CN113355322A - Photothermal control DNA folded paper synthesis method and in-situ assembly method of DNA folded paper in physiological environment - Google Patents
Photothermal control DNA folded paper synthesis method and in-situ assembly method of DNA folded paper in physiological environment Download PDFInfo
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Abstract
The invention discloses a photothermal control DNA origami synthesis method and an in-situ assembly method of DNA origami in a physiological environment, wherein a raw material chain for synthesizing the DNA origami is mixed with copper sulfide nano particles with photothermal conversion capacity, and a 980nm laser is adopted at the speed of 0.25-0.75W/cm2The power density of the DNA folding paper is irradiated for 1-6 min to form local high temperature, and then the DNA folding paper structure which is assembled correctly with high yield can be formed. According to the method, CuS nano particles are used for mediating near-infrared photothermal conversion to generate local heat so that DNA origami raw material chains in the solution are thermally denatured, the internal structure of the DNA origami raw material chains is opened, and the DNA origami is assembled according to a set structure along with the natural cooling of the solution after laser irradiation is removed. The DNA origami synthesis method is realized through the photo-thermal effect of remote control, only causes local high temperature, and therefore, the DNA origami synthesis method can be further used for in-situ assembly of a DNA origami structure in a physiological environment.
Description
Technical Field
The invention relates to the field of DNA origami synthesis, in particular to a photothermal control DNA origami synthesis method and an in-situ assembly method of DNA origami in a physiological environment.
Background
The DNA origami structure has the unique advantages of programmability, space addressability, structural complexity and the like, and is widely concerned in the biomedical fields of biosensing, bionics, biomolecule distance regulation, biological imaging, biological calculation and the like. Therefore, it is highly desirable to achieve in situ assembly of DNA origami structures in a physiological environment. Nevertheless, DNA origami nanostructures are usually composed of long single-stranded DNA scaffolds and hundreds of short DNA staple strands, and their assembly therefore requires a thermal denaturation process to open incorrect secondary structures (in kinetic traps). This process involves denaturing annealing at non-physiological temperatures (typically 90 ℃), which limits the possibility of in situ assembly of DNA origami in living systems. In recent years, the study of the synthesis of DNA/RNA nanostructures at physiological temperatures has been greatly advanced. For example, single-stranded DNA/RNA (ss-DNA/RNA) nanostructures have been designed for self-folding and even cooperative transcriptional folding; DNA binding proteins such as transcription activator-like effector proteins (TALEs) have been used to fold double-stranded DNA (ds-DNA) molecules into specific nanostructures; in phage, the RNA template is reverse transcribed into ssDNA strands, enabling the assembly of DNA Tile structures within the cell. However, the design and complexity of these DNA/RNA nanostructures is still limited compared to DNA origami structures.
Disclosure of Invention
The invention aims to provide a photothermal control DNA origami synthesis method and an in-situ assembly method of DNA origami in a physiological environment, so as to solve the problem that the prior art can not realize in-situ assembly of a DNA origami structure in the physiological environment.
According to the first aspect of the invention, the photothermal control DNA origami synthesis method is provided, the raw material chain for synthesizing the DNA origami is mixed with copper sulfide nano-particles with photothermal conversion capability, and a 980nm laser is adopted to perform the synthesis at the speed of 0.25-0.75W/cm2Irradiating for 1-6 min to form local high temperature, and then forming a high-yield correctly assembled DNA paper folding structure.
Most preferably, the copper sulfide nanoparticles have a particle size of 10nm and a hydrated particle size of 15 nm.
Most preferably, the power density of the laser irradiation is 0.5W/cm2The irradiation time was 4 min.
According to the preferred embodiment of the present invention, the DNA origami structure may be a one-dimensional six-spiral shape, a two-dimensional square shape, a cross shape, a shuttle shape, a concave shape, a three-dimensional tetrahedral shape, and the like.
