DNA tetrahedron gating logic nanometer machine and preparation method and application thereof

Abstract

The invention discloses a DNA tetrahedron gating logic nanometer machine and a preparation method and application thereof, and belongs to the technical field of biomedicine. According to the invention, a DNA tetrahedron gating logic nanometer machine DTDN is constructed, and the DNA tetrahedron gating logic nanometer machine DTDN is composed of two DNA tetrahedron nanometer structures functionalized by AS1411 aptamers. Each tetrahedron is loaded with a functional DNA hairpin structure, and the structure is composed of a stem-loop structure and an extended single-stranded tail containing an AP site. The framework gating strategy of DTDN enables efficient delivery of functional DNA hairpins, and also prevents signal leakage by blocking cohesive end sequences of HCR reactions. The cascade activated logic loop has high specificity and anti-interference capability, and can accurately activate a downstream regulation and control path. Therefore, the DTDN realizes synchronization of imaging and regulation, and a programmable, safe and accurate nucleic acid nano platform is provided for cancer diagnosis and accurate treatment based on a cGAS-STING signal channel.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a DNA tetrahedral gated logic nanomachine, its preparation method, and its application. Background Technology

[0002] The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-interferon gene stimulating factor (STING) signaling pathway is a key component of the innate immune system. As a cytoplasmic DNA sensor, cGAS recognizes and binds to various double-stranded DNA molecules, thereby activating itself and catalyzing the production of 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). This molecule subsequently activates STING, promoting the production of type I interferons (IFNs) and other inflammatory cytokines. The cGAS-STING pathway participates in the regulation of multiple biological processes, making it a highly promising therapeutic target. Various drugs targeting this pathway have shown significant therapeutic potential in a variety of diseases. However, traditional cGAS-STING agonists (chemotherapeutic drugs, non-nucleoside small molecules) are limited by off-target damage and delivery barriers; while free nucleic acid devices can activate this pathway, they suffer from insufficient stability, low bioavailability, and limited tumor selectivity. Therefore, there is a need to develop a nucleic acid nanoplatform with targeted delivery capabilities that can specifically activate the cGAS-STING pathway. Summary of the Invention

[0003] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a DNA tetrahedral gated logic nanomachine, its preparation method, and its application, thereby providing a nucleic acid nanoplatform that combines targeted delivery with cGAS-STING pathway-specific activation.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a DNA tetrahedral gated logic nanomachine DTDN is provided, including HTDN1 and HTDN2; HTDN1 is composed of an AS1411 aptamer-functionalized DNA tetrahedral nanostructure TDN1 and a functional DNA hairpin structure H1 loaded on TDN1; HTDN2 is composed of an AS1411 aptamer-functionalized DNA tetrahedral nanostructure TDN2 and a functional DNA hairpin structure H2 loaded on TDN2; wherein, TDN1 and TDN2 are respectively assembled from four different DNA strands.

[0005] Furthermore, the nucleotide sequences of the four DNA strands of TDN1 are shown in SEQ ID NO.1-4; the nucleotide sequences of the four DNA strands of TDN2 are shown in SEQ ID NO.5-8.

[0006] Furthermore, the nucleotide sequence of the functional DNA hairpin structure H1 is shown in SEQ ID NO.9; the nucleotide sequence of the functional DNA hairpin structure H2 is shown in SEQ ID NO.10.

[0007] Furthermore, the functional DNA hairpin structures H1 and H2 both consist of a stem-loop structure and an extended single-stranded tail containing an AP site.

[0008] This invention provides a method for preparing the above-mentioned DNA tetrahedral gated logic nanomachine DTDN, comprising the following steps: (1) Preparation of HTDN1: The mixture of the four DNA strands of TDN1 and the functional DNA hairpin structure H1 was heated at 95°C for 5 min and cooled to room temperature to obtain the final product; (2) Preparation of HTDN2: The mixture of the four DNA strands of TDN2 and the functional DNA hairpin structure H2 was heated at 95°C for 5 min and cooled to room temperature to obtain the final product.

