Palindromic mediated self-assembly DNA (deoxyribonucleic acid) molecule as well as preparation method and application thereof

Abstract

The invention discloses a palindromic mediated self-assembly DNA molecule as well as a preparation method and application thereof, and belongs to the technical field of biology. The palindromic mediated self-assembled DNA molecule is obtained by self-assembling an A1 chain, an L1 chain and a C1 chain; the nucleotide sequence of the chain A1 is as shown in SEQ ID NO. 1; the nucleotide sequence of the L1 chain is as shown in SEQ ID NO. 2; and the nucleotide sequence of the C1 chain is as shown in SEQ ID NO. 3. The palindromic mediated self-assembled DNA molecule A1-L1-C1 disclosed by the invention can be used for detecting miR-21, and has relatively high detection sensitivity and specificity. The A1-L1-C1 also has a drug loading function, and can target specific tumor cells and accurately deliver drugs into cancer cells. The invention provides a new technical approach for early diagnosis of tumors, detection of intracellular biomolecules and targeted drug delivery, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a palindrome-mediated self-assembly DNA molecule, its preparation method, and its applications. Background Technology

[0002] According to global cancer data released by the World Health Organization (WHO) in 2020, the number of malignant tumor cases and deaths is increasing year by year, becoming one of the leading causes of death for citizens in countries around the world. Therefore, it is urgent to control the occurrence and development of cancer to effectively reduce its incidence and mortality. To achieve the best treatment results, early detection of tumors is essential. Currently, research related to early tumor detection has become a hot topic in the medical field.

[0003] Nucleic acids, as excellent structural materials, have been widely used to construct various types of nucleic acid nanomaterial molecules. Their superior biocompatibility, biodegradability, safety, convenient automated synthesis, and good programmability make nucleic acid nanomaterials highly suitable for the biomedical field. DNA, through AT, GC-Watson-Crick base pairing, possesses programmability and addressability, thus allowing for the design and fabrication of various functional nucleic acid nanostructures by utilizing the rigidity of the DNA double strand and the flexibility of the single strand. Furthermore, DNA is an endogenous molecule in living organisms, and functional nucleic acid nanomaterials constructed from it exhibit good compatibility in both in vivo and in vitro environments and biological systems. After nearly 40 years of development in nucleic acid nanotechnology, scientists can assemble many complex and intricate two-dimensional, three-dimensional, and even curved self-assembled structures. Self-assembled nucleic acid nanostructures are easily modified; currently, many nucleic acid nanostructures can be modified with specific nucleic acid aptamers to perform specific functions.

[0004] MicroRNAs (miRNAs), as a type of exosome, are utilized by tumor cells, promoting their growth and metastasis, ultimately leading to high mortality rates in cancer patients. Therefore, using miRNAs as indicators for early cancer diagnosis and detecting changes in miRNA levels during tumor cell development has broad clinical application prospects. Studies have shown that microRNA-21 (miRNA-21 / miR-21) is abnormally expressed in almost all tumors, such as being upregulated and promoting metastasis in various cancers including lung adenocarcinoma, B-cell lymphoma, breast cancer, myeloid leukemia, lung cancer, and cholangiocarcinoma. Current literature indicates that miR-21, as an oncogenic miRNA, participates in cell growth, metastasis, and apoptosis by controlling signaling pathways and multiple target molecules, and can negatively regulate target genes by promoting the degradation of target gene mRNA or inhibiting mRNA translation. Notably, increasing evidence further confirms the close correlation between miR-21 and cancer patient diagnosis, recurrence, and prognosis prediction, suggesting that miR-21 may be a novel biomarker for tumor diagnosis and prognosis prediction.

[0005] However, current methods for detecting miR-21 generally suffer from low sensitivity and poor specificity, which limits the clinical application of miR-21 as a biomarker. Summary of the Invention

[0006] The purpose of this invention is to provide a palindrome-mediated self-assembly DNA molecule, its preparation method, and its applications, to address the problems existing in the prior art. This invention constructs a palindrome-mediated self-assembly DNA molecule, A1-L1-C1, which can be used for the detection of miR-21 with high detection sensitivity and specificity. A1-L1-C1 also possesses drug-carrying capabilities and can target specific tumor cells, precisely delivering drugs into cancer cells. This invention provides a new technical approach for early tumor diagnosis, intracellular biomolecule detection, and targeted drug delivery, and has broad application prospects.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a palindrome-mediated self-assembly DNA molecule, wherein the palindrome-mediated self-assembly DNA molecule is obtained by self-assembly of an A1 strand, an L1 strand, and a C1 strand;

[0009] The nucleotide sequence of the A1 chain is shown in SEQ ID NO.1, and its 5' end is modified with a fluorescent group;

[0010] The nucleotide sequence of the L1 chain is shown in SEQ ID NO.2, and its 3' end is modified with a quenching group;

[0011] The nucleotide sequence of the C1 chain is shown in SEQ ID NO.3; a fluorescent group is modified between the 10th and 11th bases.

[0012] The present invention also provides a method for preparing palindrome-mediated self-assembled DNA molecules as described above, wherein the A1 chain, the L1 chain and the C1 chain are mixed in a molar ratio of 1:1:0.7 and reacted at 90-98°C for 4-6 min to obtain the palindrome-mediated self-assembled DNA molecules.