According to a preferred embodiment of the present invention, there is provided a photothermal control DNA origami synthesis method comprising the steps of: copper chloride dihydrate (CuCl)2·2H2O) and thioglycolic acid (TGA) are taken as raw materials for mixing reaction, then sodium hydroxide aqueous solution is added into the suspension drop by drop, and the pH value of the solution is adjusted to 9; adding Thioacetamide (TAA) aqueous solution, reacting for 2h at 60 ℃, centrifuging after the reaction is finished, and washing with deionized water to obtain copper sulfide nano particles; configuration 10 XTAE-Mg2+A buffer solution consisting of: 400mM Tris,20mM EDTA-NH2,125mM MgAc·4H2O,200mM HAc, pH 8.0; taking a proper amount of 10 XTAE-Mg2+A buffer solution to which the copper sulfide nanoparticle solution is added; adding M13mp18 ss-DNA and DNA staple chain, mixing, and using 980nm laser at 0.25-0.75W/cm2Irradiating for 1-6 min under the power density of the copper sulfide nano particles to form a correctly assembled DNA origami structure under the photo-thermal effect of the copper sulfide nano particles.
According to a particularly preferred embodiment of the present invention, the photothermal control DNA origami synthesis method comprises the steps of: 1) adding copper chloride dihydrate (CuCl)2·2H2O, 1mM,80mL) was mixed with thioglycolic acid (TGA, 12. mu.L) and reacted for 10min, then an aqueous solution of sodium hydroxide (NaOH, 10mM) was added dropwise to the suspension to adjust the pH of the solution to 9; 2) adding Thioacetamide (TAA) aqueous solution (5mM, 20mL), reacting at 60 ℃ for 2h, centrifuging for 3 times (15000r/min,10min) after the reaction is finished, and washing with deionized water to obtain copper sulfide nanoparticles (CuS NPs); 3) mixing Tris (hydroxymethyl) aminomethane (Tris, 48.46g) and ethylenediaminetetraacetic acid (EDTA-NH)27.4448g), magnesium acetate tetrahydrate (MgAc.4H)2O, 26.806g), acetic acid (HAc, 11.4mL) were dissolved in deionized water10 XTAE-Mg is prepared in water (150mL)2+Buffer solution (400mM Tris,20mM EDTA-NH)2,125mM MgAc·4H2O, and 200mM HAc, pH 8.0,20 μ L); 4) taking 10 XTAE-Mg in 3)2+The buffer solution (10. mu.L) was dissolved in the copper sulfide nanoparticle solution (24nM, 80. mu.L) synthesized in (2); 5) dissolving M13mp18 ss-DNA (100nM, 2. mu.L) in 4) to obtain a mixed solution; 6) taking DNA staple chain (250nM, 8 uL) to dissolve in 5) to mix the solution; 7) the mixed solution in 6) is mixed evenly in a 1.5mL centrifuge tube and a 980nm laser is used at 0.5W/cm2For 4 minutes at a power density of (1); 8) after slowly cooling to room temperature, the solution was centrifuged and purified and stored at 4 ℃ before use.
According to a second aspect of the present invention, there is provided a method of in situ assembly of photothermally controlled DNA origami in a physiological environment, the method comprising: in a cell lysis solution or a cell culture solution, a raw material chain for synthesizing DNA origami is mixed with copper sulfide nano particles with photo-thermal conversion capacity, and a 980nm laser is adopted at the speed of 0.25-0.75W/cm2The power density of the DNA folding paper is irradiated for 1-6 min to form local high temperature, and then in-situ assembly of the DNA folding paper structure in a physiological environment can be realized, wherein the physiological environment comprises cell lysate or cell culture solution.
The in-situ assembly method of the DNA origami structure in the cell lysate comprises the following steps: treating HepG2 cells with trypsin, then suspending the cells in PBS solution, obtaining cell lysate through ultrasonic lysis and centrifugation, mixing the obtained cell lysate with DNA chains and copper sulfide nanoparticles needed by synthetic DNA origami to form suspension, and then mixing the suspension with the suspension at a speed of 0.25-0.75W/cm2The power density of the cell lysate is locally irradiated on the mixture solution for 1-6 min by 980nm laser, and then in-situ assembly of the DNA paper folding structure in the cell lysate can be realized.
