Construction method and application of ATP (adenosine triphosphate) responsive DNA (deoxyribonucleic acid) nanowire cluster

Abstract

The invention belongs to the technical field of DNA (Deoxyribose Nucleic Acid) nanowire clusters, and relates to a construction method of an ATP (Adenosine Triphosphate) responsive DNA nanowire cluster, which comprises the following steps: synthesizing an annular template chain containing an ATP aptamer sequence LA by using a linear template LT and the ATP aptamer sequence LA, and obtaining long single-stranded DNA containing repeated ATP aptamer sequences through RCA reaction; hybridizing a 5'end and a 3 'end of a complementary DNA chain LS with a repeated ATP aptamer sequence LA part in the long single-stranded DNA obtained in the step 1, and folding the long single-stranded DNA into a DNA nanowire cluster DNC; wherein a complementary DNA chain LS in the DNA nanowire cluster DNC has a bent configuration, and the ATP aptamer sequence LA can recognize ATP so as to induce the complementary DNA chain LS to be released from the DNA nanowire cluster DNC; the near-infrared two-region fluorescent probe disclosed by the invention has tissue penetrability and lower background interference, so that the near-infrared two-region fluorescent probe has a wide application prospect in the field of biological imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor imaging, and in particular relates to a construction method of an ATP-responsive DNA nanowire and an application thereof. Background Art

[0002] Currently, cancer is one of the most important health problems in the world and poses a huge threat to public health. Although various therapeutic interventions have been developed, the cure rate is low and the mortality rate is high due to untimely detection and cancer recurrence. Therefore, exploring accurate tumor imaging technology to achieve early diagnosis and sensitive detection of tumors is of great significance to enhance tumor treatment and improve patient prognosis. Compared with normal tissues, tumor tissues have different characteristics such as lower pH, hypoxic environment, high concentration of glutathione, high concentration of hydrogen peroxide, and increased ATP level. Taking advantage of these unique characteristics of tumors, various responsive fluorescent nanoprobes based on DNA nanotechnology, organic metal frameworks or polymer materials have been developed for tumor-specific detection and imaging.

[0003] Although fluorescence imaging has the advantages of high sensitivity, high resolution, non-invasiveness and multiplexing, traditional fluorescence detection has problems such as low penetration depth and severe background interference, which is fatal to the analysis of complex biological systems and also poses obstacles to deep tumor imaging. In contrast, the long wavelength of the second near-infrared light has excellent fluorescence penetration and weakens the biological background signal, thereby improving the signal-to-noise ratio of in vivo imaging applications. Recently, more and more near-infrared second-zone fluorescent materials have been found to achieve high-resolution biological imaging, such as quantum dots, small molecule dyes, rare earth nanoparticles and carbon nanotubes.

[0004] Organic small molecule dyes with near-infrared second-zone fluorescence characteristics have clear structures, good optical properties, and easy metabolism, and have great potential in biological imaging. However, their low cellular absorption rate and insufficient targeted accumulation hinder their application in tumor imaging and diagnosis. Therefore, people have designed a variety of dye-loaded nanocarriers to improve their ability to accumulate tumors in vivo, such as polymer micelles, metal-organic frameworks, and silica nanoparticles. As a natural biomaterial, DNA nanomaterials have the advantages of superior biocompatibility, biodegradability, and low toxicity, and therefore have been widely used in the biomedical field. In addition, the inherent programmability and deformability of DNA nanomaterials make it easy to construct stimulus-responsive detection probes for a variety of biomarkers.

[0005] Rolling circle amplification (RCA) is a simple method for preparing long single-stranded DNA with repeating units. It is relatively low-cost, simple to operate, and has mild reaction conditions. By combining with functional nucleic acids such as aptamers, i-motif structures, and G-quadruplexes, a variety of stimuli-responsive DNA nanostructures can be prepared for precise imaging and diagnosis of tumors in vivo. Considering the high expression of ATP in tumor cells, we designed an ATP-responsive DNA nanocluster based on RCA reaction to achieve fluorescence regulation of NIR-II dyes for deep tumor imaging. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an ATP-responsive DNA nanowire based on RCA reaction and its application.