[0009] Furthermore, the molar ratio of the four DNA strands of TDN1 to the functional DNA hairpin structure H1 is 1:1:1:1:1-4.

[0010] Furthermore, the molar ratio of the four DNA strands of TDN2 to the functional DNA hairpin structure H2 is 1:1:1:1:1-4.

[0011] This invention provides an application of the above-mentioned DNA tetrahedral gated logic nanomachine DTDN in detecting intracellular miRNA levels.

[0012] This invention provides an application of the above-mentioned DNA tetrahedral gated logic nanomachine DTDN in cancer cell imaging.

[0013] This invention also provides an application of the above-mentioned DNA tetrahedral gated logic nanomachine DTDN in activating the intracellular cGAS-STING pathway.

[0014] This invention offers the following advantages: It constructs a DNA tetrahedral gated logic nanomachine, DTDN, for precise imaging of cancer cells and to activate the cGAS-STING pathway guided by imaging. DTDN comprises HTDN1 and HTDN2; HTDN1 consists of an AS1411 aptamer-functionalized DNA tetrahedral nanostructure TDN1 and a functional DNA hairpin structure H1 mounted on TDN1; HTDN2 consists of an AS1411 aptamer-functionalized DNA tetrahedral nanostructure TDN2 and a functional DNA hairpin structure H2 mounted on TDN2. The structures of the functional DNA hairpin structures H1 and H2 consist of a stem-loop structure and an extended single-stranded tail containing an abase-free (AP) site. This tail sequence hybridizes complementaryly with the tetrahedral extended strand, thereby anchoring the hairpin and sealing the toehold sequence of the hybridization chain reaction (HCR), preventing premature leakage. Upon entering the target cell, the overexpressed apurine / pyrimidine endonuclease 1 (APE1) recognizes and cleaves the AP site, releasing H1 and H2 from the tetrahedron and exposing the toehold sequence. Subsequently, miRNA-21 (miR-21) acts as the initiator chain to trigger hairpin self-assembly, generating a long, nicked fluorescent double-stranded DNA (dsDNA). This dsDNA directly stimulates cGAS, thereby activating the cGAS-STING pathway. The advantages of modular DTDN include: (1) using an AS1411-modified DNA tetrahedron as a carrier, which enhances the efficiency of targeted delivery of functional DNA hairpins to cancer cells and effectively prevents signal leakage by blocking the toehold sequence of the HCR reaction; (2) the endogenous biomarker cascade activation strategy ensures specific activation of the cGAS-STING pathway in cancer cells; and (3) the generated fluorescent dsDNA enables the visualization and monitoring of the activation status of the intracellular cGAS-STING pathway. Therefore, the DTDN of this invention achieves simultaneous imaging and modulation, providing a programmable, safe and accurate nucleic acid nanoplatform for cancer diagnosis and precision treatment based on the cGAS-STING pathway. Attached Figure Description

