DNA tetrahedron self-adaptive catalytic hybridization circuit for circular RNA imaging and precise gene therapy as well as construction method and application of DNA tetrahedron self-adaptive catalytic hybridization circuit

Abstract

The invention discloses a DNA tetrahedron self-adaptive catalytic hybridization circuit for circular RNA imaging and precise gene therapy and a construction method and application of the DNA tetrahedron self-adaptive catalytic hybridization circuit. The construction method comprises the steps that a reaction solution containing T1-S2, T2, T3, T4, S2 * and an annealing buffer solution is denatured and cooled to the room temperature, and a DNA tetrahedron framework is prepared; s1, S1 * and an annealing buffer solution are mixed, heated and then cooled to the room temperature, and capture double strands (S1 / S1 *) are prepared; incubating the DNA tetrahedron framework, capturing double strands, and constructing to obtain an integrated multifunctional DNA tetrahedron; the method comprises the following steps: incubating a reaction mixture containing target circRNA with different concentrations, a DNA tetrahedron, an annealing buffer solution and BSA (Bovine Serum Albumin), and carrying out circrRNA-started DNA tetrahedron entropy-driven strand displacement hybridization reaction. According to the invention, the expression of circCDYL in cancer cells can be accurately quantified, and breast cancer patients and healthy individuals can be distinguished.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioanalysis, and in particular relates to a DNA tetrahedron adaptive catalytic hybridization circuit for circular RNA imaging and precision gene therapy, as well as a construction method and uses thereof. Background Art

[0002] Circular RNAs (circRNAs) are a class of single-stranded covalently closed RNA molecules without a 5' methylated guanine cap and a 3' polyadenylic acid tail. CircRNAs are processed by reverse splicing of precursor messenger RNA. The covalently closed circular structure gives circRNA the inherent ability to resist degradation reactions such as deadenylation, decapping and enzyme cleavage, resulting in a significantly longer half-life of circRNA compared with linear RNA. In eukaryotic cells, circRNAs have four major biological functions, including microRNA sponges, protein scaffolds, gene transcription regulation and encoding polypeptides. More and more studies have shown that circRNAs are closely involved in the occurrence and progression of various diseases by disrupting a series of cellular and physiological processes such as cell cycle, cell differentiation and death, inflammation, and matrix fibrin expression. In addition, circRNAs show specific expression profiles in cells and tissues at different developmental stages, indicating that they have great potential and clinical significance in the diagnosis, treatment and progression of malignant diseases. Therefore, circRNAs may be candidates for cancer biomarkers with clinical relevance and utility, and sensitive and accurate monitoring of circRNAs is of great significance for disease diagnosis and treatment.

[0003] Conventional techniques for circRNA detection include Northern blot hybridization, circRNA sequencing (Circ-seq), and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Northern blot hybridization can distinguish individual circRNAs based on differences in electrophoretic mobility, but is limited by harmful radioactive labels, large sample consumption, and the inability to provide quantitative data. Circ-seq can achieve high-throughput screening of circRNAs, but it is expensive, the instrument is complex, and the operation steps are cumbersome. qRT-PCR is regarded as the gold standard for circRNA quantification, but it requires strict control of thermal temperature and cycle number. To overcome the above limitations, a series of fluorescent methods combined with isothermal nucleic acid amplification techniques have been explored, including catalytic hairpin assembly / primer exchange reaction / deoxyribozyme-mediated triple amplification, three-way ligation-assisted three-letter code strand displacement amplification cascade, and T7 nuclease-assisted cyclic enzyme amplification. Despite the improved sensitivity, these detection methods still have some inevitable disadvantages: (1) The multi-enzyme amplification system requires the coordination of multiple reaction conditions such as pH, ion concentration and intensity, which inevitably increases the complexity of the experiment; (2) The multi-step operation process increases the risk of cross-contamination and easily leads to false positive / false negative results; (3) The physicochemical information within a single cell, such as spatial location and concentration distribution, can be easily overlooked by the comprehensive average measurement. Therefore, a simple and sensitive method is urgently needed to perform sensitive imaging of circRNAs in living cells.

[0004] Synergistic catalytic DNA circuits have become versatile tools for intracellular signal amplification and information processing due to their high programmability and modularity, and have been widely used in biosensing, bioimaging, nanostructure self-assembly, and molecular computing. As a typical catalytic DNA cycle, toehold-mediated strand displacement is driven by the entropy gain of the sequential hybridization reaction between two double-stranded DNA substrates with protruding tails, and thus has significant advantages in terms of simple sequence design and fast reaction kinetics. However, it is challenging to load reactive materials directly into cells, and their stability in complex biological environments is relatively low. Recently, multiple strategies have been proposed to address these issues by using inorganic nanomaterials (e.g., MnO2 nanosheets, Fe3O4 nanoparticles, and zeolitic imidazole framework-8 nanoparticles) and DNA nanoframes (e.g., spherical nucleic acids, DNA tetrahedrons, and DNA origami). Among them, DNA tetrahedrons are rigid tetrahedral DNA cages that can be rapidly internalized via caveolin-dependent receptor-mediated endocytosis and remain intact in the cytoplasm for at least 48 h, showing great value in the delivery of various biomolecules. In addition, DNA tetrahedrons with four vertices and hexagons can be used as scaffolds to co-localize reactants, which is not only conducive to regulating molecular coupling reactions and spatial conversion relationships, but also enhances the modularity and scalability of circuit design. Here, the present invention demonstrates the construction of an adaptive catalytic hybridization circuit for DNA tetrahedrons for circRNA imaging and precision gene therapy. Summary of the invention