[0007] In order to achieve the purpose of the present invention, the present invention is implemented by adopting the following technical solutions.

[0008] A method for constructing an ATP-responsive DNA nanowire comprises the following steps:

[0009] S11, synthesizing a circular template chain containing the ATP aptamer sequence LA using the linear template LT and the ATP aptamer sequence LA, and obtaining a long single-stranded DNA containing a repeated ATP aptamer sequence through an RCA reaction;

[0010] S12, using the 5' end and 3' end of the complementary DNA strand LS to partially hybridize with the repeated ATP aptamer sequence LA in the long single-stranded DNA obtained in step S11, and folding the long single-stranded DNA into a DNA nanocoil DNC; wherein:

[0011] The complementary DNA strand LS in the DNA nanocoil DNC has a bent configuration;

[0012] The ATP adaptor sequence LA in the DNA nanocoil DNC can recognize ATP to induce the complementary DNA chain LS to be released from the DNA nanocoil DNC and induce the bending configuration of the complementary DNA chain LS to collapse.

[0013] As a preferred embodiment of the present invention, both the 5' end and the 3' end of the complementary DNA chain LS have complementary fragments.

[0014] A method for constructing a DNA nanowire DNCF labeled with near-infrared second region fluorescence comprises the following steps:

[0015] S31, synthesizing a circular template chain containing the ATP aptamer sequence LA using the linear template LT and the ATP aptamer sequence LA, and obtaining a long single-stranded DNA containing a repeated ATP aptamer sequence through an RCA reaction;

[0016] S32, hybridizing the 5' end and 3' end of the DNA chain FD-LS labeled with the near-infrared region II fluorescent molecule FD with the repeated ATP aptamer sequence portion in the long single-stranded DNA obtained in step S31, and folding the long single-stranded DNA into a DNA nanowire DNCF labeled with near-infrared region II fluorescence; wherein:

[0017] The DNA chain FD-LS is formed by a click reaction between a complementary DNA chain LS modified with an azide group and a near-infrared second-zone fluorescent molecule FD-AL modified with an alkyne group;

[0018] The DNA chain FD-LS in the near-infrared II fluorescently labeled DNA nanocluster DNCF has a bent configuration, and the bent configuration makes the fluorescent dye FD modified at the end of the complementary DNA chain LS adjacent to each other, so as to induce aggregation quenching of the near-infrared II NIR-II fluorescence of FD;

[0019] The ATP aptamer sequence LA in the DNA nanocluster DNCF can recognize ATP, induce the complementary DNA chain FD-LS to be released from the DNA nanocluster DNCF, and collapse its bending configuration, so that the fluorescent reporter group FD emits 1071nm fluorescence at an excitation wavelength of 808nm, thereby achieving fluorescence enhancement in the near-infrared second region NIR-II in response to ATP.

[0020] As a preferred embodiment of the present invention, the drug is loaded into the near-infrared second region fluorescently labeled DNA nanocluster DNCF by inserting into the DNA double helix structure, and the ATP aptamer sequence LA in the near-infrared second region fluorescently labeled DNA nanocluster DNCF can recognize ATP and induce the complementary DNA chain LS to be released from the near-infrared second region fluorescently labeled DNA nanocluster DNCF, so as to achieve the delivery and release of the drug; wherein: the construction method of the near-infrared second region fluorescently labeled DNA nanocluster DNCF comprises the following steps:

[0021] S41, synthesizing a circular template chain containing the ATP aptamer sequence LA using the linear template LT and the ATP aptamer sequence LA, and obtaining a long single-stranded DNA containing a repeated ATP aptamer sequence through an RCA reaction;

[0022] S42, hybridize the 5' and 3' ends of the DNA chain FD-LS labeled with the near-infrared region II fluorescent molecule FD with the repeated ATP aptamer sequence portion in the long single-stranded DNA obtained in step S41, and fold the long single-stranded DNA into a DNA nanocluster DNCF labeled with near-infrared region II fluorescence.