[0015] Figure 1 This is a schematic diagram illustrating the use of DTDN for cancer cell-specific imaging and imaging-guided cGAS-STING pathway regulation. Figure 2 Structural diagrams of TDN1 and TDN2, DNA tetrahedral nanostructures functionalized with AS1411 aptamers; Figure 3 Structural diagrams of functional DNA hairpins H1 and H2, and control group DNA hairpins nAP-H1 and nAP-H2; Figure 4 Figure showing the results of HTDN1 synthesis and APE1-triggered H1 release; Figure 5 The dynamic light scattering measurement diagram for HTDN1; Figure 6 A schematic diagram illustrating the dsDNA self-assembly achieved by the APE1 and miR-21 cascade activation DTDN system; Figure 7 PAGE characterization of miR-21-triggered HCR; Figure 8 A schematic diagram illustrating the formation of fluorescent dsDNA triggered by "logic gating"; Figure 9 The fluorescence spectra of DTDN under different input signals; Figure 10 The fluorescence response of the DTDN system to increased miR-21 concentration is shown in the graph. Figure 11 The specific fluorescence response of DTDN to APE1 and miR-21 is shown in the figure. Figure 12 Confocal laser scanning microscopy images of DTDN and HeLa cells incubated for different times and corresponding statistics of intracellular relative fluorescence intensity; Figure 13 Confocal laser scanning microscopy images of HeLa cells treated with DTDN (a), free H1 and H2 (b), and nAP-DTDN (c), and corresponding intracellular relative fluorescence intensity statistics; Figure 14 A schematic diagram of the control group nanomachine nAP-DTDN, which has the same structure as DTDN; Figure 15 Fluorescence imaging images mediated by APE1 and miR-21 after MCF-10A and HeLa cells were incubated with nAP-DTDN or DTDN, respectively. Figure 16 for Figure 15 Statistics on relative fluorescence intensity of different cell samples and relative expression levels of miR-21 in the two cell types; Figure 17 This is a schematic diagram illustrating the inhibitory effects of APE1 inhibitors and miR-21 inhibitors on dsDNA assembly. Figure 18 Confocal laser scanning microscopy images of HeLa cells treated with blank control group, NCA group and miR-21 inhibitor group and corresponding statistics of intracellular relative fluorescence intensity; Figure 19 To measure the cell viability of different cells under different treatments; Figure 20The graph shows the expression levels of cGAMP, IFN-β mRNA, and IFN-β in MCF-10A and HeLa cells under different treatments. Detailed Implementation

[0016] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0017] This invention constructs a DNA tetrahedral gated logic nanomachine, DTDN, for precise imaging of cancer cells and to activate the cGAS-STING pathway guided by imaging. A schematic diagram illustrating its application in cancer cell-specific imaging and imaging-guided cGAS-STING pathway regulation is shown below. Figure 1 As shown.

[0018] The nucleotide sequences used in this invention are shown in Tables 1-2.

[0019] Table 1. Nucleotide sequences used in this invention

[0020] Table 2 Nucleotide sequences used in this invention

[0021] Note: Underlined bases are mismatched bases.

[0022] Example 1: Preparation of TDN and HTDN.

[0023] HTDN1 consists of five DNA strands (A1, B1, C1, D1, and hairpin H1). For the first DNA tetrahedral nanostructure (TDN1), a mixture of A1 (1 μM), B1 (1 μM), C1 (1 μM), and D1 (1 μM) was heated at 95 °C for 5 min, then cooled to room temperature. For HTDN1 (1 μM), a mixture of A1 (1 μM), B1 (1 μM), C1 (1 μM), D1 (1 μM), and H1 (3 μM) was heated at 95 °C for 5 min, then slowly cooled to room temperature. TDN2 and HTDN2 were annealed using the same procedure. The prepared HTDN1 and HTDN2 were stored at 4 °C for subsequent use (see schematic diagrams of TDN1 and TDN2). Figure 2 The structural diagrams of functional DNA hairpins H1 and H2, and control group DNA hairpins nAP-H1 and nAP-H2 are shown below. Figure 3 The sequences of nAP-H1 and nAP-H2 are shown in Table 1.

[0024] The formation of HTDN1 and the release of H1 from HTDN1 under the action of APE1 were analyzed by polyacrylamide gel electrophoresis (12% polyacrylamide gel, 100V, 100min). Simultaneously, different concentrations of miR-21 were incubated with the HCR system (H1, H2) at 37℃ for 3h to explore the feasibility of the HCR reaction. The HCR reaction products were verified by 12% polyacrylamide gel electrophoresis. All gels were finally stained with gel green dye for 5min, and images were acquired using the Bio-Rad gel imaging system.