[0005] The purpose of the present invention is to provide a DNA tetrahedron adaptive catalytic hybridization circuit for circular RNA imaging and precision gene therapy, as well as a construction method and use thereof. The constructed DNA tetrahedron adaptive catalytic hybridization circuit can accurately quantify the expression of circCDYL in cancer cells and distinguish breast cancer patients from healthy individuals, and accurately distinguish breast cancer patients from healthy individuals.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A method for constructing a DNA tetrahedron adaptive catalytic hybridization circuit for circular RNA imaging and precision gene therapy comprises the following steps:

[0008] Step 1, denaturing the reaction solution containing probes T1-S2, probe T2, probe T3, probe T4, probe S2* and annealing buffer, and then cooling to room temperature to prepare a DNA tetrahedral framework; at the same time, mixing probe S1, probe S1* and annealing buffer, heating, and then cooling to room temperature to prepare a capture double strand (S1 / S1*);

[0009] Step 2, incubating the DNA tetrahedron framework prepared in step 1 and capturing the double strands to construct an integrated multifunctional DNA tetrahedron;

[0010] Step 3, incubate the reaction mixture containing different concentrations of target circRNA, DNA tetrahedron, annealing buffer, and BSA to perform circrRNA-initiated DNA tetrahedron entropy-driven strand displacement hybridization reaction.

[0011] Among them, BSA is a globulin in bovine serum.

[0012] In the step 1, when preparing the DNA tetrahedral framework, 20 μL of a reaction solution containing 2 μM probes T1-S2, 2 μM probe T2, 2 μM probe T3, 2 μM probe T4, 2 μM probe S2* and 1× annealing buffer was denatured at 95° C. for 5 min.

[0013] In the step 1, when preparing the capture double strand (S1 / S1*), 10 μM probe S1, 10 μM probe S1* and 1× annealing buffer were mixed, heated at 95° C. for 5 min, and then gradually cooled to room temperature.

[0014] In the step 2, the prepared DNA tetrahedron framework is incubated at 37° C. and the double strands are captured at the same stoichiometric ratio for 30 minutes to construct an integrated multifunctional DNA tetrahedron.

[0015] In step 3, 30 μL of a reaction mixture containing target circRNA at different concentrations, 350 nM DNA tetrahedron, 1× annealing buffer, and 50 μg / mL BSA was incubated at 37° C. for 2 h to perform a DNA tetrahedron entropy-driven strand displacement hybridization reaction initiated by circrRNA.

[0016] The 1× annealing buffer includes 10 mM Tris-HCl, 50 mM NaCl, and 10 mM MgCl 2 .

[0017] The sequence of the probe T1-S2 is: TTC AGA CTTAGG AAT GTG CTT CCC ACG TAG TGT CGTTTT TTT TTG GAG TAC CCT GAT GAGAAC TAG CCTACT GTC CTT TCAACC TTT CCGTATTGGACC CTC GCAT.

[0018] The sequence of the probe T2 is: ACATTC CTAAGT CTGAAACATTACAGC TTG CTACAC GAGAAGAGCCGC CATAGTATT TTC TAACTC CCAAAC CCACTA.

[0019] The probe T3 sequence is: TAT CAC CAG GCAGTT GAC AGT GTAGCAAGC TGTAAT AGATGCGAG GGT CCAATAC.

[0020] The sequence of the probe T4 is: GGT GGT GGT GGT TGT GGT GGT GGT GGT TTT TTT CAA CTGCCT GGT GATAAAACGACACTACGT GGGAAT CTACTATGG CGG CTC TTC.

[0021] The sequence of the probe S2* is: GGAAAG GUU GAAAGGAUC UCAUCA GGG UAC UCC GG.

[0022] The sequence of the probe S1 is: Cy3-GGAAAG GTT GAAAGGACAGTAGGC TAG TTC TCA TCAGGGTAC TCC TTT TTT TAG TGG GTT TGG GAG TTAGA.

[0023] The sequence of the probe S1* is: GGA GUA CCC UGA UGA GAU CCU UUC AAC CUU T(BHQ2)CCCGU UAACA.