According to a preferred embodiment of the present invention, the method for in situ assembly of a DNA origami structure in a cell lysate comprises: treating HepG2 cells with trypsin, resuspending in PBS solution, ultrasonically lysing for 5min, centrifuging for 10min (10000r/min) to obtain cell lysate, mixing the cell lysate with DNA chains required by DNA paper folding and copper sulfide nanoparticles to form 100 μ L suspensionM13mp18 ss-DNA (2nM), staple chain (20nM),1 XTAE-Mg2+buffer(40mM Tris,2mM EDTA-NH2,12.5mM MgAc·4H2O, and 20mM HAc, pH 8.0), and CuS NPs (24nM), and then irradiating the mixture solution (0.5W/cm) with 980nM laser24min), 1% agarose gel at 1 XTAE-Mg2+And (3) carrying out DNA paper folding separation in a buffer solution, wherein the running voltage is 100V, the running time is 1h, the gel is dyed by gel red, and the agarose gel is subjected to gel tapping separation under the guidance of ultraviolet light. The excised samples were characterized by AFM imaging.
The in-situ assembly method of the DNA origami structure in the cell culture solution comprises the following steps: before in-situ assembly, inoculating HepG2 cells into a 24-pore plate, culturing the HepG2 cells and DMEM together for 20-28 h, and then adding a DNA chain required by the synthetic DNA origami and copper sulfide nanoparticles to obtain a mixed solution; then, the concentration of the water is controlled to be 0.25 to 0.75W/cm2The power of the method is that 980nm laser is used for locally irradiating the mixture solution for 1-6 min, and then in-situ assembly of the DNA paper folding structure in the cell culture solution can be achieved.
According to a preferred embodiment of the present invention, the method for in situ assembly of DNA origami structures in cell culture fluid comprises: before in situ assembly, HepG2 cells were seeded in 24-well plates, incubated with DMEM for 24h, and then added with DNA strands required for DNA origami synthesis and copper sulfide nanoparticles to give a 500. mu.L mixed solution consisting of M13mp18 ss-DNA (2nM), staple strands (20nM),1 XTAE-Mg2+buffer(40mM Tris,2mM EDTA-NH2,12.5mM MgAc·4H2O, and 20mM HAc, pH 8.0), copper sulfide nanoparticles (24 nM); then using a 980nm laser (0.5W/cm)2And 4min) locally irradiating the mixed solution; in addition, the operation without copper sulfide or laser irradiation was used as a control group, and the extracted product was characterized by AFM imaging.
It should be understood that the structure of the DNA origami in the present invention belongs to the prior art, and the key point of the present invention is not that, the present invention is focused on using CuS nanoparticles to mediate near infrared photothermal conversion, generating local heat to prepare strand thermal denaturation for DNA origami in solution, opening the internal structure, and after removing laser irradiation, assembling the DNA origami according to the set structure along with the natural cooling of the solution. The thermal denaturation of the DNA origami raw material strand by laser irradiation can be completed in 4 minutes and the yield of correct assembly is very high, which is also an advantage of the present invention over the prior art. Furthermore, the folding paper synthesis method used by the invention is realized by the photo-thermal effect of remote control, and only causes local high temperature, so that the folding paper can be further used for in-situ assembly of the DNA folding paper structure in a physiological environment. In conclusion, the invention not only provides a photothermal control DNA origami synthesis method, but also provides an in-situ assembly method of photothermal control DNA origami in a physiological environment.
