaacaag 7800

aaggcgaaga caggtgctta gtcgaaagaa agaaaggaag aagtttcaag gaaaggaaac 7860

aagaaggcga agacaggtgc ttagtcgaaa gaaagaaagg aagaagtttc aaggaaagga 7920

aacaagaagg cgaagacagg tgcttagtcg aaagaaagaa aggaagaagt ttcaaggaaa 7980

ggaaacaaga aggcgaagac a 8001

<210> 6

<211> 8001

<212> DNA

<213> Artificial sequences

<220>

<223> Chain B

<400> 6

agggcttggc atagacgagt tgacagagac ggaatccgac cattgtgcgc tatcttcatc 60

ttaagggctt ggcatagacg agttgacaga gacggaatcc gaccattgtg cgctatcttc 120

atcttaaggg cttggcatag acgagttgac agagacggaa tccgaccatt gtgcgctatc 180

ttcatcttaa gggcttggca tagacgagtt gacagagacg gaatccgacc attgtgcgct 240

atcttcatct taagggcttg gcatagacga gttgacagag acggaatccg accattgtgc 300

gctatcttca tcttaagggc ttggcataga cgagttgaca gagacggaat ccgaccattg 360

tgcgctatct tcatcttaag ggcttggcat agacgagttg acagagacgg aatccgacca 420

ttgtgcgcta tcttcatctt aagggcttgg catagacgag ttgacagaga cggaatccga 480

ccattgtgcg ctatcttcat cttaagggct tggcatagac gagttgacag agacggaatc 540

cgaccattgt gcgctatctt catcttaagg gcttggcata gacgagttga cagagacgga 600

atccgaccat tgtgcgctat cttcatctta agggcttggc atagacgagt tgacagagac 660

ggaatccgac cattgtgcgc tatcttcatc ttaagggctt ggcatagacg agttgacaga 720

gacggaatcc gaccattgtg cgctatcttc atcttaaggg cttggcatag acgagttgac 780

agagacggaa tccgaccatt gtgcgctatc ttcatcttaa gggcttggca tagacgagtt 840

gacagagacg gaatccgacc attgtgcgct atcttcatct taagggcttg gcatagacga 900

gttgacagag acggaatccg accattgtgc gctatcttca tcttaagggc ttggcataga 960

cgagttgaca gagacggaat ccgaccattg tgcgctatct tcatcttaag ggcttggcat 1020

agacgagttg acagagacgg aatccgacca ttgtgcgcta tcttcatctt aagggcttgg 1080

catagacgag ttgacagaga cggaatccga ccattgtgcg ctatcttcat cttaagggct 1140

tggcatagac gagttgacag agacggaatc cgaccattgt gcgctatctt catcttaagg 1200

gcttggcata gacgagttga cagagacgga atccgaccat tgtgcgctat cttcatctta 1260

agggcttggc atagacgagt tgacagagac ggaatccgac cattgtgcgc tatcttcatc 1320

ttaagggctt ggcatagacg agttgacaga gacggaatcc gaccattgtg cgctatcttc 1380

atcttaaggg cttggcatag acgagttgac agagacggaa tccgaccatt gtgcgctatc 1440

ttcatcttaa gggcttggca tagacgagtt gacagagacg gaatccgacc attgtgcgct 1500

atcttcatct taagggcttg gcatagacga gttgacagag acggaatccg accattgtgc 1560

gctatcttca tcttaagggc ttggcataga cgagttgaca gagacggaat ccgaccattg 1620

tgcgctatct tcatcttaag ggcttggcat agacgagttg acagagacgg aatccgacca 1680

ttgtgcgcta tcttcatctt aagggcttgg catagacgag ttgacagaga cggaatccga 1740

ccattgtgcg ctatcttcat cttaagggct tggcatagac gagttgacag agacggaatc 1800

cgaccattgt gcgctatctt catcttaagg gcttggcata gacgagttga cagagacgga 1860

atccgaccat tgtgcgctat cttcatctta agggcttggc atagacgagt tgacagagac 1920

ggaatccgac cattgtgcgc tatcttcatc ttaagggctt ggcatagacg agttgacaga 1980

gacggaatcc gaccattgtg cgctatcttc atcttaaggg cttggcatag acgagttgac 2040

agagacggaa tccgaccatt gtgcgctatc ttcatcttaa gggcttggca tagacgagtt 2100

gacagagacg gaatccgacc attgtgcgct atcttcatct taagggcttg gcatagacga 2160

gttgacagag acggaatccg accattgtgc gctatcttca tcttaagggc ttggcataga 2220

cgagttgaca gagacggaat ccgaccattg tgcgctatct tcatcttaag ggcttggcat 2280

agacgagttg acagagacgg aatccgacca ttgtgcgcta tcttcatctt aagggcttgg 2340

catagacgag ttgacagaga cggaatccga ccattgtgcg ctatcttcat cttaagggct 2400

tggcatagac gagttgacag agacggaatc cgaccattgt gcgctatctt catcttaagg 2460