[0024] A DNA tetrahedron adaptive catalytic hybridization circuit obtained by the construction method.

[0025] Use of the DNA tetrahedron adaptive catalytic hybridization circuit in preparing a biosensor for detecting circCDYL.

[0026] The DNA tetrahedron adaptive catalytic hybridization circuit can accurately quantify the expression of circCDYL in cancer cells and distinguish breast cancer patients from healthy individuals, with the lowest detection limit reaching 28.2aM.

[0027] Beneficial effects: The present invention constructs a DNA tetrahedron-oriented adaptive catalytic hybridization circuit for circular RNA imaging and precise gene therapy. The present invention has the following advantages: (1) The catalytic DNA circuit is composed of integrated multifunctional DNA tetrahedral probes, which have significant biocompatibility and cell delivery efficiency, meeting the prerequisites for the in vivo application of DNA nanomachines; (2) By rationally designing the four vertices and six edges of the tetrahedral structure, the integrated DNA tetrahedral probe can not only serve as a delivery carrier for the starting material, but also as a scaffold for entropy-driven catalytic hybridization reactions; (3) Local entropy-driven strand displacement hybridization provides good accuracy and sensitivity for intracellular circular RNA detection with minimal signal leakage; (4) Target stimulus-responsive biorthogonal assembly is achieved for in situ generation of three-dimensional DNA tetrahedral concatemers and on-demand release of siRNAs; (5) Compared with catalytic DNA assembly involving linear probes, the DNA tetrahedron-oriented adaptive catalytic hybridization circuit exhibits significant fluorescence stability and gene therapy efficiency. This method can sensitively detect circCDYL with a LOD of 28.2aM, accurately quantify the expression of circCDYL in cancer cells, and accurately distinguish breast cancer patients from healthy individuals. In addition, the programmable DNA tetrahedron-directed adaptive catalytic hybridization circuit can be conveniently applied to other types of RNA imaging by changing the target recognition substrate, providing a universal platform for RNA imaging and targeted gene silencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Schematic diagram of DNA tetrahedron-directed adaptive catalytic hybridization circuit for circular RNA imaging and precision gene therapy.

[0029] Figure 2 :A is a schematic diagram of the assembly of DNA tetrahedron nanowires triggered by target circular RNA; B is the assembly of the integrated multifunctional DNA tetrahedron and circular RNA-triggered DNA tetrahedron-guided adaptive catalytic hybridization circuit products analyzed by 2% AGE; C is the Cy3 fluorescence emission spectrum in the presence (red curve) and absence (blue curve) of circCDYL; D is the single-molecule detection of Cy3 spots in the presence (I) and absence (II) of circCDYL, scale bar: 10μm; E is the histogram of Cy3 counts in the presence (red column) and absence (blue column) of CircCDYL; F is the change of Cy3 fluorescence intensity with circCDYL concentration; G is the function of Cy3 counts with circCDYL concentration, different concentrations of target DNA were added to buffer, 10% FBS and 10% human serum respectively; H is Cy3 counts; 350nM integrated DNA tetrahedron probe was used in the experiment.

[0030] Figure 3:A is a schematic diagram of the proposed catalytic circuit for clinical application; B is the Cy3 counts produced by different cell lines; C is the C produced by different cell lines T Value; D is the linear correlation between MCF-7 cell number and Cy3 count in logarithmic coordinates; E and F are heat maps of circCDYL expression levels in breast tissues determined by the proposed catalytic hybridization circuit (E) and qRT-PCR (F), respectively; G and H are box plots and significance analysis of circCDYL expression levels in breast tissues determined by the proposed catalytic hybridization circuit (G) and qRT-PCR (H), respectively; I is the ROC curve of circCDYL in breast tissues detected by catalytic hybridization circuit (red curve) and qRT-PCR (blue curve); *** P < 0.001; 350 nM of the integrated DNA tetrahedron probe was used in the experiment.

[0031] Figure 4 : Fluorescence imaging of circCDYL was performed in MCF-7 cells, HeLa cells, HepG2 cells, A549 cells and MCF-10A cells, and the corresponding mean fluorescence intensity per cell (MFI) was quantitatively counted; Scale bar: 50 μm.

[0032] Figure 5 :A is the Calcein-AM (green) and PI (red) co-staining image of MCF-7 cells treated with AS1411 nucleic acid aptamer, linear assembly probe, multifunctional DNA tetrahedron and AS1411 nucleic acid aptamer / siRNA; B is the Calcein-AM and PI co-staining image of MCF-10A cells treated with multifunctional DNA tetrahedron and AS1411 aptamer / siRNA, respectively; Scale bar: 150μm; C and D are MCF-7 cells treated with AS1411 nucleic acid aptamer (a), linear assembly probe (b), multifunctional DNA tetrahedron (c) and AS1411 nucleic acid aptamer / siRNA (d), respectively, and qRT-PCR was used to detect the expression of VEGF mRNA (C), Western Blot analysis of VEGF protein levels (D); E: MCF-7 cells were treated with AS1411 nucleic acid aptamer (a), linear assembly probe (b), multifunctional DNA tetrahedron (c) and AS1411 nucleic acid aptamer / siRNA (d), and the relative cell survival rate was detected; F: Flow cytometry was used to detect the effects of AS1411 nucleic acid aptamer (a), linear assembly probe (b), multifunctional DNA tetrahedron (c) and AS1411 nucleic acid aptamer / siRNA (d) on MCF-7 cell apoptosis. DETAILED DESCRIPTION

[0033] The present invention will be further explained below in conjunction with the accompanying drawings and embodiments.