[0023] Beneficial Effects

[0024] Compared with traditional fluorescence analysis methods, the near-infrared II region NIR-II fluorescent probe of the present invention has stronger tissue penetration and lower background interference, and therefore has broad application prospects in the field of biological imaging; at the same time, the probe is constructed based on biological macromolecule DNA and has better biocompatibility; by introducing an ATP-responsive aptamer structure, the probe can achieve near-infrared II region fluorescence responsiveness to tumor cells and realize accurate imaging of deep tumor tissue in living bodies.

[0025] The present invention utilizes the superior biocompatibility and flexible programmability of DNA nanomaterials to develop an ATP-responsive DNA nanocluster for near-infrared zone II tumor fluorescence imaging and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Figure A is a schematic diagram of the synthesis of DOX-loaded DNA nanowires; Figure B is a schematic diagram of ATP-responsive NIR-II fluorescence tumor imaging and therapy;

[0027] Figure 2 Schematic diagram of the recognition and binding of the linear template and ATP in the present invention, wherein: Figure A is a schematic diagram of the folding hybridization and ATP-responsive melting of the complementary DNA chain LS and the RCA template chain; Figure B is a PAGE analysis of the release of the complementary DNA chain LS after incubation with different concentrations of ATP, wherein: Lanes 1-8 represent the linear RCA template chain (I), the complementary DNA chain LS (II), a mixture of I and II, and incubation with 0, 1, 5, 10, 50, 100 μM ATP, respectively; Figure C is the initial LS FAM / BHQ LS FAM / BHQ Fluorescence spectra of incubation with linear template LT and then incubation with ATP;

[0028] Figure 3 The formation of the DNA nanowires and the ATP-responsive unzipping in the present invention, wherein: Figure A is LS FAM / BHQ Figure 1 shows the fluorescence spectra before and after incubation with long single strands; Figure B shows the TEM image of DNA nanowires; Figure C shows the fluorescence spectra of DNA nanowires treated with different concentrations of ATP; Figure D shows the near-infrared fluorescence spectra of the main chain FD-LS modified by FD-AL and DNCF synthesized from FD-LS before and after ATP treatment;

[0029] Figure 4 Schematic diagram of DOX loading and ATP response release in the present invention, wherein: Figure A is a schematic diagram of loading DOX into a DNA nanocluster structure; Figure B is the fluorescence spectrum of DOX in the supernatant before and after incubation with DNA nanocluster; Figure C is the fluorescence spectrum of the supernatant after incubating DOX-loaded DNA nanocluster with different concentrations of ATP; Figure D is the DOX fluorescence intensity of the supernatant after incubating DOX-loaded DNA nanocluster with ATP, CTP, GTP and UTP respectively; the data error bars represent the mean ± SD (n = 3). DETAILED DESCRIPTION

[0030] The present invention will be further described with reference to the accompanying drawings and embodiments.

[0031] As an embodiment of the present invention, Figure 1 As shown, first, a circular template chain containing an ATP aptamer sequence is synthesized, and a long single-stranded DNA containing a repeated ATP aptamer sequence LA is obtained by RCA reaction. Then, the two ends of the complementary DNA chain LS are partially hybridized with the repeated ATP aptamer sequence LA, and the long single-stranded DNA is folded, thereby promoting the formation of the DNA nanocluster DNC. The fluorescent dye FD-AL is modified at both ends of the complementary DNA chain LS by a click reaction, and the bent configuration makes the fluorescent dye modified at the end of the complementary DNA chain LS adjacent, thereby inducing the near-infrared second region fluorescence quenching of FD-AL. In the presence of ATP, the aptamer sequence in the DNA nanocluster recognizes ATP, thereby releasing the complementary DNA chain LS from the long single-stranded DNA, causing the DNA nanocluster structure to disintegrate, and the FD-AL molecules separate from each other and emit 1071nm fluorescence at an excitation wavelength of 808nm, thereby achieving ATP-responsive NIR-II fluorescence enhancement.