gcttggcata gacgagttga cagagacgga atccgaccat tgtgcgctat cttcatctta 2520

agggcttggc atagacgagt tgacagagac ggaatccgac cattgtgcgc tatcttcatc 2580

ttaagggctt ggcatagacg agttgacaga gacggaatcc gaccattgtg cgctatcttc 2640

atcttaaggg cttggcatag acgagttgac agagacggaa tccgaccatt gtgcgctatc 2700

ttcatcttaa gggcttggca tagacgagtt gacagagacg gaatccgacc attgtgcgct 2760

atcttcatct taagggcttg gcatagacga gttgacagag acggaatccg accattgtgc 2820

gctatcttca tcttaagggc ttggcataga cgagttgaca gagacggaat ccgaccattg 2880

tgcgctatct tcatcttaag ggcttggcat agacgagttg acagagacgg aatccgacca 2940

ttgtgcgcta tcttcatctt aagggcttgg catagacgag ttgacagaga cggaatccga 3000

ccattgtgcg ctatcttcat cttaagggct tggcatagac gagttgacag agacggaatc 3060

cgaccattgt gcgctatctt catcttaagg gcttggcata gacgagttga cagagacgga 3120

atccgaccat tgtgcgctat cttcatctta agggcttggc atagacgagt tgacagagac 3180

ggaatccgac cattgtgcgc tatcttcatc ttaagggctt ggcatagacg agttgacaga 3240

gacggaatcc gaccattgtg cgctatcttc atcttaaggg cttggcatag acgagttgac 3300

agagacggaa tccgaccatt gtgcgctatc ttcatcttaa gggcttggca tagacgagtt 3360

gacagagacg gaatccgacc attgtgcgct atcttcatct taagggcttg gcatagacga 3420

gttgacagag acggaatccg accattgtgc gctatcttca tcttaagggc ttggcataga 3480

cgagttgaca gagacggaat ccgaccattg tgcgctatct tcatcttaag ggcttggcat 3540

agacgagttg acagagacgg aatccgacca ttgtgcgcta tcttcatctt aagggcttgg 3600

catagacgag ttgacagaga cggaatccga ccattgtgcg ctatcttcat cttaagggct 3660

tggcatagac gagttgacag agacggaatc cgaccattgt gcgctatctt catcttaagg 3720

gcttggcata gacgagttga cagagacgga atccgaccat tgtgcgctat cttcatctta 3780

agggcttggc atagacgagt tgacagagac ggaatccgac cattgtgcgc tatcttcatc 3840

ttaagggctt ggcatagacg agttgacaga gacggaatcc gaccattgtg cgctatcttc 3900

atcttaaggg cttggcatag acgagttgac agagacggaa tccgaccatt gtgcgctatc 3960

ttcatcttaa gggcttggca tagacgagtt gacagagacg gaatccgacc attgtgcgct 4020

atcttcatct taagggcttg gcatagacga gttgacagag acggaatccg accattgtgc 4080

gctatcttca tcttaagggc ttggcataga cgagttgaca gagacggaat ccgaccattg 4140

tgcgctatct tcatcttaag ggcttggcat agacgagttg acagagacgg aatccgacca 4200

ttgtgcgcta tcttcatctt aagggcttgg catagacgag ttgacagaga cggaatccga 4260

ccattgtgcg ctatcttcat cttaagggct tggcatagac gagttgacag agacggaatc 4320

cgaccattgt gcgctatctt catcttaagg gcttggcata gacgagttga cagagacgga 4380

atccgaccat tgtgcgctat cttcatctta agggcttggc atagacgagt tgacagagac 4440

ggaatccgac cattgtgcgc tatcttcatc ttaagggctt ggcatagacg agttgacaga 4500

gacggaatcc gaccattgtg cgctatcttc atcttaaggg cttggcatag acgagttgac 4560

agagacggaa tccgaccatt gtgcgctatc ttcatcttaa gggcttggca tagacgagtt 4620

gacagagacg gaatccgacc attgtgcgct atcttcatct taagggcttg gcatagacga 4680

gttgacagag acggaatccg accattgtgc gctatcttca tcttaagggc ttggcataga 4740

cgagttgaca gagacggaat ccgaccattg tgcgctatct tcatcttaag ggcttggcat 4800

agacgagttg acagagacgg aatccgacca ttgtgcgcta tcttcatctt aagggcttgg 4860

catagacgag ttgacagaga cggaatccga ccattgtgcg ctatcttcat cttaagggct 4920

tggcatagac gagttgacag agacggaatc cgaccattgt gcgctatctt catcttaagg 4980

gcttggcata gacgagttga cagagacgga atccgaccat tgtgcgctat cttcatctta 5040

agggcttggc atagacgagt tgacagagac ggaatccgac cattgtgcgc tatcttcatc 5100

ttaagggctt ggcatagacg agttgacaga gacggaatcc gaccattgtg cgctatcttc 5160

atcttaaggg cttggcatag acgagttgac agagacggaa tccgaccatt gtgcgctatc 5220