[0034] Reagents and Materials:

[0035] All oligonucleotides were synthesized and HPLC-purified by Sangon Biotech. Co., Ltd. (Shanghai, China). Ribonuclease R (RNase R) and 10× RNase R reaction buffer (0.2 M Tris-HCl, 1 M KCl, 1 mM MgCl, pH 8.0) were purchased from Beyotime (Jiangsu, China). TM 3000 transfection reagent and Ireduced-serum medium was purchased from Thermo Fisher Scientific (Massachusetts, USA). Calcein-AM / PI Cell Viability / Cytotoxicity Assay Kit and Annexin V-FITC apoptosis kit were purchased from Beyotime (Jiangsu, China). Anti-VEGFA antibody, Anti-GAPDH antibody, and GoatAnti-Rabbit IgG HL were all from Abcam (Cambridge, UK). Human breast cell line (MCF-7 cells), hepatocyte cell line (HepG2 cells), human cervical cancer cell line (HeLa cells), human lung adenocarcinoma cell line (A549 cells), and human normal breast epithelial cell line (MCF-10A cells) were from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human tissues were obtained from the Affiliated Hospital of Weifang Medical University (Weifang, Shandong, China). This study was approved by the Ethics Committee of the Affiliated Hospital of Weifang Medical University. PrimeScript TM RT reagent Kit with gDNA Eraser and TB Green Premix Ex Taq II (Tli RNaseH Plus) were purchased from Accurate Biotechnology Co., Ltd. (Hunan, China).

[0036] Fluorescence spectroscopy detection and measurement of clinical samples:

[0037] Fluorescence emission spectra were recorded using a FLS1000 steady-state transient fluorescence spectrometer (Edinburgh Instruments Ltd., Livingston, UK) with both emission and excitation slits at 3.5 nm. The excitation wavelength was 535 nm, and the Cy3 fluorescence intensity at 564 nm was used for data analysis. Total RNA was extracted from human tissue samples using the miRNeasy FFPE kit according to the manufacturer's procedure. The resulting extract was transferred to a new tube and immediately used to measure circCDYL activity.

[0038] Embodiment 1:

[0039] Step 1: Denature a 20 μL reaction volume containing 2 μM T1-S2, 2 μM T2, 2 μM T3, 2 μM T4, 2 μM S2* and 1× annealing buffer at 95°C for 5 min, then slowly cool to room temperature to prepare a DNA tetrahedral framework. At the same time, mix 10 μM S1, 10 μM S1* and 1× annealing buffer, heat at 95°C for 5 min, and then gradually cool to room temperature to prepare a capture duplex (S1 / S1*);

[0040] Step 2, incubating the prepared DNA tetrahedron framework at 37°C and capturing the double-stranded DNA at the same stoichiometric ratio for 30 min to construct an integrated multifunctional DNA tetrahedron;

[0041] Step 3: Incubate 30 μL of the reaction mixture containing different concentrations of target circRNA, 350 nM DNA tetrahedron, 1× annealing buffer, and 50 μg / mL BSA at 37 °C for 2 h to perform circrRNA-initiated DNA tetrahedron entropy-driven strand displacement hybridization reaction.

[0042] Among them, BSA is a globulin in bovine serum.

[0043] Wherein, the 1× annealing buffer includes 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, and the pH value is 7.5.

[0044] The sequence of probe T1-S2 is: TTC AGA CTTAGGAAT GTG CTT CCC ACG TAG TGT CGT TTT TTTTTG GAG TAC CCT GAT GAG AAC TAG CCTACT GTC CTT TCAACC TTT CCG TAT TGGACC CTCGCAT.

[0045] The sequence of probe T2 is: ACATTC CTAAGT CTGAAACAT TACAGC TTG CTACAC GAG AAGAGC CGCCATAGTATT TTC TAACTC CCAAAC CCACTA.

[0046] The sequence of probe T3 is: TAT CAC CAG GCAGTT GACAGT GTAGCAAGC TGTAATAGA TGC GAGGGT CCAATAC.

[0047] The sequence of probe T4 is: GGT GGT GGT GGT TGT GGT GGT GGT GGT TTT TTT CAACTG CCTGGT GATAAAACGACACTACGT GGGAAT CTACTATGG CGG CTC TTC.