Drawings
FIG. 1 is a basic representation of copper sulfide nanoparticles prepared according to the present invention;
FIG. 2 is an AFM image of a photo-thermal mediated synthesized triangular DNA origami structure of the present invention;
FIG. 3 is an agarose gel electrophoresis image of a triangular DNA origami structure synthesized by photo-thermal mediation according to the present invention;
FIG. 4 shows the photothermal effect of the copper sulfide nanoparticles prepared according to the present invention;
FIG. 5 is a diagram of a strand exchange experiment according to the present invention;
FIG. 6 is an AFM imaging diagram of gold nano-mediated photothermal effect and its mediated synthesized triangular DNA origami structure of the present invention;
FIG. 7 is an AFM image of photo-thermal mediated synthesized triangular DNA origami structures at different power densities and illumination times according to the present invention;
FIG. 8 is an AFM image and area distribution of a triangular DNA origami structure synthesized by a thermal incubation method for a positive control group according to the present invention;
FIG. 9 shows the present invention at 0.25W/cm2AFM imaging graph and area distribution of the photo-thermal mediated synthesized triangular DNA origami structure under the condition of 1-6 min;
FIG. 10 shows the present invention at 0.5W/cm2AFM imaging graph and area distribution of the photo-thermal mediated synthesized triangular DNA origami structure under the condition of 1-6 min;
FIG. 11 shows the present invention at 0.75W/cm2AFM imaging graph of photo-thermal mediated synthesized triangular DNA origami structure under 1-6 min conditionAnd area distribution;
FIG. 12 is a thermal map of the yield of correctly folded triangular DNA signatures of the present invention at different power densities and irradiation times;
FIG. 13 is a thermal map of solution temperature at different power densities and exposure times in accordance with the present invention;
FIG. 14 is a schematic diagram of site-directed modification of a FRET pair on a triangular DNA origami designed by the present invention;
FIG. 15 is a detailed cross-sectional view of a FRET pair designed according to the present invention for site-directed modification on a triangular DNA origami;
FIG. 16 is a single molecule imaging plot of photothermal mediated synthesized FRET versus modified triangular DNA origami structures of the present invention;
FIG. 17 is a single molecule imaging quantitation plot of photothermal mediated synthetic FRET versus modified triangular DNA origami structures of the invention;
FIG. 18 is a photograph of a fluorescence image of a photothermal mediated synthesized FRET pair modified triangular DNA origami structure of the present invention;
FIG. 19 is a diagram of photo-thermal mediated dynamic assembly fluorescence instant imaging of triangular DNA origami structures of the present invention;
FIG. 20 is an AFM imaging of the photothermal mediated dynamic assembly of triangular DNA origami structures of the present invention;
FIG. 21 is an agarose gel electrophoresis image of the photothermal mediated dynamic assembly process of triangular DNA origami structures of the present invention;
FIG. 22 is a cross-sectional view of a shuttle folded paper structure of the present design;
FIG. 23 is a cross-sectional view of a female origami structure of the present invention;
FIG. 24 is an AFM image and an agarose gel electrophoresis of 6 different DNA origami structures synthesized by photothermal mediation in accordance with the present invention;
FIG. 25 is a schematic diagram of the photothermal mediated in situ synthesis of a triangular DNA origami structure of the present invention in a cell lysis solution;
FIG. 26 is an AFM image of the photo-thermal mediated triangular DNA origami structure of the present invention synthesized in situ in a cell lysate;
FIG. 27 is an agarose gel electrophoresis image of the photothermal mediated triangular DNA origami structure of the present invention synthesized in situ in a cell lysis solution;
FIG. 28 is a schematic diagram of the photothermal mediated in situ synthesis of a triangular DNA origami structure of the present invention in cell culture;
FIG. 29 is an AFM image of the photo-thermal mediated triangular DNA origami structure of the present invention synthesized in situ in cell culture;
FIG. 30 is a graph showing cell death and staining after in-situ synthesis of the photo-thermal mediated triangular DNA origami structure of the present invention in cell culture solution.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings.
Example 1 Synthesis of DNA origami Structure
1. Preparation of copper sulfide nanoparticle solution:
adding copper chloride dihydrate (CuCl)2·2H2O, 1mM,80mL) was mixed with thioglycolic acid (TGA, 12. mu.L) and reacted for 10min, then an aqueous solution of sodium hydroxide (NaOH, 10mM) was added dropwise to the suspension to adjust the pH of the solution to 9; an aqueous Thioacetamide (TAA) (5mM, 20mL) solution was added and the reaction was carried out at 60 ℃ for 2 hours. After the reaction, the reaction mixture was centrifuged 3 times (15000r/min,10min), and washed with deionized water to obtain copper sulfide nanoparticles (CuS NPs).
As shown in fig. 1, TEM results show that the as-synthesized copper sulfide nano-scale morphology uniform size is about 10 nm; DLS results show that the hydrated particle size of the synthesized copper sulfide nano material is about 15 nm; XRD verifies that the copper sulfide nano material is successfully synthesized; the UV-VIR-NIR spectrum result shows that the synthesized copper sulfide nano material has strong absorption in the infrared region and has very good photo-thermal conversion prospect.