[0013] The present invention also provides the application of palindromic-mediated self-assembled DNA molecules as described above in the preparation of products for detecting miR-21, said products including reagents, kits, or chips.

[0014] The present invention also provides a product for detecting miR-21, the product comprising the above-mentioned palindrome-mediated self-assembled DNA molecule.

[0015] This invention also provides the application of palindromic-mediated self-assembly of DNA molecules as described above in the preparation of tumor-targeted drug delivery systems.

[0016] The present invention also provides a method for preparing a tumor-targeted drug delivery system, wherein the above-mentioned palindromic self-assembled DNA molecules are mixed with drugs and incubated to obtain the tumor-targeted drug delivery system.

[0017] Optionally, the incubation temperature is 20-25℃ and the time is 10-14h.

[0018] The present invention also provides a tumor-targeting drug delivery system prepared by the above preparation method.

[0019] The present invention also provides the application of the tumor-targeting drug delivery system described above in the preparation of drugs for treating tumors.

[0020] The present invention also provides the application of palindromic-mediated self-assembly of DNA molecules as described above in the preparation of products for tumor imaging.

[0021] The present invention discloses the following technical effects:

[0022] This invention constructs a palindrome-mediated self-assembly DNA molecule, A1-L1-C1, which can be used for the detection of miR-21 with high sensitivity and specificity, and a detection limit of up to 200 pM. By linking to the AS1411 aptamer, A1-L1-C1 achieves active targeting of specific tumor cells. Using A1-L1-C1 to load drugs allows for precise delivery of drugs into cancer cells without affecting normal cells. The A1-L1-C1 of this invention can not only be used for imaging detection of intracellular tumor markers but also as a biosensor to detect miR-21 levels, serving as a drug delivery platform for targeted cancer therapy. This invention provides a new technical approach for early tumor diagnosis, intracellular biomolecule detection, and targeted drug delivery, with broad application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 A schematic diagram illustrating the mechanism by which the A1-L1-C1 system targets tumor cells and releases fluorescent signals;

[0025] Figure 2 Here are agarose gel electrophoresis images; where A is the assembly screening diagram of the backbone chain A1-L1, lane a: A2 + L2; lane b: A2 + L2-1; lane c: A3 + L3; lane d: A3 + L3-1; lane e: A1 + L1; lane f: A1 + L1-1; B is the assembly diagram of A1-L1-C1, lane a: A1-L1-C1;

[0026] Figure 3 The results of the feasibility analysis of the A1-L1-C1 detection system are shown below. A is the feasibility electrophoresis diagram under 12% natural polyacrylamide gel, lane a: A1; lane b: L1; lane c: C1; lane d: miR-21; lane e: C1 + miR-21; lane f: A1 + L1; lane g: A1-L1-C1; lane h: A1-L1-C1 + miR-21. B is the fluorescence spectrum of the feasibility study, with samples a~h representing the same samples as in A. C is the feasibility electrophoresis diagram under 3% agarose gel, with all lanes representing the same samples as in A.

[0027] Figure 4The image shows the optimized A1-L1 ratio results; A is a 12% natural polyacrylamide gel electrophoresis image; B is a 3% agarose gel electrophoresis image; the letters in A and B have the same meaning, lane a:A1:L1=2:1; lane b:A1:L1=1.5:1; lane c:A1:L1=1:1; lane d:A1:L1=1:1.5; lane e:A1:L1=1:2;

[0028] Figure 5 The results show the optimized ratio of (A1-L1) to C1; where A is the signal-to-noise ratio obtained by assembling C1-FAM and A1-L1 at different concentrations; and B is the fluorescence spectrum of assembling C1-FAM and A1-L1 at different concentrations.

[0029] Figure 6 Results of miR-21 reaction time optimization;

[0030] Figure 7 The results show the stability analysis of A1-L1-C1 in 10% FBS; where A is the gel electrophoresis image of A1-L1-C1 after incubation in 10% FBS; B is the relative quantitative change curve of band fluorescence intensity over time; C is the degradation rate curve at different times after incubation in 10% FBS by detecting fluorescence signal; and D is the fluorescence signal intensity detected at different times.

[0031] Figure 8 The results show the sensitivity analysis of the A1-L1-C1 detection system; where A represents the fluorescence spectra generated by the A1-L1-C1 detection system at different concentrations of miR-21; B represents the linear relationship between high concentration (nM) of miR-21 and fluorescence intensity; and C represents the linear relationship between low concentration (pM) of miR-21 and fluorescence intensity.

[0032] Figure 9 The results represent the specificity analysis of the A1-L1-C1 detection system.

[0033] Figure 10 The results show the cell targeting analysis of A1-L1-C1; where A represents the flow cytometry analysis of A1-L1-C1 after incubation with 293T cells and HELF cells; and B represents the flow cytometry analysis of A1-L1-C1 after incubation with A549 cells and Huh7 cells.

