Biological preparation method and application of DNA (deoxyribonucleic acid) nanostructure with aptamer for targeting tumor cells

Abstract

The invention discloses a preparation method and application of an aptamer functionalized DNA nano-structure targeting tumor cells, the DNA nano-structure is prepared by performing enzyme digestion, annealing and origami on loop single-stranded DNA produced by a biological method, and particularly relates to optimization of deoxyribozyme in phagocytids, widening of pH range of cut loop single-stranded DNA and shortening of enzyme digestion time. The single-stranded DNA capable of being automatically folded into the DNA nanostructure is efficiently prepared; according to the present invention, the nucleic acid aptamer is inserted into the customized sequence so as to functionalize the customized sequence, and the produced and prepared functionalized DNA nano-structure has characteristics of no toxicity on cells, drug loading, specific tumor cell targeting, and cell toxicity stronger than the free drug, and can achieve the specific targeting and killing effects on the tumor cells; therefore, efficient preparation of the DNA nanostructure can be realized, and functional targeted tumor cell drug release can be carried out.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to the preparation and application of an aptamer-functionalized DNA nanostructure targeting tumor cells. Background Art

[0002] DNA nanostructures have the advantages of precisely controllable size and structure, programmability, easy modification of functional groups, and good biocompatibility, and can be widely applied in multiple fields such as bioimaging, sensing, drug delivery, etc. However, due to the immature synthesis and preparation technology of DNA nanostructures, it is difficult to produce them on a large scale. Therefore, the efficient synthesis of the raw material single-stranded DNA (ssDNA) for preparing DNA nanostructures has become a key problem to be solved urgently.

[0003] The M13 phage method is a widely used method for producing ssDNA, which uses traditional DNA origami technology to fold DNA into various different nanostructures. However, the M13 phage method has a great limitation, that is, the sequence and length of the generated ssDNA are basically fixed and cannot be customized according to the needs of users. Phagemid (phasmid) is a new type of vector constructed to solve the problem of expressing customized DNA. This plasmid is constructed by combining a part of the sequence of the M13 genome with plasmid DNA. During the replication and production process, the host Escherichia coli needs to be infected by a helper phage or assisted by a helper plasmid to obtain the necessary replicase and protein coat, generate circular single-stranded DNA (cssDNA) through rolling circle replication, and be packaged and secreted extracellularly together with the protein coat. After extracting cssDNA from the packaged and secreted phage and removing the redundant fragments, customized single-stranded DNA is obtained. The advantages of the phagemid method lie not only in its user-defined characteristics but also in the expansion of its application prospects. The ssDNA produced by the phagemid method can not only be used to construct simple DNA origami structures but also be applied to the construction of more complex DNA nanostructures.

[0004] Deoxyribozyme (DNAzyme) is a ssDNA fragment with catalytic function and has high recognition and catalytic ability. By combining deoxyribozyme with phagemid to produce ssDNA, customized sequences with up to several thousand bases can be produced. Incubating the phage cssDNA with Zn 2+ together can trigger the programmed DNA hydrolysis of DNAzyme at specific sites, and can effectively and economically separate the target ssDNA fragment from the phagemid vector. In principle, various DNAzymes can be obtained by simply changing the sequence. Therefore, it can be expected to utilize this function to cut cssDNA to obtain customized ssDNA.

[0005] An aptamer is a short oligonucleotide sequence (RNA or ssDNA) that can fold into a specific three-dimensional structure and bind to a target with high specificity and high affinity. Its unique advantages have made it an ideal tool for tumor research in recent years. Combining the molecular recognition properties of aptamers with DNA nanostructures can integrate characteristics such as targeted recognition, bioimaging, and drug delivery. In the field of life science research, it can provide a more effective and low-side-effect method for tumor diagnosis and treatment, and has good application prospects in the field of tumors.