[0032] As an embodiment of the present invention, the specific process of constructing ATP-responsive DNA nanowires is as follows:

[0033] 1. ATP-induced conformational changes

[0034] ATP is a key biomarker for diagnosing tumors. Therefore, we used ATP aptamers as specific signal response modules to achieve precise directional imaging of tumors in vivo. First, we used polyacrylamide gel electrophoresis (PAGE) experiments to verify the ability of ATP aptamers to recognize and selectively bind to ATP. Figure 2As shown in Figure B, after the ATP aptamer was incubated with the lock strand containing the complementary sequence, a band with a lower mobility was observed in lane 3, indicating that the two DNA strands were hybridized. Then, after adding different concentrations of ATP, the ATP-responsive splitting of the double strand was verified by PAGE imaging. With the increase of ATP concentration (1μM, 3μM, 5μM), the two bands of the ATP aptamer and the complementary strand became clearer and brighter, as shown in Figure 3. Figure 2 Lanes 4, 5, and 6 described in panel B in FIG. 5 , indicate successful binding of ATP to the aptamer and that ATP responsively disassembles the double helical conformation.

[0035] In order to form a bent configuration, we designed the complementary DNA strand LS to have complementary fragments at both the 5′ and 3′ ends, which can hybridize with the ATP aptamer sequence in the RCA template strand, e.g. Figure 1 As shown in Figure A. We first explored the separation of the RCA template strand and the complementary DNA strand LS induced by ATP by polyacrylamide gel electrophoresis PAGE, as shown in Figure A. Figure 1 As shown in Figure B, the band mobility of lane 3 is low, indicating that the RCA template chain containing the ATP aptamer hybridizes with the complementary DNA chain to produce a new band. As the ATP concentration increases, this hybridization gradually disintegrates, producing two bands, the RCA template chain and the complementary DNA chain LS. When the concentration reaches 100μM, the hybrid chain is completely opened. The experimental results show that the template chain also has good ATP responsiveness and loosens, as shown in Figure 2. Figure 1 As shown in Figure C. Before adding the template strand, the fluorescence intensity of FAM on the complementary DNA strand LS is very strong, as shown in Figure C. Figure 1 The blue line shown in Figure C shows that after hybridization with the template chain, the fluorescence intensity of FAM decreased significantly due to fluorescence quenching caused by the proximity of FAM and BHQ1, indicating the formation of a bent conformation. After co-incubation with ATP, the fluorescence intensity of FAM recovered again. The above results demonstrate the folded conformation and ATP responsiveness of the complementary DNA chain LS connected to the RCA template.

[0036] Considering that the length of the complementary segment between the RCA template strand and the backbone strand may affect the hybridization efficiency and ATP responsiveness, we optimized their complementary base numbers by the change in fluorescence signal. Figure 2 As shown in Figures A and B in , when the number of complementary bases reaches 10, the fluorescence signal-to-noise ratio reaches the best. Therefore, we selected the template chain and main chain with 10 complementary bases as the most suitable candidate materials for subsequent experimental research and further characterization.

[0037] 2. Synthesis and characterization of ATP-responsive DNA nanowires

[0038] Using the template strand for RCA reaction, a long single-stranded structure with a repeating sequence including the ATP aptamer sequence was synthesized, e.g. Figure 3 As shown in Figure A, and verified by PAGE imaging. Figure 3 As shown in Figure B, the template strand is first incubated with the connecting strand to obtain a circular template. After the excess unreacted linear oligonucleotide is degraded by Exo I / III treatment, a single band can be observed, such as Figure 3 Lane 3 shown in Figure B, which means that the DNA circle for subsequent reactions was successfully generated.

[0039] Subsequently, the generation of long single strands was also verified by PAGE, and after incubating the circular template with primer DNA and performing RCA reaction, a band with lower mobility appeared, e.g. Figure 3 Lane 5 shown in panel B.