[0034] Figure 11 The image shows the fluorescence spectrum of drug-loaded A1-L1-C1. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Example 1

[0041] 1. Experimental Materials and Methods

[0042] 1.1 Experimental Materials

[0043] 1.1.1 Nucleic acid sequence

[0044] All nucleic acid sequences used in this experiment were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and purified using high-performance liquid chromatography (HPLC). Sequence information is shown in Table 1. Base sequences capable of complementary pairing to form double-stranded DNA are indicated by the same label. The underlined AS1411 is a deoxyribonucleic acid aptamer sequence that specifically recognizes nucleolar proteins on tumor cell membranes. The bolded sequence is a palindromic sequence, which promotes assembly into macromolecules. Mutant sequences are represented by uppercase M, with the mutation site indicated by the lowercase letter corresponding to the mutated base. M1 to M3 indicate that the number of bases at the mutation site has increased from 1 to 3. Because DNA strands are easily synthesized, not easily degraded, convenient for experimental operation, and do not affect experimental results, all miRNA sequences used in the experiment were synthesized using deoxyribonucleotides.

[0045] Table 1 Nucleic Acid Sequences

[0046]

[0047] 1.1.2 Cell lines

[0048] Normal human renal epithelial cells (293T cells), normal human embryonic lung fibroblasts (HELF cells), human liver cancer cells (Huh7 cells), and human alveolar basal epithelial lung cancer cells (A549 cells) were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0049] 1.1.3 Reagents

[0050] All chemical reagents used in this experiment were of analytical grade; all solutions prepared by ourselves used ultrapure water (ddH2O) as the solvent, and all were treated by a Milli-Q ultrapure water system. The main reagents used and their manufacturers are shown in Table 2.

[0051] Table 2 Reagents and Manufacturers

[0052]

[0053] 1.1.4 Instruments

[0054] The instruments used in this experiment are mainly used for nucleic acid nanomaterial characterization, fluorescence detection, gel electrophoresis, nucleic acid sample preparation, cell targeting and other experiments, as detailed in Table 3.

[0055] Table 3 Instruments and Manufacturers

[0056]

[0057] 1.2 Experimental Methods

[0058] 1.2.1 Nucleic acid sequence lysis

[0059] Store the prepared nucleic acid at -20°C. Before use, centrifuge at 13000 rpm for 1 min, remove the tube smoothly, and slowly open the cap. Add TE buffer slowly along the tube wall to achieve a final concentration of 10 μM. Vortex the solution to ensure complete dissolution of the nucleic acid powder. Centrifuge at 8000 rpm for 30 seconds twice. Finally, use a low-speed centrifuge to remove any remaining liquid from the tube wall. Use immediately or store at 4°C.

[0060] 1.2.2 Preparation of solutions used in the experiment

[0061] (1) Preparation of 5×TBE electrophoresis buffer: Tris 54.00 g, Na2EDTA·2H2O 3.72 g, boric acid 27.50 g, and ultrapure water to a final volume of 1 L.

[0062] Use NaOH and HCl solutions, adjust the pH of the solution to 7.4 using a PHS-3C precision pH meter, then pour it into a clean glass bottle for storage. Depending on the needs of subsequent experiments, it can be diluted with ultrapure water to prepare a 0.5×TBE electrophoresis buffer for natural polyacrylamide gel electrophoresis experiments.

[0063] (3) Preparation of 1×TE buffer: Tris 0.121 g, Na2EDTA 0.336 g, and ultrapure water to a final volume of 100 mL.

[0064] Adjust the pH of the solution to 8.0 and use immediately or store in a refrigerator at 4°C.

[0065] (4) Preparation of 10% ammonium persulfate (APS) solution: Weigh 5 g of ammonium persulfate on an electronic balance AR224CN, then put it into a beaker containing 50 mL of ultrapure water, stir with a glass rod until completely dissolved, and then pour it into a 50 mL centrifuge tube for immediate use or storage in a 4°C refrigerator.

[0066] (5) Preparation of 12% natural polyacrylamide gel: 2.4 mL of 30% acrylamide, 1.2 mL of 5×TBE buffer, 100 µL of 10% APS, 5 µL of TEMED, and 2.4 mL of ddH2O.

[0067] (6) Preparation of 3% agarose gel: Weigh 1.5 g of agarose and add it to an Erlenmeyer flask. Measure 50 mL of 0.5×TBE and add it to the Erlenmeyer flask. Place the flask in a microwave oven and heat for 1-2 minutes. Remove the flask every 15-20 seconds and shake well. After boiling, remove the flask and rinse it with water while shaking to cool it to about 60°C. Pour the gel into a gel holder, insert a comb with a central hole, and gently shake it back and forth to remove air bubbles. Wait 30 minutes for the gel to form.

[0068] 1.2.3 Gel electrophoresis experiment

[0069] (1) Composition and preparation of the samples used in the experiment: Each sample system consists of 2 µL of sequence with 1×TM added to 20 µL, denatured at 90℃ for 5 min, and annealed to room temperature.

[0070] (2) 12% natural polyacrylamide gel electrophoresis (native-PAGE)

[0071] Prepare Marker: 7 μL ddH2O+1 μL Low DNA ladder+2 μL 6×Loading buffer+2 μL 10×SYBR Green I.

[0072] Prepare the gel electrophoresis system sample: 8 μL sample solution + 2 μL 6× Loading buffer + 2 μL 10× SYBR Green I.

[0073] After all samples were thoroughly mixed, 10 μL of the sample was loaded. 0.5×TBE electrophoresis buffer was used, with a voltage of 80 V and a time of 70 min. After gel electrophoresis analysis on a BIO-RAD electrophoresis system (USA), the gel images were exposed on a ChemiDoc XRS imaging system and analyzed using Image Lab software.