[0006] The present invention combines aptamers with the phagemid method to produce functionalized ssDNA, and realizes the simple and efficient preparation of functionalized DNA nanostructures by optimizing the DNAzyme sequence. With the targeting property of the aptamer, it realizes the function of specific targeted drug release of drug-loaded DNA nanostructures to tumor cells, providing a technical basis and reference for the functional application of DNA nanostructures. Summary of the Invention

[0007] The object of the present invention is to optimize the DNAzyme sequence, broaden the working pH range of DNAzyme, and perform enzymatic cleavage with high efficiency; and provide the preparation and application of a nucleic acid aptamer-functionalized DNA nanodevice that can specifically target tumor cells.

[0008] Before elaborating on the content of the present invention, the terms used herein are defined as follows:

[0009] The term "ssDNA" refers to: single-stranded DNA.

[0010] The term "DNAzyme" refers to: deoxyribozyme.

[0011] The term "AS1411" refers to: an aptamer that recognizes and targets nucleolin, with the sequence 5'-3' being -GGTGGTGGTGGTTGTGGTGGTGGTGGT-, and can enter the cell nucleus by relying on the shuttle effect of nucleolin in the cell.

[0012] The term "S2.2" refers to: an aptamer that specifically binds to the glycoprotein MUC1 widely expressed on the cell membranes of various tumors, with the sequence 5'-3' being -GCAGTTGATCCTTTGGATACCCTGG-.

[0013] The term "DOX" refers to: doxorubicin, an anti-tumor drug applicable to acute leukemia, malignant lymphoma, breast cancer, etc.

[0014] The first object of the present invention is to produce ssDNA using the phagemid method, optimize the DNAzyme sequence, and perform enzymatic cleavage of ssDNA with a wide pH range and high efficiency.

[0015] Among them, the produced ssDNA can self-fold into triangular DNA nanostructures through base complementary pairing.

[0016] Preferably, ssDNA is produced by co-culturing helper phage and Escherichia coli.

[0017] Preferably, the designed phagemid contains an optimized DNAzyme sequence and can be self-cleaved by Zn 2+ to obtain ssDNA.

[0018] Preferably, the pH range for the cleavage of DNAzyme is broadened.

[0019] Preferably, the time for the complete self-cleavage of DNAzyme is shortened.

[0020] The second object of the present invention is to prepare a nucleic acid aptamer-functionalized DNA nanostructure that can specifically target tumor cells.

[0021] Preferably, a functionalized DNA nanostructure is produced by constructing a phagemid containing an aptamer sequence.

[0022] Preferably, functional hairpin rectangles and triangular DNA nanostructures are folded by high-temperature annealing. The annealing program is from 85°C to 60°C, decreasing by 1°C every 10 minutes; from 60°C to 40°C, decreasing by 1°C every 30 minutes; from 40°C to 25°C, decreasing by 1°C every 15 minutes.

[0023] The third object of the present invention is to provide an application for targeted detection of tumor cells, including triangular DNA nanostructures, AS1411 aptamers that specifically recognize nucleolin, S2.2 aptamers that recognize MUC1 protein, and DOX that binds to the DNA nanostructure and can be detected by fluorescence.

[0024] Furthermore, the ability of the functionalized triangular DNA nanostructure to load the drug DOX is detected.

[0025] Furthermore, the biosafety of the functionalized triangular DNA nanostructure is detected.

[0026] Furthermore, DOX that can be detected with red fluorescence is bound to the aptamer-bearing functionalized triangular DNA nanostructure, and its cytotoxicity to tumor cells is detected.

[0027] Furthermore, the specific targeting of the functional triangular DNA nanostructure to tumor cells is detected.

[0028] The present invention has the following advantages:

[0029] The present invention optimizes the DNAzyme sequence, combines it into a phagemid, and through Zn 2+Induce the DNAzyme to undergo a cleavage reaction, which has a wide pH range for the cleavage reaction and a fast reaction rate; the present invention constructs and produces functional triangular and hairpin rectangular DNA nanostructures carrying aptamers through the phagemid method, and uses the triangular DNA nanostructure to specifically target cancer cells for drug release. The combined drug doxorubicin can be detected by fluorescence, so the diagnosis and treatment integration of target tumor cells can be achieved. Description of the Drawings

[0030] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 A is the electrophoresis pattern of the cleavage results of DNAzyme at different pH values. Lane M is the 2000 DNA Marker.