[0040] The main chain hybridizes with the repeating sequence in the obtained long single strand, causing multiple folding transformations and finally forming a compact globular structure. FAM / BHQ1 The fluorescence quenching of the main chain confirmed the bending configuration of the main chain. The FAM fluorescence intensity decreased significantly after the complementary DNA chain LS reacted with the RCA long single chain. Figure 2 As shown in Figure A, it shows the formation of a folded structure. Subsequently, transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to further characterize the formed spherical nanostructures. According to DLS analysis, the hydrated diameter of this DNA nanochip was measured to be about 50 nanometers, while the monodispersed particle size in the TEM image was about 40 nanometers, as shown in Figure 1. Figure 2 These results indicate that the long single strands obtained from RCA can be transformed into spherical nanostructures through hybridization with the backbone.

[0041] 3. ATP responsiveness of DNA nanocluster near-infrared fluorescence in the second region

[0042] First, use LS FAM / BHQ1 The synthesized self-quenched DNC verifies whether ATP can disrupt the assembly of DNA nanowires. Under the action of different concentrations of ATP, the fluorescence recovery of FAM was monitored. The results showed that with the increase of ATP concentration, the fluorescence intensity of FAM increased significantly, such as Figure 2 As shown in Figure C, this shows that LS FAM / BHQ1 Released from the RCA backbone, the DNA nanocluster structure disintegrates.

[0043] In order to achieve tumor-targeted near-infrared second-zone fluorescence imaging, we selected a commonly used dye FD-1080 with excellent near-infrared second-zone fluorescence properties as the fluorescent dye for designing DNA nanowires. Considering the shortcomings and difficulties of FD-1080 in conjugating with high molecular weight polymers, we synthesized the alkyne-modified probe (FD-AL) through a four-step reaction and 1 HNMR, 1 It was characterized by C NMR and high-resolution mass spectrometry. After covalently linking with the azide-labeled complementary DNA chain LS through a click reaction, the FD-AL-modified complementary DNA chain LS (FD-LS) showed the same characteristic emission peak of 1071 nm as FD-AL under 808 nm excitation, indicating that the modification of the NIR-II fluorescent FD-AL with the DNA chain was successful. After assembling with the long single chain to form a nano-spherical structure, due to the aggregation quenching effect, e.g. Figure 2 The red line in Figure D shows that the fluorescence intensity at 1071 nm is suppressed compared to that of the original FD-LS. Figure 2 The red line shown in Figure D. In the presence of ATP, the formed FD-AL modified DNA nanowires (DNCF) unzip under the recognition and binding of ATP and show the restored near-infrared second region fluorescence signal, as shown in Figure 2. Figure 2 The blue line shown in Figure D indicates that the nanowire cluster has the potential to achieve ATP-responsive near-infrared zone II tumor imaging.

[0044] 4. DOX loading and ATP response release

[0045] In order to achieve drug delivery and controlled release for precise tumor treatment, we loaded the anticancer drug DOX (doxorubicin) into DNCF by inserting it into the DNA double helix structure, such as Figure 3 As shown in Figure A. The successful loading of DOX was verified by detecting the fluorescence change of DOX. Figure 3 As shown in Figure B. After DNCF was co-incubated with DOX for 1 hour, the fluorescence intensity of DOX in the supernatant was lower than that of the original DOX solution, indicating that DOX was successfully loaded on DNCF. Then, different concentrations of ATP were incubated to monitor the drug release after reaction with ATP. As the ATP concentration increased, the fluorescence intensity of DOX in the supernatant continued to increase compared with the control group without ATP treatment, indicating that the synthesized DNCFD has ATP-responsive drug release, showing its potential in specific tumor treatment, such as Figure 3 As shown in Figure C.