[0025] 100 μL of HTDN1 (25 nM) was dropped onto a mica sheet, allowed to stand for 1 min, rinsed with deionized water, dried with nitrogen, and then imaged using an atomic force microscope (AFM).

[0026] Example 2: Test Method (1) Dynamic study on enzyme response characteristics of in vitro DTDN system Cy3-labeled H1 strands (Cy3-H1, sequence shown in Table 2) were hybridized with BHQ1-labeled DNA tetrahedral nanostructures (BHQ1-TDN1) to form the Cy3-HTDN1-BHQ1 hybrid product. 100 nM Cy3-HTDN1-BHQ1 was placed in PBS buffer containing APE1 (1.0 U / mL) and incubated at 37°C for 3 h. The fluorescence intensity of the resulting solution was then recorded using a fluorescence spectrometer.

[0027] (2) In vitro fluorescence assay To evaluate the feasibility of DTDN forming fluorescent dsDNA under endogenous stimulation, a series of fluorescence experiments were conducted. In the feasibility verification experiment of DTDN for APE1 and miR-21, 100 nM DTDN (HTDN1 and HTDN2) and 100 nM miR-21 were incubated in PBS buffer containing APE1 (1.0 U / mL) at 37 °C for 4 h. The fluorescence signal of dsDNA in the range of 640-750 nm was recorded at an excitation wavelength of 600 nm.

[0028] After verifying the feasibility of dsDNA self-assembly, the response of DTDN to different concentrations of miR-21 was evaluated. Solutions containing 100 nM DTDN were mixed with different concentrations of miR-21 and incubated for 4 h in PBS buffer containing APE1 (1.0 U / mL); the fluorescence spectra of all samples were then recorded. To evaluate the selectivity of the DTDN system, different nucleases and negative control miRNAs were also selected, incubated under the above conditions, and fluorescence intensity was recorded.

[0029] (3) Cell culture and fluorescence imaging All cells were seeded in confocal culture dishes and cultured for 24 h in a cell culture incubator at 37°C and 5% CO2. Cells were then incubated with 100 nM DTDN for different times (2, 3, 4, and 5 h). Cells were then gently washed with PBS. All cell fluorescence images were acquired using a confocal microscope (excitation wavelength 600 nm, emission spectrum collection range 640–780 nm). Statistical analysis and graphing were performed using Graphpad Prism 8.0 software (GraphPad Software). Experimental data are presented as mean ± standard deviation.

[0030] (4) MTT test Cell viability after incubation with DTDN or nAP-DTDN was assessed using the MTT assay. First, human cervical cancer cell line (HeLa) and human normal breast cell line (MCF-10A) were seeded in 96-well plates and cultured at 37°C for 24 h. Then, the cells were treated with Durbeco modified Eagle medium (DMEM) containing different concentrations (0, 50, 100, and 200 nM) of DTDN and nAP-DTDN for 36 h. 50 μL of 3-(4,5-dimethylthiazolium)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well, and the cells were incubated at 37°C for 4 h. The MTT solution was then discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. Finally, the absorbance at 490 nm was measured.

[0031] (5) Calcein-AM / PI staining analysis HeLa cells were co-incubated with 100 nM DTDN and 100 nM nAP-DTDN for 24 h, respectively, and then treated with live-cell green fluorescent dye (Calcein-AM) and dead-cell red fluorescent dye (Propidium Iodide, PI) for 20 min. Imaging was performed using confocal microscopy. Fluorescence signals of Calcein-AM (495-545 nm) and PI (562-620 nm) were acquired, respectively. Cells not treated with DTDN or nAP-DTDN served as a blank control group.