[0032] Figure 1 B is the cleavage efficiency curve at the corresponding pH in 1A.

[0033] Figure 1 C is the electrophoresis pattern of the cleavage results of DNAzyme when the reaction is carried out for 1 h. Lane M is the 2000 DNA Marker.

[0034] Figure 1 D is the atomic force microscopy (AFM) characterization diagram of the triangular DNA nanostructure.

[0035] Figure 2 A is the growth curve of the recombinant bacteria containing three types of triangular phagemids carrying aptamers.

[0036] Figure 2 B is the production curve of the three recombinant bacteria for producing functional triangular circular single-stranded DNA.

[0037] Figure 2 C is the agarose gel electrophoresis pattern of the functional triangular circular single-stranded DNA extracted at different times. Lane M is the 5000 DNA Marker. In the following electrophoresis patterns, Lane M is the 5000 Marker.

[0038] Figure 3 A is the comparison diagram before and after the aptamer-carrying triangular circular single-stranded DNA induces the self-cleavage of deoxyribozyme.

[0039] Figure 3 B is the AFM characterization diagram of the aptamer-carrying triangular DNA nanostructure.

[0040] Figure 4A shows the growth curve of a recombinant bacterium containing a hairpin rectangular phagemid with three kinds of aptamers.

[0041] Figure 4 B shows the production curve of functional hairpin rectangular loop single-stranded DNA by three kinds of recombinant bacteria.

[0042] Figure 4 C shows the agarose gel electrophoresis pattern of functional hairpin rectangular loop single-stranded DNA extracted at different times.

[0043] Figure 5 A shows the comparison diagram before and after the self-cleavage of deoxyribozyme induced by aptamer-containing hairpin rectangular loop single-stranded DNA.

[0044] Figure 5 B shows the AFM characterization of the aptamer-containing hairpin rectangular DNA nanostructure.

[0045] Figure 6 A shows the toxicity curve of the DNA nanostructure against MCF-7 cells.

[0046] Figure 6 B shows the toxicity curve of the DNA nanostructure loaded with DOX against MCF-7 cells.

[0047] Figure 7 A shows the laser confocal microscopy imaging results of the incubation of the DNA nanostructure with MCF-7 cells. The scale bar is 50 μm. The excitation wavelengths of Hoechst and Dox are 405 nm and 488 nm respectively, and the emission wavelengths are 450 nm and 525 nm respectively.

[0048] Figure 7 B shows the fluorescence analysis results in 7A, where F D / F H is the fluorescence rate of DOX / the fluorescence rate of Hoechst, ***p < 0.001. Detailed implementation manners

[0049] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present application.

[0050] The descriptions of the strains involved in the embodiments of the present application are shown in Table 1 below:

[0051] Table 1

[0052]

[0053] The technical solutions and beneficial effects of the present application will be further described below in conjunction with embodiments.

[0054] Example 1 Optimize the deoxyribozyme sequence, with a wide reaction pH range and short reaction time

[0055] In this case, based on the DNAzyme optimized by the Dietz team (Biotechnological mass production of DNA origami[J].Nature,2017), the fifth base A of the catalytic core was replaced with G to obtain the optimized DNAzyme. The DNAzyme was ligated into the phagemid by PCR to construct recombinant Escherichia coli. The recombinant bacteria produced phage particles with the assistance of helper phages, and circular single-stranded DNA was obtained by alkaline lysis.

[0056] Add cleavage reaction buffers with different pH values (100 mM HEPES, 200 mM NaCl, 8 mM ZnCl2, adjusted pH) to the obtained circular single-stranded DNA, and measure the cleavage at pH 6 - 8. After adding cleavage reaction buffers with different pH values, react overnight at 37°C, and perform agarose gel electrophoresis on the results of different groups. The results are as Figure 1 shown in A and B. The pH range of the constructed DNAzyme cleavage reaction is relatively wide, and 100% cleavage can be completed between 6.4 - 7.6.

[0057] Measure the cleavage efficiency of the DNAzyme, react at different pH values of the cleavage reaction buffer, and the results are as Figure 1 shown in C. The optimized DNAzyme can complete all cleavages in only 1 h within a wide pH range, improving the cleavage efficiency.