[0074] (3) 3% agarose gel electrophoresis

[0075] The preparation process for agarose gel electrophoresis samples is the same as that for PAGE electrophoresis. 0.5×TBE electrophoresis buffer was used, with a voltage of 100 V and a time of 45 min. After gel electrophoresis analysis on the electrophoresis apparatus, the gel images were exposed on a ChemiDoc XRS imaging system and analyzed using ImageLab software.

[0076] 1.2.4 Assembly Experiment

[0077] (1) Backbone screening experiment: The sequences A1, L1 / L1-1; A2, L2 / L2-1; A3, L3 / L3-1 (2 µL each, concentration of 10 µM) were added to 1×TM buffer (total volume 20 µL), denatured at 95℃ for 5 min in a metal bath, annealed, and gradually cooled to room temperature. Suitable backbones were screened by agarose gel electrophoresis.

[0078] (2) A1-L1-C1 assembly experiment: Add 2 µL of each of the A1, L1 and C1 sequences (10 µM concentration) to 1×TM buffer (total volume 20 µL), denature in a 95℃ metal bath for 5 min, and gradually cool to room temperature at room temperature (annealing). After assembly, perform agarose gel electrophoresis to observe whether the assembly was successful and whether there were any impurities.

[0079] 1.2.5 Feasibility Experiment of the Detection System

[0080] (1) Sample preparation: After the sequence A1-L1-C1 (20 µL, 3 µM) was assembled in 1×TM buffer, it was mixed with the target miR-21 (2 µL, 10 µM) or an equal volume of 1×TM buffer (2 µL, blank control) and reacted at room temperature for 1.5 h. Then, all samples were diluted to 200 µL with 1×TM buffer. To verify the feasibility of A1-L1-C1 in the in vitro detection process, the fluorescence intensity of solutions with and without miR-21 was compared and analyzed.

[0081] (2) Set the detection parameters of the F-7000 fluorescence spectrometer: Turn on the fluorescence spectrometer in advance and warm it up for 30 min. Open the corresponding software on the computer and set the following parameters: select wavelength scan, set the excitation light wavelength to 492 nm (set 5 nm grating), the emission light wavelength collection range to 500 nm-600 nm, the emission light slit and excitation light to 5 nm, select the scan speed of 240 nm / min, the response time to 0.5 s, and the photomultiplier tube voltage to 600 V.

[0082] (3) Sample loading and fluorescence intensity detection: Use a pipette to mix the solution in the EP tube, then take 180µL and slowly add it to the fluorescence cuvette along the side wall, taking care to prevent air bubbles from forming during this operation. Finally, place the fluorescence cuvette in the sample slot of the instrument to begin detection. After the detection is completed, discard the sample solution, and wash the fluorescence cuvette three times each with ultrapure water and anhydrous ethanol in sequence. After drying, measure the next sample solution.

[0083] 1.2.6 Proportion Optimization Experiment

[0084] (1) Optimization experiment of A1:L1 ratio: Multiple groups of A1:L1 with different ratios were set up and gel electrophoresis analysis was performed on the electrophoresis apparatus. The gel images were recorded and the optimal ratio was compared and analyzed.

[0085] (2) (A1-L1):C1 ratio optimization experiment: set up multiple groups of (A1+L1):C1 with different ratios, place them on the electrophoresis apparatus for gel electrophoresis analysis, record the gel images, and compare and analyze the optimal ratio.

[0086] 1.2.7 Optimization of miR-21 reaction time

[0087] Optimization of reaction time for miR-21: The prepared A1-L1-C1 was mixed with the target miR-21 (final concentrations of 10 nM, 20 nM and 50 nM) or an equal amount of 1×TM and reacted for 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h and 3 h, respectively. The total volume was adjusted to 200 μL with 1×TM buffer solution. Then the fluorescence intensity of the sample solution with and without the target miR-21 was measured at different reaction times.

[0088] 1.2.8 Stability testing in FBS

[0089] The stability of A1-L1-C1 in 10% FBS was investigated using a 12% natural polyacrylamide gel electrophoresis assay: A1-L1-C1 and FBS were mixed to a final FBS concentration of 10%, and then incubated at 37°C for 0 h, 4 h, 8 h, 12 h, 24 h, and 48 h, respectively. Once the samples reached the corresponding reaction time, gel electrophoresis was immediately initiated.

[0090] Fluorescence detection: Al-L1-C1-FAM modified with fluorescent groups and FBS were mixed, and the samples were treated according to the steps described above for natural polyacrylamide gel. Fluorescence was detected using an F-7000 fluorescence spectrometer. The fluorescence intensity of each sample was compared at different incubation times to verify the stability of this detection system under complex environmental conditions.

[0091] 1.2.9 Sensitivity Analysis of miR-21 Detection

[0092] A1-L1-C1 (20 µL, containing 1 μM A1, 1 μM L1, and 0.7 μM C1) was mixed with different concentrations of target miR-21 (final concentration range: 0–180 nM) or an equal volume of 1×TM buffer. The final volume of each sample group was then adjusted to 200 μL using 1×TM buffer solution. After incubation at room temperature for 1.5 h, the fluorescence intensity of the A1-L1-C1 detection system at different concentrations of target miR-21 was measured using an F-7000 fluorescence spectrometer.