[0046] 5. Specificity and stability of DNA nanoclusters

[0047] Using CTP, GTP and UTP as controls, the specificity of DNCFD was studied by detecting the release of DOX. After DNCFD was co-incubated with ATP, the fluorescence signal was significantly restored compared with that of the untreated nanowires, such as Figure 3 As shown in Figure D, the fluorescence enhancement after co-incubation with CTP, GTP or UTP was negligible compared to the fluorescence signal of the control group. The fluorescence intensity of DNC remained relatively stable, indicating the stability of this spherical DNA assembly. In addition, the hydration diameter of DNC was further monitored by DLS analysis, and the hydration diameter of DNC changed slightly during the 12-hour incubation with PBS or FBS. These results indicate that this ATP-responsive DNA nanocluster has excellent specificity and satisfactory stability.

[0048] 6. Conclusion

[0049] In the present invention, we designed a debondable DNA nanocluster to achieve ATP-triggered NIR-II fluorescence enhancement for early tumor imaging and treatment. The ATP-responsive conformational transition was achieved by integrating the ATP aptamer sequence into the long single-stranded DNA backbone. The curved complementary strands modified with FD-AL at both ends helped to form a spherical structure and induced the NIR-II fluorescence quenching effect, which could be restored after binding with ATP. In vitro experiments verified the "turn-on" of NIR-II fluorescence under ATP triggering, while tumor imaging in dimethylbenzanthracene-induced breast cancer mice demonstrated the feasibility of this DNA nanocluster for early tumor diagnosis. In addition, the anticancer drug DOX in the DNA double helix can be effectively released after the disintegration of the DNA nanocluster, indicating its potential application in targeted drug delivery and tumor treatment. This ATP-responsive DNA nanocluster conformational reversal achieves accurate NIR-II fluorescence imaging of early tumors, providing more opportunities for the clinical application of DNA nanotechnology in tumor diagnosis and precision medicine.

[0050] As an embodiment of the present invention, the specific test method is:

[0051] 1. Materials: Sodium chloride, solid ammonium persulfate, and tetramethylethylenediamine (TEMED) were purchased from Huijiecheng Biotechnology Co., Ltd. (Nanjing, China); calcium chloride, NaN3, sucrose, methyl-β-cyclodextrin, wortmannin, oligomycin, ATP, GTP, CTP, and UTP were purchased from Liangwei Technology Co., Ltd. (Nanjing, China). T4 DNA ligase, exonuclease 1, exonuclease 3, phi29 DNA polymerase, 10% bovine embryonic serum, DMEM, and double antigen were purchased from Shanghai Yisheng Technology Co., Ltd. The DNA strands and modified DNA strands used in this article were purchased from Shanghai Sangong Bioengineering Co., Ltd. The DNA sequences are detailed in Table 1.

[0052] 2. Instruments: Polyacrylamide electrophoresis (PAGE) was imaged by a Bio-Rad imaging system (Hercules, California, USA), and intracellular fluorescence images were obtained by a TCS SP5 confocal laser scanning microscope (CLSM) (Leica, Germany). Fluorescence spectra were recorded by an F-4700 spectrophotometer (Hitachi, Japan), and UV-visible absorption spectra were recorded by a UV-3600N UV / VIS spectrophotometer (Shimadzu, Japan). Zeta potential analysis and dynamic light scattering (DLS) analysis were performed on a ZetaPlus 90Plus / BI-MAS (Brookhaven, USA). Transmission electron microscopy (TEM) images were taken by a JEM-2800 transmission electron microscope (JEOL Co., Ltd., Japan).

[0053] 3. Verification of ATP reactivity: Take a medium concentration of ATP aptamer chain (2μL, 10μM) and react with complementary DNA chain LS (2μL, 10μM) in 20μL of 1×PBS buffer solution, and add 2μL 30μM Na+ to the solution. The two react in a 95°C water bath for 5 minutes, and then slowly cool to 37°C. After annealing, add different concentrations of ATP (1μM, 3μM, 5μM) and react in a 37°C water bath for 2 hours. Prepare a 15% polyacrylamide gel, place the polyacrylamide gel in an electrophoresis tank containing 1×TBE buffer, and electrophoresed at 100V for 90 minutes. Take the template chain (2μL, 10μM) and react with the same concentration of complementary DNA chain LS (2μL, 10μM) in a 20μL system. The buffer solution is 1×PBS, and 2μL 30μM Na+ is added. + The two were reacted in a 95°C water bath for 5 minutes and then slowly cooled to 37°C. After annealing, different concentrations of ATP (1μM, 5μM, 10μM, 50μM, 100μM) were added and reacted in a 37°C water bath for 2 hours. A 15% polyacrylamide gel was prepared and placed in an electrophoresis tank containing 1×TBE buffer and electrophoresed at 100V for 90 minutes.