[0058] After inducing the self-cleavage of the DNAzyme at 37°C, separate and purify it by agarose gel electrophoresis to remove the redundant vector sequence and deoxyribozyme sequence, and obtain the target ssDNA. Add the origami system buffer with a final concentration of 1×TAE / Mg 2+ (40 mmol·L - 1 Tris-HCl (pH 8.0 at 22°C), 2 mmol·L -1 EDTA, 12.5 mmol·L -1 MgCl2) to make the final concentration of ssDNA 1 - 4 nmol·L -1 . Use a PCR instrument to anneal and fold the ssDNA. The annealing program is 90°C for 3 minutes, 75°C for 5 minutes, 60°C for 5 minutes, 45°C for 5 minutes, and 22°C for 5 minutes. Scan and image the sample after the annealing and folding process with an atomic microscope. The results are as Figure 1As shown in D, the obtained ssDNA can be successfully folded into triangular DNA nanostructures, indicating that single-stranded DNA for folding triangular structures can be prepared after inserting the DNAzyme sequence.

[0059] Example 2 Preparation of Functionalized Triangular DNA Nanostructures

[0060] Construct recombinant bacteria J525-A, J525-S, J525-A+S, and measure their growth and production of cssDNA: As Figure 2 shown in A and B, the maximum yield of cssDNA ranges from 17 to 20 mg / L, and the yield of ssDNA for preparing functional triangular nanostructures ranges from 2.6 to 3.1 mg / L.

[0061] After producing functionalized triangular cssDNA, DNAzyme self-cleavage was carried out according to the method in Case 1. The results of agarose gel electrophoresis are shown in Figure 3 A. Each cssDNA was cleaved into a triangular and a vector fragment. The band at 250 bp in the electrophoresis pattern is the target triangular band. The triangular ssDNA was obtained by agarose gel recovery and 1× TAE / Mg 2+ buffer was added. Subsequently, a thermal cycler was used for programmed annealing. The annealing program was from 85°C to 60°C, decreasing 1°C every 10 minutes; from 60°C to 40°C, decreasing 1°C every 30 minutes; from 40°C to 25°C, decreasing 1°C every 15 minutes.

[0062] The prepared triangular DNA nanostructures were characterized using atomic force microscopy (AFM). The results are shown in Figure 3 B. All three functional ssDNAs produced can be successfully folded into triangles.

[0063] Example 3 Preparation of Functionalized Hairpin Rectangular DNA Nanostructures

[0064] Construct recombinant bacteria J3898-A, J3898-S, J3898-A+S, and measure their growth and production of cssDNA: As Figure 4 shown in A and B, the maximum yield of cssDNA is 6 - 7 mg / L, and the yield of ssDNA for preparing functional hairpin rectangular DNA nanostructures is 3 - 3.7 mg / L.

[0065] After producing functionalized hairpin rectangular cssDNA, DNAzyme self-cleavage was carried out according to the method in Case 1. The results of agarose gel electrophoresis are shown in Figure 5 A. Each cssDNA was cleaved into a hairpin rectangle and a vector fragment. The band around 1500 bp in the electrophoresis pattern is the target band. The ssDNA was obtained by agarose gel recovery, and 1× TAE / Mg 2+Buffer was used for program annealing, and the prepared hairpin rectangular DNA nanostructures were characterized by AFM. The results are as Figure 5 shown in B. The produced functional ssDNA can be successfully folded into hairpin rectangles.

[0066] Example 4 Detection of the drug-loading capacity of functional triangular DNA nanostructures

[0067] By comparing the production of hairpin rectangles and triangular cssDNA, it was found that although the yields of functional triangular ssDNA and hairpin rectangular ssDNA were similar, the number of triangular ssDNA was larger. Therefore, functional triangular DNA nanostructures were selected for the application of targeting tumor cells.