ttcatcttaa gggcttggca tagacgagtt gacagagacg gaatccgacc attgtgcgct 5280

atcttcatct taagggcttg gcatagacga gttgacagag acggaatccg accattgtgc 5340

gctatcttca tcttaagggc ttggcataga cgagttgaca gagacggaat ccgaccattg 5400

tgcgctatct tcatcttaag ggcttggcat agacgagttg acagagacgg aatccgacca 5460

ttgtgcgcta tcttcatctt aagggcttgg catagacgag ttgacagaga cggaatccga 5520

ccattgtgcg ctatcttcat cttaagggct tggcatagac gagttgacag agacggaatc 5580

cgaccattgt gcgctatctt catcttaagg gcttggcata gacgagttga cagagacgga 5640

atccgaccat tgtgcgctat cttcatctta agggcttggc atagacgagt tgacagagac 5700

ggaatccgac cattgtgcgc tatcttcatc ttaagggctt ggcatagacg agttgacaga 5760

gacggaatcc gaccattgtg cgctatcttc atcttaaggg cttggcatag acgagttgac 5820

agagacggaa tccgaccatt gtgcgctatc ttcatcttaa gggcttggca tagacgagtt 5880

gacagagacg gaatccgacc attgtgcgct atcttcatct taagggcttg gcatagacga 5940

gttgacagag acggaatccg accattgtgc gctatcttca tcttaagggc ttggcataga 6000

cgagttgaca gagacggaat ccgaccattg tgcgctatct tcatcttaag ggcttggcat 6060

agacgagttg acagagacgg aatccgacca ttgtgcgcta tcttcatctt aagggcttgg 6120

catagacgag ttgacagaga cggaatccga ccattgtgcg ctatcttcat cttaagggct 6180

tggcatagac gagttgacag agacggaatc cgaccattgt gcgctatctt catcttaagg 6240

gcttggcata gacgagttga cagagacgga atccgaccat tgtgcgctat cttcatctta 6300

agggcttggc atagacgagt tgacagagac ggaatccgac cattgtgcgc tatcttcatc 6360

ttaagggctt ggcatagacg agttgacaga gacggaatcc gaccattgtg cgctatcttc 6420

atcttaaggg cttggcatag acgagttgac agagacggaa tccgaccatt gtgcgctatc 6480

ttcatcttaa gggcttggca tagacgagtt gacagagacg gaatccgacc attgtgcgct 6540

atcttcatct taagggcttg gcatagacga gttgacagag acggaatccg accattgtgc 6600

gctatcttca tcttaagggc ttggcataga cgagttgaca gagacggaat ccgaccattg 6660

tgcgctatct tcatcttaag ggcttggcat agacgagttg acagagacgg aatccgacca 6720

ttgtgcgcta tcttcatctt aagggcttgg catagacgag ttgacagaga cggaatccga 6780

ccattgtgcg ctatcttcat cttaagggct tggcatagac gagttgacag agacggaatc 6840

cgaccattgt gcgctatctt catcttaagg gcttggcata gacgagttga cagagacgga 6900

atccgaccat tgtgcgctat cttcatctta agggcttggc atagacgagt tgacagagac 6960

ggaatccgac cattgtgcgc tatcttcatc ttaagggctt ggcatagacg agttgacaga 7020

gacggaatcc gaccattgtg cgctatcttc atcttaaggg cttggcatag acgagttgac 7080

agagacggaa tccgaccatt gtgcgctatc ttcatcttaa gggcttggca tagacgagtt 7140

gacagagacg gaatccgacc attgtgcgct atcttcatct taagggcttg gcatagacga 7200

gttgacagag acggaatccg accattgtgc gctatcttca tcttaagggc ttggcataga 7260

cgagttgaca gagacggaat ccgaccattg tgcgctatct tcatcttaag ggcttggcat 7320

agacgagttg acagagacgg aatccgacca ttgtgcgcta tcttcatctt aagggcttgg 7380

catagacgag ttgacagaga cggaatccga ccattgtgcg ctatcttcat cttaagggct 7440

tggcatagac gagttgacag agacggaatc cgaccattgt gcgctatctt catcttaagg 7500

gcttggcata gacgagttga cagagacgga atccgaccat tgtgcgctat cttcatctta 7560

agggcttggc atagacgagt tgacagagac ggaatccgac cattgtgcgc tatcttcatc 7620

ttaagggctt ggcatagacg agttgacaga gacggaatcc gaccattgtg cgctatcttc 7680

atcttaaggg cttggcatag acgagttgac agagacggaa tccgaccatt gtgcgctatc 7740

ttcatcttaa gggcttggca tagacgagtt gacagagacg gaatccgacc attgtgcgct 7800

atcttcatct taagggcttg gcatagacga gttgacagag acggaatccg accattgtgc 7860

gctatcttca tcttaagggc ttggcataga cgagttgaca gagacggaat ccgaccattg 7920

tgcgctatct tcatcttaag ggcttggcat agacgagttg acagagacgg aatccgacca 7980