[0032] (6) cGAMP and IFN-β were analyzed by enzyme-linked immunosorbent assay (ELISA). To detect cGAMP levels, MCF-10A cells and HeLa cells treated with DTDN or nAP-DTDN for 6 h were collected, respectively. cGAMP was quantified using a 2'3'-cGAMP competitive ELISA kit (Dongguan Enzyme-Linked Biotechnology Co., Ltd.) according to the manufacturer's instructions. Absorbance at 450 nm was measured using a Thermo Scientific Varioskan Flash (Thermo Fisher Scientific, Inc.). After adding DTDN and nAP-DTDN to MCF-10A and HeLa cells, respectively, and incubating at 37°C for 16 h, IFN-β was quantified using a human IFN-β ELISA kit (Dongguan Enzyme-Linked Biotechnology Co., Ltd.).

[0033] (7) RT-qPCR detection To determine mRNA levels, HeLa cells were treated with DTDN and nAP-DTDN for 8 hours, respectively. Cells were then collected using RNAiso Plus (Trizol), and total RNA was extracted according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using the Vazyme RT kit. Gene expression was quantified using the AceQ qPCR SYBR Green Master Mix kit (Vazyme, Nanjing, China).

[0034] Results analysis: (1) Assembly and disassembly of HTDN Successfully synthesizing HTDN with a three-dimensional structure is fundamental to achieving imaging-guided cGAS-STING pathway modulation. Therefore, the preparation process of HTDN was characterized using various techniques. Figure 4 A illustrates the assembly of HTDN1 and the dissociation of hairpin H1. First, A1, B1, C1, D1, and H1 were mixed in a molar ratio of 1:1:1:1:3. Then, according to the Watson-Crick base pairing rule, the mixture was slowly cooled from 95°C to room temperature to form HTDN1. The synthesis of HTDN1 was detected by non-denaturing polyacrylamide gel electrophoresis (Native-PAGE) and atomic force microscopy (AFM). The results are as follows... Figure 4As shown in B, samples in lanes 1-4 represent A1, A1+B1, A1+B1+C1, and TDN1 (A1+B1+C1+D1), respectively. With the addition of the sequence to the reaction system, the migration rate of the reaction product gradually decreased, confirming the formation of TDN. Compared to lane 4, lane 5 showed even lower migration, which is due to the formation of a larger DNA nanostructure HTDN1 between H1 and TDN after the addition of strand H1. The addition of APE1 (lane 6) accelerated the migration rate of the HTDN1 band, indicating that the hairpin dissociated from HTDN1. Native-PAGE experimental results confirmed the feasibility of the method for preparing HTDN and the enzyme activation strategy of this invention.

[0035] The morphology of HTDN1 was evaluated using AFM. The results are as follows: Figure 4 As shown in C, HTDN1 exhibits a uniformly sized monodisperse state, which is consistent with the results of dynamic light scattering (DLS) experiments (see Figure C). Figure 5 The results were consistent. Next, a fluorescence experiment (see...) was conducted... Figure 4 D) The feasibility of unloading hairpin H1 in the presence of APE1 was evaluated. Cy3-labeled H1 binds to BHQ1-labeled DNA tetrahedrons via its complementary sequence to form Cy3-HTDN1-BHQ1, causing Cy3 fluorescence quenching. A significant fluorescence signal was detected in the presence of APE1, indicating the release of Cy3-H1 from Cy3-HTDN1-BHQ1.

[0036] (2) Verification of DTDN induction performance Human APE1 and miR-21 are widely considered to be overexpressed during the occurrence and development of cancer cells. Therefore, this invention selects APE1 to trigger the release of hairpin H1 and H2, and the HCR reaction activated by the miR-21 cascade drives the alternating hybridization of H1 and H2 to form long fluorescently nicked double-stranded DNA (dsDNA, see...) Figure 6 ).Depend on Figure 7 It can be seen that no new bands appeared when miR-21 was deleted, which proves the stability of hairpins H1 and H2. When only miR-21 and H1 were present, a certain degree of reaction occurred and new DNA products were produced. Figure 7 The 6th band in the image). In the presence of miR-21, H1 and H2 self-assemble to form dsDNA of different lengths ( Figure 7 (The 7th and 8th bands in the image) confirm that HCR is successfully activated and functions in the presence of miR-21.