In-situ assembly of DNA origami:
mixing Tris (hydroxymethyl) aminomethane (Tris, 48.46g) and ethylenediaminetetraacetic acid (EDTA-NH)27.4448g), magnesium acetate tetrahydrate (MgAc.4H)2O, 26.806g), acetic acid (HAc, 11.4mL) were dissolved in deionized water (150mL) to prepare 10 XTAE-Mg2+Buffer solution (400mM Tris,20mM EDTA-NH)2,125mM MgAc·4H2O,and 200mM HAc,pH 8.0,20μL)；
Taking 10 XTAE-Mg2+Dissolving the buffer solution (10 μ L) in copper sulfide nanoparticle solution (24nM, 80 μ L), and dissolving M13mp18 ss-DNA (100nM,2 μ L); add DNA staple chain (250nM, 8. mu.L); the mixed solution was mixed well in a 1.5mL centrifuge tube and applied to a 980nm laser at 0.5W/cm2For 4 minutes at a power density of (1); after slowly cooling to room temperature, the solution was centrifuged and purified and stored at 4 ℃ before use. The DNA staple chain used therein can be referred to in the literature (Rothemund, P.W.K.folding DNA to create nanoscales flaps and patterns. Nature 2006,440(7082), 297. 302.).
As shown in FIGS. 2 and 3, AFM imaging and agarose gel electrophoresis image results prove that the photothermal effect of the copper sulfide successfully realizes the correct assembly of the triangular paper folding structure.
As shown in fig. 4-6, thermal imaging, chain exchange experiments, and AFM imaging have demonstrated that copper sulfide with a strong photothermal conversion effect in the near infrared region can generate local high temperature, and can open the secondary structure of the raw material chain of the origami to realize the assembly of DNA origami. Gold nanoparticles with poor photo-thermal conversion effect in a near-infrared region can only generate low temperature, and the assembly of the DNA origami cannot be realized. It is stated that the assembly of the origami is done by thermal effects, not by the nanomaterial itself.
Example 2 optimization of DNA origami Synthesis efficiency
In this example 2, the power density and irradiation time of the 980nm laser used in the above-mentioned copper sulfide photothermal effect-mediated DNA origami synthesis method were adjusted based on example 1 to obtain the best synthesis efficiency, wherein the power density was 0.25W/cm2，0.50W/cm2，0.75W/cm2The irradiation time is 1min, 2min, 3min, 4min, 5min and 6min respectively.
As shown in FIGS. 7-13, the AFM imaging and thermal maps showed a laser power density of 0.50W/cm2The illumination time of 4 minutes is a critical condition, and the folding synthetic yield is more than 80%. Beyond this power density and irradiation time, the synthetic yield of the folded paper is not significantly increased, while below this power density and irradiation time, the synthetic yield of the folded paper is not significantly increasedThe rate is greatly reduced. Therefore, the present invention uses (0.5W/cm)24min) was used for the subsequent studies as the optimal synthesis conditions.
Example 3 study of DNA origami Synthesis dynamic Assembly Process
We labeled A43, B43, C43 used in the synthesis of triangular DNA origami with 5' -Cy3(Cy 3-ACTAGAAATATATAACTATATGTACGCTGAGA; Cy 3-CACGCATAAGAAAGGAACAACTAAGTCTTTCC; Cy 3-ACGTTGTATTCCGGCACCGCTTCTGGCGCATC). A43, B43, C43 were labeled with 3' -Cy5(TCAATAATAGGGCTTAATTGAGAATCATAATT-Cy 5; ATTGTGTCTCAGCAGCGAAAGACACCATCGCC-Cy 5; CCAGGGTGGCTCGAATTCGTAATCCAGTCACG-Cy5) to study the dynamic assembly process of DNA origami.
As shown in FIGS. 14-21, the single molecule TIRF imaging, fluorescence imaging, AFM imaging and agarose gel electrophoresis images together reflect the dynamic assembly process of DNA origami. Within 4 minutes of laser irradiation, the origami is not formed, and the local high temperature helps the thermal denaturation of the DNA origami raw material chain to open the wrong internal structure; within 4 minutes of natural cooling, the paper folding starts to assemble slowly; after cooling for 4 minutes, the DNA origami assembly is essentially complete and we can see a high yield of correctly assembled triangular DNA origami structures.
Example 4 Universal study on the Synthesis of different DNA origami structures
In addition to the triangular DNA origami structure, we have designed 6 different origami structures such as six-helix (one-dimensional), square (two-dimensional), cross (two-dimensional), shuttle (two-dimensional), concave (two-dimensional) and tetrahedron (three-dimensional) for studying the universality of the copper sulfide photothermal effect mediated DNA origami synthesis method, it should be understood that the design of these different shapes of DNA origami structures can be realized by the conventional technical means for those skilled in the art, and the design of these different shapes of DNA origami structures does not belong to the innovation point of the present invention, and therefore, it is not described herein again.