ttgtgcgcta tcttcatctt a 8001

<210> 7

<211> 42

<212> DNA

<213> Artificial sequences

<220>

<223> Short Chain 1

<400> 7

tgtcttcgcc ttcttgtttc cacggtacgt atcggactat tg 42

<210> 8

<211> 42

<212> DNA

<213> Artificial sequences

<220>

<223> Short Chain 2

<400> 8

tttccttgaa acttcttcct tgactgtcga ttacccgtta at 42

<210> 9

<211> 42

<212> DNA

<213> Artificial sequences

<220>

<223> Short Chain 3

<400> 9

tctttctttc gactaagcac cgcagtatga catcgaccgg at 42

<210> 10

<211> 42

<212> DNA

<213> Artificial sequences

<220>

<223> Short Chain 4

<400> 10

tggtcggatt ccgtctctgt ccaatagtcc gatacgtacc gt 42

<210> 11

<211> 42

<212> DNA

<213> Artificial sequences

<220>

<223> Short Chain 5

<400> 11

taagatgaag atagcgcaca aattaacggg taatcgacag tc 42

<210> 12

<211> 83

<212> DNA

<213> Artificial sequences

<220>

<223> Short Chain 6

<400> 12

atctaactgc tgcgccgccg ggaaaatact gtacggttag aaactcgtct atgccaagcc 60

ctatccggtc gatgtcatac tgc 83

<210> 13

<211> 63

<212> DNA

<213> Artificial sequences

<220>

<223> Long single-stranded DNA molecules

<400> 13

ggtgcttagt cgaaagaaag aaaggaagaa gtttcaagga aaggaaacaa gaaggcgaag 60

aca 63

<210> 14

<211> 63

<212> DNA

<213> Artificial sequences

<220>

<223> Long single-stranded DNA molecules

<400> 14

agggcttggc atagacgagt tgacagagac ggaatccgac cattgtgcgc tatcttcatc 60

tta 63

Claims (12)

1. A multivalent aptamer DNA nanostring, comprising m tandem repeat units comprising two long single-stranded DNA molecules and 2n short single-stranded DNA molecules, wherein m is 127; n is a number of 3, and,

wherein, in the repeating unit, the sequences of the two long single-strand DNA molecules are different; at least one short single-stranded DNA molecule in the 2n short single-stranded DNA molecules is connected with an adapter, each short single-stranded DNA molecule is provided with two sections of hybridization regions, one of the two sections of hybridization regions is hybridized with one part of the long single-stranded DNA molecule, the other hybridization region is hybridized with one part of the other short single-stranded DNA molecule to form a double-helix structure, and therefore the two long single-stranded DNA molecules and the 2n short single-stranded DNA molecules are self-assembled into a DNA nano ladder,

wherein the aptamer is acute lymphoblastic leukemia T lymphocyte aptamer Sgc8,

wherein, in the repeating unit, the two long single-stranded DNA molecules are shown as SEQ ID NO. 13 and SEQ ID NO. 14, and the 2n short single-stranded DNA molecules are shown as SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12.

2. The multivalent aptamer DNA nanoslide of claim 1, wherein the aptamer is labeled with a fluorophore at the 5' end.

3. The method of preparing a multivalent aptamer DNA nano ladder according to claim 1 or 2, comprising the steps of:

(1) synthesizing two long single-stranded DNAs each having a nucleotide sequence of m long single-stranded DNA molecules connected in series by Rolling Circle Amplification (RCA) reactions, respectively;

(2) and (2) constructing the DNA nano ladder by combining m multiplied by 2n short single-stranded DNA molecules by taking the two long single-stranded DNAs synthesized in the step (1) as scaffold chains based on DNA origami.