[0048] The sequence of probe S1 is: Cy3-GGAAAG GTT GAAAGGACAGTAGGC TAG TTC TCATCA GGG TACTCC TTT TTT TAG TGG GTT TGG GAG TTAGA.

[0049] The sequence of probe S1* is: GGA GUA CCC UGA UGA GAU CCU UUC AAC CUU T(BHQ2)CC CGUUAACA.

[0050] The sequence of probe S2* is: GGAAAG GUU GAAAGGAUC UCAUCAGGG UAC UCC GG.

[0051] Example 2: Probe design and overall process Figure 1 shown.

[0052] As a proof of concept, circCDYL was selected as a model target. In this strategy, the present inventors cleverly designed a multifunctional DNA tetrahedron that can serve as both a delivery vehicle for reactants and a scaffold for an entropy-driven catalytic circuit. The designed DNA tetrahedron has an AS1411 aptamer (i.e., a tumor-targeting aptamer that binds to nuclear proteins overexpressed on cancer cell membranes) at one vertex, a capture duplex (S1 / S1*) at the opposite vertex, and a fuel duplex (T1-S2 / S2*) at the adjacent edge. S1 / S1* and T1-S2 / S2* are DNA / RNA hybrids with a protruding toehold site at the tail and a protruding toehold site at the stem. S1 and S1* were modified with a Cy3 fluorophore and a BHQ2 quencher, respectively. The successful assembly of the DNA tetrahedron brings Cy3 and BHQ2 into close proximity, resulting in efficient fluorescence quenching. When circCDYL is present, it binds to the toehold that captures the double-stranded protrusion, initiating the strand migration of S1*, resulting in the release of the initially locked S1 to form a circCDYL / S1* hybrid. At the same time, the dissociation of S1 / S1* leads to an increase in the spatial distance between Cy3 and BHQ2, thereby restoring the fluorescence of Cy3. Subsequently, the released S1 can interact with T1-S2 through the protrusion toe-mediated strand migration to form a S1 / T1-S2 double-stranded structure accompanied by the release of S2*. The hybridization between S1 and T1-S2 leads to the self-stacking of DNA tetrahedrons. The released S2* then interacts with the circCDYL / S1* hybrid to generate a S1* / S2* RNA duplex and release the target circCDYL. Notably, the generated S1* / S2* can essentially function as a small interfering RNA (siRNA) to induce the degradation of vascular endothelial growth factor (VEGF)-related messenger RNA (mRNA) in tumor cells, thereby achieving on-site targeted stimulation and on-demand gene therapy. Furthermore, the released target circCDYL can be recycled to trigger a new round of strand displacement hybridization reaction, resulting in long tandem DNA tetrahedral nanowires and accumulated siRNAs. In contrast, when the target circCDYL is absent, the entropy-driven strand displacement reaction does not occur, leading to neither the assembly of DNA tetrahedral nanowires nor the generation of siRNAs.

[0053] Example 3: Feasibility of verification experiment

[0054] 2% agarose gel electrophoresis (AGE) analysis was used to verify the step-by-step assembly of the integrated DNA tetrahedron and its responsiveness to the target circCDYL. Figure 2 Middle B, lane 1), T2 ( Figure 2 Middle B, lane 2), T3 ( Figure 2 Middle B, lane 3), T4 ( Figure 2Middle B, lane 4), S2*( Figure 2 Middle B, lane 5), S1 / S1*( Figure 2 The electrophoretic mobility of the bands gradually decreased with the addition of the target circCDYL (Fig. 5B, lane 6), indicating that the integrated multifunctional DNA tetrahedron was successfully assembled. An obvious complete DNA tetrahedron band was observed in lane 6. When the target circCDYL was added, a large molecular weight Lander-like band was observed, which was generated by the generated siRNA ( Figure 2 In contrast, when the target circCDYL was missing, only the original band of the DNA tetrahedron ( Figure 2 Middle B, lane 8), indicating that there is neither target-triggered assembly of DNA tetrahedral nanowires nor production of siRNA.

[0055] Fluorescence emission spectroscopy measurements were further performed to verify the feasibility of the circRNA-triggered DNA tetrahedron-guided adaptive catalytic hybridization circuit. In the presence of circCDYL, a significantly enhanced Cy3 signal was observed ( Figure 2 C, red curve), indicating that target-triggered DNA tetrahedral nanowire assembly occurred through toehold-mediated strand displacement hybridization. In contrast, when circCDYL was missing ( Figure 2 In C, blue curve), a weak Cy3 fluorescence signal was observed. In addition, single-molecule imaging was performed. Compared with fluorescence measurement, single-molecule imaging has significant advantages such as less sample usage, high sensitivity, and less background interference. In the absence of circCDYL, no ( Figure 2 The Cy3 spots in (D, II) indicate that neither branch-mediated strand displacement hybridization nor assembly of DNA tetrahedral nanowires occurs. When circCDYL is present, a clear Cy3 signal can be obtained ( Figure 2 Notably, the Cy3 signal induced by circCDYL was 25.6-fold higher than that generated by the control group without circCDYL ( Figure 2 These results demonstrate the feasibility of the proposed adaptive catalytic circuit for DNA tetrahedron for circRNA detection.