[0068] The drug doxorubicin (DOX) was selected to study the characteristics of triangular DNA nanostructures and used as a drug carrier for experiments. Before delivering drugs with DNA nanostructures, it was necessary to determine that the DNA nanostructures themselves were safe for cells. Therefore, the CCK8 method was used to measure the cytotoxicity of triangular DNA nanostructures. Different concentrations of triangular DNA nanostructures were incubated with MCF-7 breast cancer cells for 24 h. The results are as Figure 6 shown in A. In MCF-7 cells, the single DNA nanostructure had almost no cytotoxicity, and its inhibitory effect was less than 5% like that of the control group (DNA nanostructure was 0), indicating that triangular DNA nanostructures were suitable as drug carriers.

[0069] The CCK8 method was used to evaluate the killing ability of DOX@DNA nanostructures on tumor cells and compare it with free DOX. As Figure 6 shown in B, after treating MCF-7 cells with different concentrations of DOX and DOX@DNA nanostructures for 48 h respectively, it can be seen that the killing effect of DOX@DNA nanostructures on MCF-7 cells was enhanced, and the lethality rate was higher than that of free DOX.

[0070] The experimental results proved that the DNA nanostructures prepared by the present invention were suitable as drug carriers, and the inhibitory effect on cells after carrying drugs was stronger than that of free DOX, which was beneficial to inhibiting the activity of cancer cells.

[0071] Example 5 Specific detection of functional triangular DNA nanostructures targeting tumor cells

[0072] The aptamer AS1411 specifically recognizes nucleolin on the surface of tumor cell membranes and can enter the nucleus relying on the shuttle effect of nucleolin in cells; the aptamer S2.2 can specifically bind to glycoprotein MUC1 on the membranes of various tumors. These two aptamers were modified on triangular DNA nanostructures to double-target breast cancer cells MCF-7, realizing the detection of cancer cells with strong specificity, high sensitivity and simple operation.

[0073] In this case, in order to determine the targeted cancer cell characteristics of the functional triangular DNA nanostructure, a comparative experiment was carried out: Triangular DNA nanostructures without aptamers carrying DOX and triangular DNA nanostructures with aptamers at the same concentration were incubated with MCF-7 cells for 1 h respectively. After discarding the culture medium, the nuclei were stained with Hoechst for 10 min, then the liquid was aspirated and fixed with 4% paraformaldehyde for 5 min. The accumulation of DOX@DNA nanostructures and DOX@A+S-DNA nanostructures in MCF-7 cells was explored by a laser confocal microscope. As Figure 7 shown in A, it can be seen that at the same concentration, compared with the triangular structure without aptamer, more amounts of the aptamer-modified DNA nanostructures are internalized into MCF-7 cells. It can also be seen from the fluorescence analysis results, namely Figure 7 in B, that the fluorescence of the carried drug DOX is increased by 2.6 times. It can be concluded that the aptamer-modified DNA nanostructures can specifically target MCF-7, that is, tumor cells with high expression of nucleolin or MUC1 protein on the cell membrane surface.

[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a long single-stranded DNA with aptamers, including a deoxyribozyme sequence and nucleic acid aptamer sequences, wherein the deoxyribozyme sequence is an optimized deoxyribozyme with base changes at specific positions, and the nucleic acid aptamers include aptamers targeting nucleolin and MUC1 protein respectively.

2. According to the preparation method described in claim 1, wherein The optimized deoxyribozyme can cleave circular single-stranded DNA within a wide pH range.

3. According to the preparation method described in claim 1, characterized in that, The optimized deoxyribozyme can rapidly cleave circular single-stranded DNA.

4. According to the preparation method described in claim 1, wherein Insert the aptamer sequences into the sequences of phagemid synthetic DNA nanostructures.

5. According to the preparation method described in claim 1, characterized in that, Obtain single-stranded DNA for folding into aptamer-bearing DNA nanostructures through self-cleavage of the deoxyribozyme.

6. According to the preparation method described in claim 5, wherein, The single-stranded DNA directly forms functional triangular and hairpin rectangular DNA nanostructures by high-temperature annealing, including but not limited to triangular and hairpin rectangular DNA nanostructures.

7. The functional DNA nanostructure according to claim 6, wherein The functional DNA nanostructures can load anti-cancer drugs.

8. The functional DNA nanostructure according to claim 6, wherein The functional DNA nanostructures can specifically target tumor cells.

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