As shown in FIGS. 22-24, the AFM image and the agarose gel electrophoresis image together demonstrate that the copper sulfide photothermal effect mediated DNA origami synthesis method has universality.
Example 5 copper sulfide photothermal Effect mediated in situ Synthesis of DNA origami in cellular Environment
1. In-situ assembly method of DNA paper folding structure in cell lysate
HepG2 cells were trypsinized and resuspended in PBS solution, lysed by 5min sonication and centrifugation at 10min (10000r/min), and the lysate was then mixed with DNA strands needed for DNA origami synthesis and copper sulfide nanoparticles to form a 100. mu.L suspension containing M13mp18 ss-DNA (2nM), staple strands (20nM),1 XTAE-Mg2+buffer(40mM Tris,2mM EDTA-NH2,12.5mM MgAc·4H2O, and 20mM HAc, pH 8.0), and CuS NPs (24nM), and then irradiating the mixture solution (0.5W/cm) with 980nM laser24min), 1% agarose gel at 1 XTAE-Mg2+And (3) carrying out DNA paper folding separation in a buffer solution, wherein the running voltage is 100V, the running time is 1h, the gel is dyed by gel red, and the agarose gel is subjected to gel tapping separation under the guidance of ultraviolet light. The excised samples were characterized by AFM imaging.
2. In-situ assembly method of DNA paper folding structure in cell culture solution
Before in situ assembly, HepG2 cells were seeded in 24-well plates, incubated with DMEM for 24h, and then added with DNA strands required for DNA origami synthesis and copper sulfide nanoparticles to give a 500. mu.L mixed solution consisting of M13mp18 ss-DNA (2nM), staple strands (20nM),1 XTAE-Mg2+buffer(40mM Tris,2mM EDTA-NH2,12.5mM MgAc·4H2O, and 20mM HAc, pH 8.0), copper sulfide nanoparticles (24 nM); then using a 980nm laser (0.5W/cm)2And 4min) locally irradiating the mixed solution; in addition, the operation without copper sulfide or laser irradiation was used as a control group, and the extracted product was characterized by AFM imaging.
As shown in FIGS. 25-27, AFM images and agarose gel electrophoresis images together demonstrate that copper sulfide photothermal effect can realize in-situ synthesis of DNA origami in cell lysate.
As shown in FIGS. 28-30, AFM imaging and cell death and survival experiments prove that the copper sulfide photothermal effect can realize in-situ synthesis of the DNA origami in the cell culture solution, and the synthesis method only induces local cell damage and is relatively safe to the overall physiological environment.
The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent application. The invention has not been described in detail in order to avoid obscuring the invention.
Claims (10)
1. The photothermal control DNA origami synthesis method is characterized in that a raw material chain for synthesizing the DNA origami is mixed with copper sulfide nano particles with photothermal conversion capacity, and a 980nm laser is adopted to perform the steps of 0.25-0.75W/cm2Irradiating for 1-6 min to form local high temperature, and then forming a high-yield correctly assembled DNA paper folding structure.
2. The method for synthesizing DNA origami according to claim 1, wherein the copper sulfide nanoparticles have a particle size of 10nm and a hydrated particle size of 15 nm.
3. The method for synthesizing DNA origami according to claim 1, wherein the power density of laser irradiation is 0.5W/cm2The irradiation time was 4 min.
4. The DNA origami synthesis method according to claim 1, wherein the DNA origami structure comprises: one-dimensional six-helix, two-dimensional square, cross, shuttle, concave, and three-dimensional tetrahedral.