[0056] Example 4: Sensitivity Detection

[0057] Under the optimal experimental conditions, the detection sensitivity of the proposed catalytic DNA circuit was investigated by measuring the response changes of circCDYL to Cy3 signals at different concentrations. -15 to 1×10 -9 M( Figure 2In the concentration range of F (inset), the logarithm of Cy3 fluorescence intensity (F) and circCDYL concentration (C) showed a good linear relationship, and the regression equation was F = 273506.07 + 16905.82lg C (R 2 =0.9997), and the detection limit (LOD) was 4.17×10 -16 In addition, the present invention performed single-molecule imaging to monitor the effect of different concentrations of circCDYL on Cy3 counts. When the concentration of circCDYL increased from 1×10 -16 M increases to 1×10 -7 M, the Cy3 count (N) and the logarithmic concentration (C) of circCDYL showed a good linear correlation ( Figure 2 The regression equation is N = 686.68 + 39.65lg C (R 2 =0.9997). The calculated LOD is 2.82×10- 17 M, which is 14.79 times higher than the LOD obtained from fluorescence measurement.

[0058] Example 5: Anti-interference capability analysis

[0059] To evaluate the anti-interference ability of this method in different complex matrices, different concentrations of circCDYL were added to 10% FBS and 10% human serum, respectively. -7 M( Figure 2 Middle H, green column), 1×10 -9 M( Figure 2 Medium H, red column) or 1×10 -12 M( Figure 2 When the Cy3 signal obtained from the mixture of FBS and human serum was analyzed (Figure 5H, blue column), it was basically consistent with the signal generated by the target alone, which confirmed that the proposed method has good anti-interference ability.

[0060] Example 6: Actual sample analysis

[0061] In order to demonstrate the applicability of this method in the analysis of actual biological samples, the expression of circCDYL in MCF-7 cells, HepG2 cells, HeLa cells, A549 cells and MCF-10A cells was detected. Figure 3 In B, the weak Cy3 signal obtained from Amaranth column) is compared to that from MCF-7 cells ( Figure 3 Middle B, purple column), HepG2 cells ( Figure 3 Middle B, blue column), HeLa cells ( Figure 3 B, orange columns) and A549 cells ( Figure 3The Cy3 signal obtained in (B, green column) was significantly enhanced, indicating that circCDYL is upregulated in tumor cells. This result was further verified by qRT-PCR ( Figure 3 In addition, the expression level of circCDYL in different numbers of MCF-7 cells was determined. Cy3 counts (N) increased with the number of MCF-7 cells (X) from 1 to 1,000,000, and there was a good linear correlation between N and the logarithm of X from 1 to 1,000,000. The regulatory equation was N = 62.16 + 39.06 lg X (R 2 =0.9987), LOD is determined to be 1 cell ( Figure 3 (middle D).

[0062] Example 7: Clinical sample analysis

[0063] The potential application of the proposed method in clinical diagnosis was further evaluated by monitoring the expression of circCDYL in 8 pairs of breast cancer tissues and healthy adjacent tissues. Figure 3 As shown in Figure E, the Cy3 signal generated by breast cancer tissue is much higher than that of healthy adjacent cancer tissue. The average Cy3 signal in breast cancer tissue was measured to be 180.23±21.40, which is higher than that of healthy adjacent cancer tissue (78.01±9.23) (t test, P<0.001; Figure 3 The expression of circCDYL in breast cancer tissue was upregulated by 2.31 times. T The value is much higher than that in healthy adjacent cancer tissue ( Figure 3 C obtained in F, H) T This is consistent with the results measured by the proposed adaptive catalytic hybridization circuit for DNA tetrahedrons. In addition, the accuracy of the proposed method in distinguishing breast cancer patients from healthy subjects was evaluated by receiver operating curve (ROC) analysis. The area under the curve (AUC) of the proposed method was calculated to be 1, which is consistent with the AUC value measured by qRT-PCR ( Figure 3 These results indicate that the proposed method can distinguish breast cancer tissue from healthy adjacent tissue with high accuracy.