[0054] 4. Complementary DNA chain LS folding verification: Take the template and mix it with equal concentrations of LS modified with FAM and BHQ1 at both ends respectively. FAM / BHQ1 Chain reaction, the buffer solution was 1× PBS, with 30 μM Na + The two were reacted in a 95°C water bath for 5 minutes and then slowly cooled to 37°C. After annealing, different concentrations of ATP (0.1μM, 0.5μM, 1μM, 5μM, 10μM) were added, and after reacting in a 37°C water bath for 2 hours, the fluorescence intensity was measured under an enzyme-labeled instrument.

[0055] 5. Synthesis of RCA: Take p-linear (4 μL, 100 μM) and lingation DNA (4 μL, 100 μM), react in a 95°C water bath for 5 minutes, and then slowly cool to 37°C. After annealing, add T4 ligase (1 μL, 400 U / μL) and 10×T4 ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH=7.5) (2 μL), react in a 25°C water bath for 16 hours, and then heat at 65°C for 10 minutes to inactivate T4 ligase. Take 8 μL of the above template chain and react with DNA primer (8 μL, 10 μM) in a 95°C water bath for 5 minutes, and then slowly cool to 37°C. Take 10 μL of the above solution, add phi29 DNA polymerase (1 μL, 0.2U), 10×phi29 DNA polymerase buffer (2 μL) (50 mM Tris-HCl, 10 μM MgCl2, 10 μM (NH4)2SO4, 4 μM DTT, pH = 7.5), dNTPs (2 μL, 0.1 mM), BSA (1 μL, 0.4 μg / μL) and 4 μL Millin-Q ultrapure water, react in a 37°C water bath for 4 hours, then heat at 65°C for 10 minutes to inactivate the phi29 DNA polymerase to prepare the RCA product.

[0056] 6. Synthesis and verification of DNA nanowires: Synthesized RCA long single strands and modified LS FAM / BHQ1 The two ends of the chain reacted in a ratio of 1:15 to produce DNA nanoclusters. The fluorescence changes of the synthesized nanofragments were then measured to verify their conformational changes, and then the particle size and morphology of the DNA nanoclusters were measured by DLS and TEM. The specific synthesis route of alkynyl-modified FD-AL can be found in the supporting information. To prepare a complementary DNA chain LS modified with a fluorescent material, the azide-modified DNA chain and the alkynyl-modified FD-AL were mixed in a concentration ratio of 1:10. The reaction was carried out at 50°C for 24 hours under the action of catalysts copper sulfate and sodium ascorbate. Then, DNA nanoclusters containing the fluorescent material FD-AL were prepared according to the above ratio, such as Figure 4 As shown in Figure A.

[0057] 7. DOX loading and verification: DOX was incubated with DNA nanowires at 37 °C for 1.5 h, and the fluorescence was detected and the loading amount of DOX was verified, e.g. Figure 4 As shown in Figure B. Then, it was incubated with different concentrations of ATP (0μM, 5μM, 10μM, 50μM, 100μM, 200μM) for 2 hours to detect the fluorescence change of DOX. After that, it was incubated with equal concentrations (100μM) of ATP, CTP, GTP and UTP for 2 hours to detect the specific release of ATP and measure the fluorescence intensity change of DOX, as shown in Figure B. Figure 4 As shown in Figures C and D.

[0058] The preferred embodiments of the present invention are described above with reference to the accompanying drawings, but the scope of the rights of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by a person skilled in the art without departing from the scope and essence of the present invention should be within the scope of the rights of the present invention.