5. The DNA origami synthesis method according to claim 1, comprising the steps of:
s1: preparing copper sulfide nano particles: mixing and reacting copper chloride dihydrate and thioglycolic acid serving as raw materials, then dropwise adding a sodium hydroxide aqueous solution into the suspension, and adjusting the pH of the solution to 8-9; adding a thioacetamide aqueous solution into the mixture, reacting for 1.5-2.5 h at the temperature of 58-62 ℃, centrifuging after the reaction is finished, and washing with deionized water to obtain a copper sulfide nano particle solution;
s2: preparing 10 XTAE-Mg2+Buffer solution, taking a proper amount of 10 XTAE-Mg2+A buffer solution to which the copper sulfide nanoparticle solution is added;
s3: adding M13mp18 ss-DNA and a DNA staple chain into the solution obtained in the step S2, uniformly mixing, and using a 980nm laser at 0.25-0.75W/cm2Irradiating for 1-6 min under the power density of the copper sulfide nano particles to form a correctly assembled DNA origami structure under the photo-thermal effect of the copper sulfide nano particles.
6. The DNA origami synthesis method according to claim 5, comprising the steps of:
1) 80mL of copper chloride dihydrate (1 mM) and 12 mu L of thioglycolic acid are mixed and reacted for 10min, then 10mM sodium hydroxide aqueous solution is added dropwise into the suspension, and the pH of the solution is adjusted to 9;
2) adding 20mL of 5mM thioacetamide aqueous solution, reacting at 60 ℃ for 2h, centrifuging for 3 times at 15000r/min for 10min after the reaction is finished, and washing with deionized water to obtain copper sulfide nano particles;
3) preparing 10 XTAE-Mg2+A buffer solution consisting of 400mM Tris,20mM EDTA-NH2,125mM MgAc·4H2O,and 200mM HAc,pH 8.0,20μL；
4) Taking 10 mu L of 10 XTAE-Mg in the step 3)2+Dissolving the buffer solution in 80 mu L of 24nM copper sulfide nano particle solution synthesized in the step 2);
5) dissolving 2 μ L of 100nM M13mp18 ss-DNA in the mixed solution in step 4);
6) taking 8 mu L of DNA staple chain with 250nM to dissolve in the mixed solution in the step 5);
7) uniformly mixing the mixed solution obtained in the step 6) in a 1.5mL centrifuge tube, and using a 980nm laser at 0.5W/cm2For 4 minutes at a power density of (1);
8) after slowly cooling to room temperature, the solution was centrifuged and purified and stored at 4 ℃ before use.
7. The in-situ assembling method of the photothermal control DNA origami in the physiological environment is characterized in that in a cell lysis solution or a cell culture solution, a raw material chain for synthesizing the DNA origami is mixed with copper sulfide nano particles with photothermal conversion capacity, and a 980nm laser is adopted to perform in-situ assembling on the DNA origami in a range of 0.25-0.75W/cm2The power density of the DNA folding paper is irradiated for 1-6 min to form local high temperature, and then in-situ assembly of the DNA folding paper structure in a physiological environment can be realized, wherein the physiological environment comprises cell lysate or cell culture solution.
8. The method of claim 7, wherein the method for in situ assembly of the DNA origami structure in the cell lysate comprises: treating HepG2 cells with trypsin, then suspending the cells in PBS solution, obtaining cell lysate through ultrasonic lysis and centrifugation, mixing the obtained cell lysate with DNA chains and copper sulfide nanoparticles needed by synthetic DNA origami to form suspension, and then mixing the suspension with the suspension at a speed of 0.25-0.75W/cm2The power density of the cell lysate is locally irradiated on the mixture solution for 1-6 min by 980nm laser, and then in-situ assembly of the DNA paper folding structure in the cell lysate can be realized.
9. The method of claim 7, wherein the method for in situ assembly of the DNA origami structure in the cell culture fluid comprises: before in-situ assembly, inoculating HepG2 cells into a 24-pore plate, culturing the HepG2 cells and DMEM together for 20-28 h, and then adding a DNA chain required by the synthetic DNA origami and copper sulfide nanoparticles to obtain a mixed solution; then, the concentration of the water is controlled to be 0.25 to 0.75W/cm2The power density of the method is that 980nm laser is used for locally irradiating the mixture solution for 1-6 min, and then in-situ assembly of the DNA paper folding structure in the cell culture solution can be achieved.
10. The method of claim 8 or 9, wherein the DNA origami structure is assembled in situ using a 1% agarose gel on 1 XTAE-Mg2+Separating DNA in buffer solution at running voltage of 100V for 1 hr, staining gel with gel red, separating agarose gel by ultraviolet light guide gel tapping, and separating the extracted product by AFMAnd (4) characterizing by the image.
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