[0064] Example 8: Cell imaging analysis

[0065] MCF-7 cells were used to investigate the feasibility of the proposed catalytic hybridization circuit for intracellular circular RNA imaging. The designed multifunctional DNA tetrahedrons could be transported into living cells via nucleolar / AS1411 aptamer-mediated microendocytosis. After incubation with the multifunctionalized DNA tetrahedrons, a time-dependent fluorescence enhancement was observed in MCF-7 cells, and the fluorescence intensity of Cy3 was studied as a plateau at 4 h. Next, the proposed method was used to measure the dynamic changes of circCDYL concentration in different cell types. Figure 4 As shown, MCF-7 cells, HepG2 cells, and HeLa cells produced significantly high Cy3 fluorescence signals, while MCF-10A cells produced extremely low fluorescence, indicating that this method can accurately measure the expression of circRNAs in living cells.

[0066] Example 9: Gene silencing analysis

[0067] The gene silencing effect and therapeutic efficacy of this method were further studied at the cellular level. A linear assembly probe was designed for comparison. MCF-7 cells and MCF-10A cells were incubated with AS1411 aptamer (negative control), linear assembly probe, multifunctional DNA tetrahedron and AS1411 aptamer / siRNA (positive control), respectively, and co-stained with Calcein-AM and propidium iodide (PI). Figure 5 As shown in Figure A, different PI signals were obtained in MCF-7 cells treated with integrated DNA tetrahedrons, indicating that gene therapy targeting circCDYL stimulation occurred. In addition, the PI signal of MCF-7 cells treated with integrated DNA tetrahedrons was significantly higher than that of MCF-7 cells treated with linear assembly probes. The improved gene therapy efficiency of integrated DNA tetrahedrons may be due to the inherent resistance to enzyme cleavage of its tetrahedral-like spatial structure. In addition, the PI signal of MCF-7 cells treated with integrated DNA tetrahedrons was comparable to that of MCF-7 cells treated with AS1411 aptamer / siRNA, indicating that the presence of target circCDYL can effectively initiate the entropy-driven assembly of DNA tetrahedron nanowires and the release of siRNA. In contrast, a clear calcium-AM signal was observed in MCF-10A cells treated with DNA tetrahedrons, and the PI signal was negligible, indicating that the DNA tetrahedron probe has good biocompatibility and low toxicity to normal cells ( Figure 5 In B), the present invention further uses qRT-PCR to detect VEGF mRNA levels. Figure 5As shown in Figure C, VEGF mRNA expression was significantly downregulated in MCF-7 cells treated with DNA tetrahedrons (55.51%), which was not significantly different from that in MCF-7 cells treated with AS1411 aptamer / siRNA (42.15%), indicating that the designed multifunctional DNA tetrahedrons exhibited good stability in cells and promoted the target-stimulated release of siRNAs. In contrast, the linearly assembled probes showed a weakened inhibitory effect on the expression of VEGF mRNA compared with the integrated DNA tetrahedrons, because linear DNA is more easily degraded by exonucleases in cells. Protein immunoblot analysis showed that the target-stimulated DNA tetrahedron-directed adaptive catalytic hybridization circuit greatly inhibited the expression of VEGF protein ( Figure 5 In addition, the present invention evaluates the therapeutic effect of genes by evaluating cell viability. Figure 5 As shown in Figure E, incubation of MCF-7 cells with linear assembly probes, multifunctional DNA tetrahedrons, and AS1411 aptamer / siRNA induced 23.20%, 41.39%, and 52.85% of cell apoptosis, respectively, while AS1411 aptamer did not induce obvious apoptotic signals. These results were further verified by flow cytometry using the Annexin V-FITC / PI apoptosis kit ( Figure 5 Middle F).

[0068] The present invention discloses a DNA tetrahedron adaptive catalytic hybridization circuit for circular RNA imaging and precision gene therapy, and a construction method and use thereof, which have the following advantages: (1) The catalytic DNA circuit is composed of an integrated multifunctional DNA tetrahedron probe, which has significant biocompatibility and cell delivery efficiency, and meets the prerequisites for the in vivo application of DNA nanomachines; (2) By rationally designing the four vertices and six edges of the tetrahedron structure, the integrated DNA tetrahedron probe can not only serve as a delivery carrier for the starting material, but also as a scaffold for the entropy-driven catalytic hybridization reaction; (3) Local entropy-driven chain displacement hybridization provides good accuracy and sensitivity for intracellular circular RNA detection with minimal signal leakage; (4) Target stimulus-responsive dual orthogonal assembly is achieved for in situ generation of three-dimensional DNA tetrahedron concatemers and on-demand release of siRNAs; (5) Compared with the catalytic DNA assembly involving linear probes, the DNA tetrahedron directional adaptive catalytic hybridization circuit exhibits significant fluorescence stability and gene therapy efficiency. This method can sensitively detect circCDYL with a LOD of 28.2aM, accurately quantify the expression of circCDYL in cancer cells, and accurately distinguish breast cancer patients from healthy individuals. In addition, the programmable DNA tetrahedron-directed adaptive catalytic hybridization circuit can be conveniently applied to other types of RNA imaging by changing the target recognition substrate, providing a universal platform for RNA imaging and targeted gene silencing.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a DNA tetrahedron adaptive catalytic hybridization circuit for circular RNA imaging and precision gene therapy, characterized in that: The following steps are involved: Step 1, denaturing the reaction solution containing probes T1-S2, probe T2, probe T3, probe T4, probe S2* and annealing buffer, and then cooling to room temperature to prepare a DNA tetrahedral framework; at the same time, mixing probe S1, probe S1* and annealing buffer, heating, and then cooling to room temperature to prepare a capture double strand (S1 / S1*); Step 2, incubating the DNA tetrahedron framework prepared in step 1 and capturing the double strands to construct an integrated multifunctional DNA tetrahedron; Step 3, incubate the reaction mixture containing different concentrations of target circRNA, DNA tetrahedron, annealing buffer, and BSA to perform circrRNA-initiated DNA tetrahedron entropy-driven strand displacement hybridization reaction.