[0037] (3) Sensing capability of DTDN The sensing capability of DTDN was further evaluated and verified using fluorescence detection technology. The hairpin stem of H1 was modified with the fluorescent group Cy5 and the quencher group BHQ2, respectively (see Table 2). After the addition of APE1, DTDN unloaded the hairpin structures H1 and H2, exposing the sticky terminal sequence of HCR. Upon the addition of the promoter miR-21, the HCR reaction was triggered and the hairpin H1 structure was gradually opened, leading to spatial separation of Cy5 and BHQ2, while simultaneously enhancing the fluorescence signal of Cy5 (see Table 2). Figure 8 ).Depend on Figure 9 It was observed that a strong fluorescence signal was clearly visible when APE1 and miR-21 were present simultaneously. However, the detected signal was weaker when APE1 or miR-21 was added alone, or neither was added. These results indicate that the cascade recognition of APE1 and miR-21 activates the formation of dsDNA from DTDN.

[0038] (4) DTDN’s ability to recognize promoters of different concentrations Depend on Figure 10 A and 10B show that the fluorescence intensity steadily increases with increasing miR-21 concentration until it reaches a plateau. Figure 10 C further indicates that within the range of 0.01–1 nM, fluorescence intensity is linearly correlated with miR-21 concentration (R0). 2 =0.998).

[0039] Furthermore, the specific responses of DTDN to APE1 and miR-21 were investigated. Compared with other enzymes, Cy5 signaling was significantly enhanced in the APE1 response (see...). Figure 11 A). In the presence of other common enzymes, the fluorescence signal showed almost no change compared to the control group. This confirms that DTDN has high specificity for APE1 and can effectively avoid potential interference from other enzymes. The selectivity of DTDN was investigated by introducing mismatched sequences and other negative miRNAs. The results are as follows: Figure 11 As shown in Figure B, a significant Cy5 signal only appears when miR-21 is present, indicating that DTDN has good selectivity for miR-21.

[0040] (5) Performance evaluation of DTAN in cancer cell imaging Based on in vitro experiments, the ability of DTDN to image cancer cells was further evaluated. First, this invention investigated the optimal incubation time for DTDN. The results are as follows... Figure 12 As shown in Figure A, DTDN was incubated with cells for different times (2, 3, 4, and 5 hours), and the fluorescence intensity of the cells in the confocal images was extracted using Image-J software. Figure 12As shown in Figure B, the fluorescence intensity of HeLa cells incubated with DTDN increased with increasing incubation time, reaching its maximum at approximately 4 hours. Therefore, 4 hours was chosen as the incubation duration for subsequent experiments. To verify the ability of DTDN to deliver functional nucleic acids, a free system (H1+H2) was used as a control. Figure 13 It can be seen that, compared with the free system (sample b), HeLa cells incubated with DTDN (sample a) showed obvious Cy5 fluorescence, indicating that DTDN achieved excellent cellular uptake efficiency due to the AS1411 aptamer and the tetrahedral structure of DNA.

[0041] To verify the ability of DTDN to generate long-chain linear fluorescent products in cancer cells through hierarchical activation, this invention compared and analyzed DTDN and nAP-DTDN in HeLa cells and MCF-10A cells. In the nAP-DTDN system lacking the AP site, the base sequences of nAP-H1 and nAP-H2 were identical to those of H1 and H2 in DTDN (see...). Figure 3 and Figure 14 Clearly, nAP-H1 and nAP-H2 cannot be cleaved by APE1 and cannot hybridize with miR-21, therefore they cannot produce fluorescent signals (samples a and b). Figure 15 Fluorescence imaging results showed that only HeLa cells treated with DTDN (sample d) exhibited a strong Cy5 signal. No significant signal was detected in MCF-10A cells treated with DTDN (sample c). Quantitative summarization of these confocal imaging results confirmed a significant difference in fluorescence signals between HeLa cells treated with DTDN (see [link to article]). Figure 16 A). Furthermore, the relative levels of miR-21 in the two cell types were determined by RT-PCR (see [reference needed]). Figure 16 (B) indicates that miR-21 is overexpressed in HeLa cells. Overexpression of APE1 and miR-21 in cancer cells can effectively activate DTDN to form dsDNA, confirming that the DTDN system can distinguish different cells based on significantly different fluorescence signals, particularly enabling the differentiation between cancer cells and normal cells.