[0093] 1.2.10 Specificity analysis of miR-21 detection

[0094] A1-L1-C1 (20 µL, containing 1 μM A1, 1 μM L1, and 0.7 μM C1) was mixed with target miR-21, sequences with different mismatched bases, and several other microRNA family members (miRNA-26a, miRNA-31, miRNA-141, and miRNA-122) to achieve a final concentration of 50 nM for each sequence. The final volume of each sample was then adjusted to 200 μL using 1×TM buffer. After incubation at room temperature for 1.5 h, the fluorescence intensity of different groups was detected using an F-7000 fluorescence spectrometer to assess the specificity of A1-L1-C1 detection.

[0095] 1.2.11 Cell Culture

[0096] 293T cells, HELF cells, A549 cells, and Huh7 cells were cultured in DMEM medium containing 1% streptomycin / penicillin and 10% FBS in an incubator at 37°C and 5% CO2. The medium was typically changed every 2 days, and cells were passaged every 3-4 days.

[0097] (1) Cell thawing: Take out the cryovials of 293T cells, HELF cells, A549 cells and Huh7 cells stored in a -80℃ freezer or liquid nitrogen tank, and immediately place them in a preheated 37℃ water bath and gently shake for about 1 min. After thawing, transfer them to a biosafety cabinet, gently transfer the cell solution into a 1.5 mL centrifuge tube, seal it with sealing film, centrifuge at 1200 rpm for 3 min, discard the supernatant, gently add 1 mL of complete DMEM medium to resuspend the cells, then transfer them to a culture dish, add 3 mL of medium and gently pipette to mix, and mark the medium used, date, culturer's name and cell name on the dish with a pen, and then culture in a cell culture incubator.

[0098] (2) Cell medium change: Take out the culture dish, being careful to keep the movement gentle and level, check the cell density and state under the microscope, transfer the cells that need medium change to the biosafety cabinet, pour out the culture medium in the culture dish, wash three times with PBS, and finally add 3 mL of complete culture medium. Take it back to the cell culture incubator and continue culturing at 37°C and 5% CO2.

[0099] (3) Cell passage: Remove the culture dish, being careful to handle it gently and horizontally. Observe the state and density of the cells under a microscope. When the cell density is observed to be above 80%, cell passage can be performed. Transfer the cells to be passaged to the biosafety cabinet. All cells used in the experiment are adherent cells, and the treatment method is the same: discard the old culture medium, wash three times with PBS, and then add trypsin containing EDTA for digestion. When the cells can be blown off the bottom of the dish, discard the trypsin, immediately add DMEM culture medium, and gently blow off the cells from the bottom of the dish and resuspend and mix well. Take a small amount into the cell culture dish, add the whole culture medium and mix well. Mix using the "cross" shaking method and then observe the cell density under a microscope to avoid too many or too few cells. Incubate in a 37°C, 5% CO2 cell culture incubator.

[0100] (4) Cell cryopreservation: Before cryopreservation, the cells need to be checked under a microscope to ensure they are in good condition and that the cell density is above 80%. Remove the programmed cooling box in advance. For adherent cells: digest the cells according to the cell passage method and collect them in 1.5 mL centrifuge tubes. Centrifuge at 1200 rpm for 3 minutes, then prepare fresh cryopreservation solution (culture medium: FBS: DMSO = 5:4:1) in a 5:4:1 ratio. Resuspend the cells in the prepared cryopreservation solution, aliquot the cell suspension into 1 mL cell cryopreservation tubes, seal them with sealing film, and place them in a programmed cooling box. Place the programmed cooling box in a -80℃ freezer for gradient cooling. Store for short-term at -80℃ or transfer to liquid nitrogen for long-term storage within 3 days.

[0101] 1.2.12 Flow cytometry targeting detection

[0102] Aptamer targeting assay: A 6-well plate pre-cultured with cells was washed three times with PBS. The prepared sample (Cy5-A1-L1-C1) and cells were then incubated in the dark for 4 hours. After 4 hours, the plate was washed three times with PBS. 200 µL of EDTA-free trypsin was added to each well for digestion. After digestion, 200 µL of DMEM was used to stop the digestion. The cells were then pipetted from the bottom well and transferred to 1.5 mL EP tubes. After centrifugation at 1200 rpm for 3 min, the cells were washed three times with PBS. Finally, the cells were resuspended in 400 µL of PBS and filtered into flow cytometry tubes. Each tube of cells was analyzed using flow cytometry.

[0103] 1.2.13 Assessment of drug loading in A1-L1-C1

[0104] A1-L1-C1 stock solution (550 µL, containing 27.5 μM A1, 27.5 μM L1, and 19.25 μM C1) was prepared in advance. A fixed amount of Dox (2 µL, 200 µM) was added to different volumes (20 µL, 60 µL, 80 µL, 100 µL, 120 µL, 140 µL) of A1-L1-C1 stock solution, and the volume was brought up to 200 µL with 1×TM buffer. The final Dox concentration was 2 µM in all cases. After shaking and mixing, the mixture was incubated at room temperature for 12 h, and the fluorescence of each experimental group was detected using an F-7000 fluorescence spectrometer.