4. The production method according to claim 3,

in the step (1), respectively taking the circular DNA shown as SEQ ID NO. 1 and SEQ ID NO. 2 as a template, adding Phi29 DNA polymerase in the presence of DNA primers shown as SEQ ID NO. 3 and SEQ ID NO. 4, Phi29 DNA polymerase buffer solution and dNTPs to initiate reaction, incubating at 30 ℃ for 10min, raising the temperature to 65 ℃ to inactivate Phi29 DNA polymerase, and preparing the long single-strand DNA shown as SEQ ID NO. 5 and SEQ ID NO. 6,

in the step (2), the long single-stranded DNA shown as SEQ ID NO. 5 and SEQ ID NO. 6 and the DNA are taken in sequenceThe short single-stranded DNA molecules shown as SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12 were added to a centrifuge tube containing an annealing buffer solution containing 12mM Mg²⁺The TAE buffer solution is evenly mixed, properly centrifuged at low speed, thermally treated at 95 ℃ for 5min, naturally annealed to 25 ℃, and kept overnight to prepare the DNA nano ladder.

5. The method of claim 3, comprising the steps of:

1) preparation of Long Single-stranded DNA:

respectively using the circular DNA templates A, B shown as SEQ ID NO. 1 and SEQ ID NO. 2 to carry out Rolling Circle Amplification (RCA) reaction, sequentially adding 2 muL of primers shown as SEQ ID NO. 3 and SEQ ID NO. 4 with the concentration of 10 muM, 2 muL of circular DNA template with the concentration of 10 muM, 2 muL of 10 xPhi 29 DNA polymerase buffer solution and 2 muL of dNTPs with the concentration of 7mM in each reaction system, and supplementing ddH₂O to the total volume of 20 mu L, adding 0.8 mu L of Phi29 DNA polymerase of 10U/. mu.L to initiate reaction, incubating at 30 ℃ for 10min, and then raising to 65 ℃ to inactivate the Phi29 DNA polymerase to prepare Rolling Circle Amplification (RCA) products of a chain A and a chain B shown as SEQ ID NO. 5 and SEQ ID NO. 6;

2) preparing a DNA nano ladder:

in step 1), 1 μ L of each of the product chain A and chain B of the Rolling Circle Amplification (RCA), 1 μ L of each of the short chains 1-6 shown as SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12 with the concentration of 100 μ M are added into a centrifuge tube containing an annealing buffer solution containing 12mM Mg²⁺TAE buffer of (5), supplement of ddH₂O to 100 mu L, mixing evenly, centrifuging at a proper low speed, carrying out heat treatment at 95 ℃ for 5min, naturally annealing to 25 ℃, and standing overnight to obtain the DNA nano ladder.

6. A polyvalent aptamer DNA nano ladder-antitumor drug complex, wherein the polyvalent aptamer DNA nano ladder of claim 1 or 2 is used as a drug carrier to carry an antitumor drug.

7. The polyvalent aptamer DNA nano ladder-antitumor drug complex of claim 6, wherein said antitumor drug is inserted between DNA double strands by means of physical binding to form a DNA nano ladder-antitumor drug complex; alternatively, the genetic anti-tumor drug is anchored by extending at least one of the short single-stranded DNA molecules to which the aptamer is not linked out of the cohesive end.

8. The polyvalent aptamer DNA nano ladder-antitumor drug complex of claim 7, wherein the antitumor drug is inserted between DNA double strands by means of physical binding to form a DNA nano ladder-antitumor drug complex, and the antitumor drug is an anthracycline anticancer drug.

9. The polyvalent aptamer DNA nanosladder-antitumor drug complex of claim 8, wherein said antitumor drug is selected from doxorubicin hydrochloride (Dox), Daunorubicin (DNR) and Epirubicin (EPR).

10. The multivalent aptamer DNA nanosladder-antineoplastic drug complex of claim 7, wherein the genotypic antineoplastic drug is selected from the group consisting of small interfering rna (sirna) and cytosine-phosphate-guanosine (CPG).

11. A method of preparing a polyvalent aptamer DNA nano ladder-antitumor drug complex as claimed in any one of claims 6-10, comprising the step of loading an antitumor drug using the polyvalent aptamer DNA nano ladder as claimed in claim 1 or 2 as a drug carrier.

12. Use of the DNA ladder of claim 1 or 2 for the preparation of a carrier for an anti-tumor drug.

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