2. The method for constructing a DNA tetrahedron adaptive catalytic hybridization circuit for circular RNA imaging and precision gene therapy according to claim 1, characterized in that: In the step 1, when preparing the DNA tetrahedral framework, 20 μL of a reaction solution containing 2 μM probes T1-S2, 2 μM probe T2, 2 μM probe T3, 2 μM probe T4, 2 μM probe S2* and 1× annealing buffer was denatured at 95° C. for 5 min.

3. The method for constructing a DNA tetrahedron adaptive catalytic hybridization circuit for circular RNA imaging and precision gene therapy according to claim 1, characterized in that: In the step 1, when preparing the capture double strand (S1 / S1*), 10 μM probe S1, 10 μM probe S1* and 1× annealing buffer were mixed, heated at 95° C. for 5 min, and then gradually cooled to room temperature.

4. The method for constructing a DNA tetrahedron adaptive catalytic hybridization circuit for circular RNA imaging and precision gene therapy according to claim 1, characterized in that: In the step 2, the prepared DNA tetrahedron framework is incubated at 37° C. and the double strands are captured at the same stoichiometric ratio for 30 minutes to construct an integrated multifunctional DNA tetrahedron.

5. The method for constructing a DNA tetrahedron adaptive catalytic hybridization circuit for circular RNA imaging and precision gene therapy according to claim 1, characterized in that: In step 3, 30 μL of a reaction mixture containing target circRNA at different concentrations, 350 nM DNA tetrahedron, 1× annealing buffer, and 50 μg / mL BSA was incubated at 37° C. for 2 h to perform a DNA tetrahedron entropy-driven strand displacement hybridization reaction initiated by circrRNA.

6. A method for constructing a DNA tetrahedron adaptive catalytic hybridization circuit for circular RNA imaging and precision gene therapy according to any one of claims 2, 3 or 5, characterized in that: The 1× annealing buffer includes 10 mM Tris-HCl, 50 mM NaCl, and 10 mM MgCl 2 .

7. The method for constructing a DNA tetrahedron adaptive catalytic hybridization circuit for circular RNA imaging and precision gene therapy according to claim 1, characterized in that: The sequence of the probe T1-S2 is: TTCAGACTT AGGAAT GTG CTTCCC ACG TAG TGT CGT TTT TTT TTG GAG TAC CCT GAT GAG AAC TAG CCTACT GTC CTTTCAACC TTT CCG TATTGGACC CTC GCAT; The probe T2 sequence is: ACATTC CTAAGT CTGAAACATTACAGC TTG CTACAC GAGAAGAGC CGCCATAGTATT TTC TAACTC CCAAAC CCACTA; The probe T3 sequence is: TAT CAC CAG GCAGTT GAC AGT GTAGCAAGC TGTAAT AGATGC GAGGGT CCAATAC; The probe T4 sequence is: GGT GGT GGT GGT TGT GGT GGT GGT GGT TTT TTT CAA CTG CCTGGT GATAAAACGACACTACGT GGGAAT CTACTATGG CGG CTC TTC; The sequence of the probe S2* is: GGAAAG GUU GAAAGGAUC UCAUCA GGG UAC UCC GG; The sequence of the probe S1 is: Cy3-GGAAAG GTT GAAAGGACAGTAGGC TAG TTC TCA TCAGGG TACTCC TTT TTT TAG TGG GTT TGG GAG TTAGA; The sequence of the probe S1* is: GGA GUA CCC UGA UGA GAU CCU UUC AAC CUU T(BHQ2)CC CGUUAACA.

8. A DNA tetrahedron adaptive catalytic hybridization circuit obtained by the construction method of claim 1.

9. Use of the DNA tetrahedron adaptive catalytic hybridization circuit according to claim 8 in preparing a biosensor for detecting circCDYL.

10. The use according to claim 9, characterized in that: The DNA tetrahedron adaptive catalytic hybridization circuit can accurately quantify the expression of circCDYL in cancer cells and distinguish breast cancer patients from healthy individuals, with the minimum detection limit reaching 28.2aM.

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