[0105] Parameter settings: excitation wavelength of 488 nm (with a 5 nm grating), emission wavelength collection range of 500 nm-750 nm, emission slit and excitation slit of 10 nm, scan speed of 240 nm / min, response time of 0.5 s, and photomultiplier tube voltage of 700 V.

[0106] 2. Experimental Results

[0107] 2.1 Assembly and Detection Signal Release Principle of A1-L1-C1

[0108] Figure 1 This diagram illustrates the assembly and signal release of the A1-L1-C1 sequence. This invention constructs a nucleic acid chain A1 containing the aptamer AS1411, which can specifically recognize and bind to nucleolar proteins on the membrane surface. This allows the nucleic acid nanomaterials to target tumor cells without entering normal cells, thereby achieving the purpose of detecting specific target cells. Furthermore, this invention designs a nucleic acid chain L1 with a quencher group at one end and a C1 sequence modified with a fluorescent group that is completely complementary to miR-21. Figure 1 As shown, the end of L1 with the quencher group is partially complementary to C1. When it encounters the target miR-21, C1 is completely replaced. When the fluorescent group FAM moves a certain distance away from the quencher group BHQ2, it releases fluorescence and can be quantitatively detected by the corresponding instrument, thus realizing the quantitative detection of miR-21. In addition, drug delivery (e.g., Dox) can be carried between the bases assembled from A1 and L1, thereby achieving targeted drug delivery and targeted therapy.

[0109] 2.2 Gel electrophoresis analysis of A1-L1-C1 assembly screening

[0110] Taking into account factors such as the potential steric hindrance and base number of the constructed A1-L1 structure, 3% agarose gel electrophoresis was used for detection. The experimental results are as follows: Figure 2As shown in Figure A, the experimental results show that A1-L1 has the best assembly effect, the main band position is the highest, its molecular weight is larger, and there are fewer impurity bands. Therefore, A1 and L1 were selected as the main chains. Figure 2 The B band is the result of the assembly of the three chains A1-L1-C1, with a distinct bright band and a molecular weight of about 400 bp.

[0111] 2.3 Feasibility Analysis of the A1-L1-C1 Detection System

[0112] The feasibility of detecting miR-21 using A1-L1-C1 was analyzed using 12% native-PAGE. Figure 3 As shown in Figure A, the band in lane e is positioned higher than the bands in lanes c and d and has no extraneous bands, indicating that C1 and miR-21 can complementarily pair. Lanes f to h show obvious pore-clogging, indicating a larger assembled molecular weight. Lane h, except for pore clogging, shows the same position and height as lane e and almost the same brightness, indicating that the addition of miR-21 successfully competitively replaced the C1 chain. (Agarose gel electrophoresis results are shown.) Figure 3 As can be seen from C), the molecular assembly in lane h showed obvious destructive phenomena after the addition of miR-21, and the band darkened significantly, thus verifying the feasibility of the detection system. Since agarose is used to distinguish macromolecules, and the chains of C1 and miR-21 are relatively short, there are no obvious bands in lanes c and d.

[0113] Fluorescence measurement results as follows Figure 3 As shown in Figure B, curve c, representing C1-FAM modified with a fluorescent group, exhibits higher fluorescence intensity. Curve e, where miR-21 and C1 are completely complementary, shows higher fluorescence intensity than curves c and h alone. This may be because the C1-FAM sequence is not fully unfolded in curves c and h, and certain spatial structures formed may hinder its detection. Curve g shows very low fluorescence intensity, indicating that L1-BHQ2 modified with a quencher group can effectively quench the fluorescence of C1-FAM, resulting in low fluorescence background during the experiment. Curve h, with the target analyte miR-21 added to curve g, shows strong fluorescence intensity, indicating that miR-21 successfully competitively displaces C1-FAM, moving it away from the quencher group and spatially separating it, leading to fluorescence recovery and detection. The fluorescence intensity of other control groups is very low. Calculations show that the experimental group has a signal-to-noise ratio of approximately 20. The experimental results show a relatively clear phenomenon, indicating that this detection system A1-L1-C1 can be used to detect miR-21.

[0114] 2.4 A1-L1 Proportion Optimization

[0115] Based on the successful experiment in the feasibility analysis, the ratio of A1 and L1 used in the experiment was optimized. Figure 4 To optimize the experimental results using 12% natural polyacrylamide gel and 3% agarose gel electrophoresis respectively, lanes a to e represented the A1:L1 ratio gradually adjusted from 2:1 to 1:2. As shown in the figure, lane c exhibited clear and bright bands with fewer impurities in both the 12% natural polyacrylamide gel and 3% agarose gel electrophoresis images, indicating good assembly. Therefore, a molar ratio of 1:1 was selected as the optimal A1:L1 ratio for the experiment.

[0116] 2.5 C1 Proportion Optimization

[0117] Because some L1 sequences in nanomaterial molecules assembled from A1 and L1 are completely hidden inside the molecule, they cannot perform base pairing assembly with C1. Therefore, the proportion of C1 needs to be optimized to minimize fluorescence background during experiments while meeting the detection requirements of the target analyte. Figure 5 As shown in A, the signal-to-noise ratio is highest and the detection effect is best when the C1-FAM concentration is 70 nM. Figure 5 As can be seen from B, the fluorescence background value is low when the C1-FAM concentration is 70 nM. Therefore, 70 nM was selected as the optimal experimental concentration of C1 (i.e., the molar ratio of A1:L1:C1 is 1:1:0.7) for the experiment.