[0042] (6) The role of APE1 and miR-21 in DTDN activation Pretreatment of cells with an APE1 inhibitor (7-nitroindole-2-carboxylic acid, NCA) resulted in HeLa cells with downregulated APE1 expression (see schematic diagram). Figure 17 These cells were then co-incubated with DTDN. Figure 18It was found that the fluorescence signal of HeLa cells treated with NCA was significantly lower than that of the control group, which was due to the inhibition of the dissociation process of H1 and H2. Furthermore, the ability of DTDN to monitor miR-21 expression levels in cells was investigated. HeLa model cells were pretreated with miR-21 mimics and miR-21 inhibitors, respectively, to achieve upregulation and downregulation of miR-21. DTDN was then co-incubated with these pretreated cells. Compared with untreated cells (blank control group), cells pretreated with miR-21 mimics showed significantly enhanced fluorescence intensity, while cells pretreated with miR-21 inhibitors showed a significantly weakened fluorescence signal. This indicates that different concentrations of miR-21 can induce different degrees of fluorescent dsDNA assembly. These results suggest that APE1 and miR-21 play an indispensable role in activating DTDN to form dsDNA.

[0043] (7) DTDN activates the cGAS-STING pathway in living cells. Based on the above results, this study further investigated whether DTDN can activate the cGAS-STING pathway in living cells through specifically assembled dsDNA structures, thereby regulating cell fate. This invention compared and analyzed the changes in MCF-10A cells and HeLa cells after incubation with DTDN or nAP-DTDN. Prior to this analysis, the effects of DTDN and nAP-DTDN on the cell viability of MCF-10A and HeLa cells were assessed using MTT assay. The results are as follows... Figure 19 As shown in A and 19B, in MCF-10A and HeLa cells, cell viability remained almost unchanged with increasing nAP-DTDN concentration, indicating that the DNA structure itself does not possess regulatory function. However, when DTDN entered HeLa cells, dose-dependent cytotoxicity was observed, but DTDN had almost no effect on normal MCF-10A cells. These results indicate that the DTDN system can recognize the APE1 and miR-21 environment in cancer cells and induce cell death by selectively generating dsDNA. The ability of DTDN to induce cancer cell death was further validated by live / dead cell double staining analysis using Calcein-AM / PI. Compared with other groups (blank control group, nAP-DTDN group), the DTDN group showed large-area red fluorescence and significantly increased cancer cell mortality (see...). Figure 19 C).

[0044] After co-incubating cells with DTDN, the activation of the cGAS-STING signaling pathway was assessed by detecting intracellular cGAMP levels, cytokine mRNA levels, and IFN-β secretion in the culture medium (see [link to relevant documentation]). Figure 20 A). By Figure 20As shown in Figure B, compared with the baseline levels detected in the untreated blank control group, the cGAMP content in HeLa cells incubated with nAP-DTDN did not increase significantly, while the cGAMP level in the DTDN-treated group was significantly higher than that in the blank control group. Further RT-qPCR and ELISA experiments demonstrated that IFN-β mRNA expression in DTDN-treated HeLa cells (see Figure B) was significantly higher than that in the blank control group. Figure 20 C) and IFN-β protein secretion (see C) Figure 20 D) All were significantly enhanced. In contrast, no significant changes were observed in MCF-10A cells or cells treated with nAP-DTDN. These results indicate that the DTDN system of the present invention can specifically activate the cGAS-STING pathway in cancer cells, representing a promising targeted anti-tumor strategy.