Claims

1. A method for constructing an ATP-responsive DNA nanocluster, characterized in that: The steps include: S11, synthesizing a circular template chain containing the ATP aptamer sequence LA using the linear template LT and the ATP aptamer sequence LA, and obtaining a long single-stranded DNA containing a repeated ATP aptamer sequence through an RCA reaction; S12, using the 5' end and 3' end of the complementary DNA strand LS to partially hybridize with the repeated ATP aptamer sequence LA in the long single-stranded DNA obtained in step S11, and folding the long single-stranded DNA into a DNA nanocoil DNC; wherein: The complementary DNA strand LS in the DNA nanocoil DNC has a bent configuration; The ATP adaptor sequence LA in the DNA nanocoil DNC can recognize ATP to induce the complementary DNA chain LS to be released from the DNA nanocoil DNC and induce the bending configuration of the complementary DNA chain LS to collapse.

2. The method for constructing an ATP-responsive DNA nanowire according to claim 1, characterized in that: The complementary DNA chain LS has complementary fragments at both the 5' end and the 3' end.

3. Application of a DNA nanowire DNCF labeled with near-infrared second region fluorescence in tumor-targeted near-infrared second region fluorescence imaging, characterized in that: The method for constructing the DNA nanowire DN CF labeled with near-infrared second-region fluorescence comprises the following steps: S31, synthesizing a circular template chain containing the ATP aptamer sequence LA using the linear template LT and the ATP aptamer sequence LA, and obtaining a long single-stranded DNA containing a repeated ATP aptamer sequence through an RCA reaction; S32, hybridizing the 5' end and 3' end of the DNA chain FD-LS labeled with the near-infrared region II fluorescent molecule FD with the repeated ATP aptamer sequence portion in the long single-stranded DNA obtained in step S31, and folding the long single-stranded DNA into a DNA nanowire DNCF labeled with near-infrared region II fluorescence; wherein: The DNA chain FD-LS is formed by a click reaction between a complementary DNA chain LS modified with an azide group and a near-infrared second-zone fluorescent molecule FD-AL modified with an alkyne group; The DNA chain FD-LS in the near-infrared II fluorescently labeled DNA nanocluster DNCF has a bent configuration, and the bent configuration makes the fluorescent dye FD modified at the end of the complementary DNA chain LS adjacent to each other, so as to induce aggregation quenching of the near-infrared II NIR-II fluorescence of FD; The ATP aptamer sequence LA in the DNA nanocluster DNCF can recognize ATP, induce the complementary DNA chain FD-LS to be released from the DNA nanocluster DNCF, and collapse its bending configuration, so that the fluorescent reporter group FD emits 1071nm fluorescence at an excitation wavelength of 808nm, thereby achieving fluorescence enhancement in the near-infrared second region NIR-II in response to ATP.

4. Application of a DNA nanowire DNCF labeled with near-infrared second region fluorescence in drug delivery and controlled release, characterized in that: The drug is loaded into the near-infrared second region fluorescently labeled DNA nanocluster DNCF by inserting into the DNA double helix structure, and the ATP aptamer sequence LA in the near-infrared second region fluorescently labeled DNA nanocluster DNCF can recognize ATP and induce the complementary DNA chain LS to be released from the near-infrared second region fluorescently labeled DNA nanocluster DNCF, so as to achieve the delivery and release of the drug; wherein: the construction method of the near-infrared second region fluorescently labeled DNA nanocluster DNCF comprises the following steps: S41, synthesizing a circular template chain containing the ATP aptamer sequence LA using the linear template LT and the ATP aptamer sequence LA, and obtaining a long single-stranded DNA containing a repeated ATP aptamer sequence through an RCA reaction; S42, hybridize the 5' and 3' ends of the DNA chain FD-LS labeled with the near-infrared region II fluorescent molecule FD with the repeated ATP aptamer sequence portion in the long single-stranded DNA obtained in step S41, and fold the long single-stranded DNA into a DNA nanocluster DNCF labeled with near-infrared region II fluorescence.

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