[0118] 2.6 miR-21 reaction time optimization

[0119] When miR-21 is added to the detection system A1-L1-C1, the reaction time of miR-21 is optimized to maximize the displacement of C1 and thus enable the detection of fluorescence signals, thereby achieving a higher signal-to-noise ratio and better detection performance. The results are as follows: Figure 6 As shown in the figure, different concentrations of miR-21 have a corresponding maximum fluorescence intensity when the miR-21 reaction is 1.5 h. Therefore, 1.5 h is selected as the optimal reaction time for miR-21.

[0120] 2.7 Stability study of A1-L1-C1 in 10% FBS

[0121] The stability of A1-L1-C1 under complex environmental conditions was investigated using a 10% FBS anti-degradation experiment. Multiple A1-L1-C1 samples were prepared and FBS was added. The FBS concentration in each system was adjusted to 10%, and the samples were incubated for 0 h, 4 h, 8 h, 12 h, 24 h, and 48 h, respectively, followed by 12% native-PAGE electrophoresis. The results are as follows: Figure 7 As shown, Figure 7In A, the position and brightness of the band A1-L1-C1 remained almost unchanged from 0 h to 24 h, indicating that A1-L1-C1 was minimally degraded within 24 hours, and a clear and distinct band still existed at 48 h; Figure 7 In sample B, it can be seen that A1-L1-C1 largely remains intact over time, with approximately 70% of A1-L1-C1 still present after 48 hours. Figure 7 C in the diagram represents C1-FAM modified with a fluorescent group. After incubation with 10% FBS, the degradation rate of A1-L1-C1 at different time points was determined by detecting the FAM fluorescence signal. Even after 48 hours, the material degradation rate remained very low. Figure 7 The fluorescence intensity was also found to be very low in D, indicating that only a small portion of it degraded. All of the above demonstrates that the assembled nanomaterial molecule A1-L1-C1 can resist the degradation of FBS for a relatively long period, and the assembled structure is relatively robust. Therefore, A1-L1-C1 exhibits excellent biological stability in 10% FBS.

[0122] 2.8 Sensitivity Study of the A1-L1-C1 Detection System

[0123] To evaluate the detection capability of the A1-L1-C1 detection system, different concentrations of the target miR-21 were added to the system under the same detection conditions for testing. The experimental results are as follows: Figure 8 As shown in Figure A, as the concentration of miR-21 increases, its corresponding fluorescence intensity also increases. However, when the concentration reaches 140 nM and 160 nM, the increase in fluorescence intensity is very small, indicating that A1-L1-C1 is approaching its detection limit. In the inset, it can be observed that at a miR-21 concentration of 200 pM, the fluorescence value is significantly different from the background fluorescence value (0 pM) of the blank control group. As the miR-21 concentration continues to decrease, the detected fluorescence value becomes less distinct from the background fluorescence intensity and is not very stable, thus indicating that the detection limit of the A1-L1-C1 detection system is 200 pM.

[0124] Figure 8 Figures B and C show a linear relationship between miR-21 at high and low concentrations, respectively. Figure 8 C is a dot plot of the fluorescence intensity of miR-21 at low concentration (pM) and corresponding concentration. The linear regression equation is F=28.04+0.0123C, with a correlation coefficient of 0.9954. Its linear response range is from 200 pM to 1 nM. Figure 8Figure B shows a scatter plot of the fluorescence intensity and concentration of miR-21 at a high concentration (nM). The linear regression equation is F = 124.0 + 5.91C, with a correlation coefficient of 0.9990. The linear response range is from 20 nM to 120 nM. Here, F and C represent the fluorescence value at 519 nm and the concentration of the target miR-21, respectively. The dose points for higher concentrations of target miR-21 (e.g., 140 nM) exceed the linear range.

[0125] 2.9 Specificity Study of the A1-L1-C1 Detection System

[0126] The target miR-21 of this invention has only 22 bases, and its homologous miRNAs show very high similarity, some differing by only one or a few bases; therefore, detecting miR-21 is very difficult. Thus, to achieve the goal of detecting only the target miR-21, it is necessary to ensure the specificity and stability of the detection method. Homologs miR-26a, miR-31, miR-141, and miR-122 were selected as controls for experiments to study the specificity of the A1-L1-C1 detection system. Figure 9 As shown, the fluorescence intensity produced by A1-L1-C1 when detecting miR-21 is the highest, and much higher than that of other homologous miRNAs. This shows that A1-L1-C1 can easily distinguish miR-21 from the other four homologous miRNAs and has very good detection specificity for miR-21.

[0127] In addition, experiments were conducted with different mismatched bases: one mismatched base (M1), two mismatched bases (M2), and three mismatched bases (M3) to further investigate the specificity of A1-L1-C1 detection. The results are as follows... Figure 9 As shown, the relative fluorescence intensity decreased with increasing number of mismatched bases. M3 exhibited very low relative fluorescence intensity, while M1 and M2 showed relatively high relative fluorescence intensity, but were still clearly distinguishable from miR-21. When the fluorescence intensity of miR-21 was defined as 1, the corresponding relative fluorescence intensities of M1, M2, and M3 were 0.61, 0.07, and 0.01, respectively. Numerically, the different mismatched bases showed very significant differences, indicating that A1-L1-C1 can specifically detect single or multiple mutated bases, further demonstrating the high detection specificity of the A1-L1-C1 detection system for miR-21.