[0045] In summary, this invention constructs a DNA tetrahedral gated logic nanomachine (DTDN) by integrating an endogenous biomarker cascade activation strategy and a DNA tetrahedral gating strategy to achieve selective imaging of cancer cells and precise activation of the cGAS-STING pathway. The DTDN constructed in this invention has three significant advantages: (1) using AS1411-modified DNA tetrahedrons as a carrier enhances the efficiency of targeted delivery of functional DNA hairpins to cancer cells and effectively prevents signal leakage by blocking the toehold sequence of the HCR reaction; (2) the endogenous biomarker cascade activation strategy ensures specific activation of the cGAS-STING pathway in cancer cells; and (3) the generated fluorescent dsDNA enables visual monitoring of the activation state of the intracellular cGAS-STING pathway. Therefore, this nucleic acid nanomachine, which can specifically stimulate the cGAS-STING pathway in cancer cells, has great potential in disease diagnosis and treatment.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A DNA tetrahedron-gated logic nanomachine (DTDN), characterized in that, The DTDN comprises HTDN1 and HTDN2; the HTDN1 is composed of a DNA tetrahedron nanostructure TDN1 functionalized by AS1411 aptamer and a functional DNA hairpin structure H1 loaded on the TDN1; the HTDN2 is composed of a DNA tetrahedron nanostructure TDN2 functionalized by AS1411 aptamer and a functional DNA hairpin structure H2 loaded on the TDN2; wherein the TDN1 and the TDN2 are assembled by four different DNA chains respectively.

2. The DNA tetrahedron-gated logic nanomachine (DTDN) according to claim 1, wherein, The nucleotide sequences of the four DNA chains of the TDN1 are shown as SEQ ID NO. 1-4; the nucleotide sequences of the four DNA chains of the TDN2 are shown as SEQ ID NO. 5-8.

3. The DNA tetrahedron-gated logic nanomachine (DTDN) according to claim 1, wherein, The nucleotide sequence of the functional DNA hairpin structure H1 is shown as SEQ ID NO. 9; the nucleotide sequence of the functional DNA hairpin structure H2 is shown as SEQ ID NO.

10.

4. The DNA tetrahedron-gated logic nanomachine (DTDN) according to claim 1, wherein, The structures of the functional DNA hairpin structures H1 and H2 are both composed of a stem-loop structure and an extended single-stranded tail containing an AP site.

5. The method for preparing DNA tetrahedral gate logic nanomachine DTDN according to any one of claims 1-4, characterized in that, The method comprises the following steps: (1) preparing the HTDN1: heating a mixture of the four DNA chains of the TDN1 and the functional DNA hairpin structure H1 at 95℃ for 5 min, and cooling to room temperature to obtain the HTDN1; (2) preparing the HTDN2: heating a mixture of the four DNA chains of the TDN2 and the functional DNA hairpin structure H2 at 95℃ for 5 min, and cooling to room temperature to obtain the HTDN2.

6. The production method according to claim 5, wherein The molar ratio of the four DNA chains of the TDN1 and the functional DNA hairpin structure H1 is 1:1:1:1:1-4.

7. The preparation method according to claim 5, characterized in that, The molar ratio of the four DNA chains of the TDN2 and the functional DNA hairpin structure H2 is 1:1:1:1:1-4.

8. Use of the DNA tetrahedron gate logic nanomachine DTDN in any one of claims 1-4 in detecting the level of miRNA in cells.

9. Use of the DNA tetrahedron gate logic nanomachine DTDN in any one of claims 1-4 in imaging cancer cells.

10. Use of the DNA tetrahedron gate logic nanomachine DTDN in any one of claims 1-4 in activating the cGAS-STING pathway in cells.

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