[0128] 2.10 Preliminary analysis of cell targeting

[0129] We used 293T cells, HELF cells, A549 cells, and Huh7 cells to investigate the ability of A1-L1-C1 to target and enter different cells. Following the experimental procedures, the prepared sample (Cy5-A1-L1-C1) was incubated with different cells for 4 hours, and then analyzed by flow cytometry to obtain the fluorescence intensity peaks inside different cells. Figure 10 As can be seen, after the addition of the material, the fluorescence peaks of A549 cells and Huh7 cells showed a significant rightward shift, while the fluorescence peaks of 293T cells and HELF cells showed almost no shift. This is because 293T cells and HELF cells lack nucleolin proteins on their surface that can specifically recognize and bind to AS1411, while A549 cells and Huh7 cells do have nucleolin proteins on their surface. Therefore, the sample cannot specifically target 293T cells and HELF cells, but can enter the cells in small amounts through infiltration or endocytosis. The experiment shows that Cy5-A1-L1-C1 can specifically target A549 cells and Huh7 cells, but not 293T cells and HELF cells, thus indicating that the A1-L1-C1 nucleic acid material molecule modified with AS1411 has significant cell targeting ability.

[0130] 2.11 Preliminary Study and Analysis of Drug Loading in A1-L1-C1

[0131] A1-L1-C1 can target and enter tumor cells and detect the intracellular target miR-21. Based on this characteristic, this invention further investigated its drug-loading performance. Doxorubicin (Dox) is a drug that kills tumor cells. When targeted and loaded into tumor cells, it can specifically kill tumor cells and exert a targeted drug-loaded therapeutic effect. Dox can be embedded in the GC base pairs of a double-stranded DNA DNA, and its fluorescence will be quenched. This property was used to study the drug-loading capacity of A1-L1-C1. The results are as follows: Figure 11 As shown, the fluorescence intensity of Dox decreased continuously with the increase of A1-L1-C1, indicating that Dox could be successfully loaded into A1-L1-C1. However, when the amount of A1-L1-C1 reached a certain level, 120 µL (i.e., 1.62 µM), even with further increases in the amount, the fluorescence of Dox did not decrease further, indicating that almost all of Dox was loaded into A1-L1-C1. This study demonstrates that A1-L1-C1 can be used for drug loading, laying the foundation for future research on targeted therapy.

[0132] Based on the above, this invention successfully designed and constructed a palindrome-mediated self-assembly DNA molecule, A1-L1-C1, which exhibits feasibility as expected and demonstrates excellent stability in 10% FBS, with very little degradation within 48 hours. In in vitro stability testing, it exhibits low fluorescence background and strong anti-degradation ability. During in vitro miR-21 detection, the fluorescence emitted by A1-L1-C1 shows a good linear relationship with the target concentration within a certain concentration range and exhibits high detection sensitivity, with a linear range of 0.2-120 nM, a detection limit of up to 200 pM, and a correlation coefficient of 0.9990. A1-L1-C1 can specifically recognize target miR-21; in the absence of target miR-21, its fluorescence background is very low, while a significant fluorescence signal is visible when target miR-21 is present. It can also clearly distinguish other homologous miRNAs and effectively differentiate single or multiple base site mutations. A1 is linked to an aptamer, enabling it to specifically target corresponding tumor cells. After incubating A1-L1-C1 with cells for 4 hours, flow cytometry revealed that the fluorescence peaks of 293T cells and HELF cells showed almost no shift, while the fluorescence peaks of A549 cells and Huh7 cells shifted significantly to the right. This is because 293T cells and HELF cells do not have nucleolin proteins that can specifically recognize and bind to AS1411, while A549 cells and Huh7 cells do have corresponding nucleolin proteins. This indicates that A1-L1-C1 cannot enter 293T cells and HELF cells. Using A1-L1-C1 for drug delivery, such as Dox, can achieve drug delivery and targeted therapy of tumor cells.

[0133] In short, A1-L1-C1 can not only successfully detect targets in vitro, but also target and enter living cells to achieve intracellular detection, imaging, and drug delivery for tumor marker miRNAs. Using the AS1411 aptamer allows it to target tumor cells for detection without affecting normal cells, creating conditions for subsequent targeted drug delivery and treatment of specific tumor cells. This invention's palindromic-mediated self-assembled DNA molecule A1-L1-C1 has broad applications and significant reference value in various aspects such as intracellular miR-21 detection and imaging, early cancer detection and diagnosis, and tumor cell-targeted drug delivery and treatment.

[0134] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A palindrome-mediated self-assembly DNA molecule, characterized in that, The palindromic-mediated self-assembly DNA molecule is obtained by self-assembly of the A1 strand, L1 strand and C1 strand; The nucleotide sequence of the A1 chain is shown in SEQ ID NO.1, and its 5' end is modified with a fluorescent group; The nucleotide sequence of the L1 chain is shown in SEQ ID NO.2, and its 3' end is modified with a quenching group; The nucleotide sequence of the C1 chain is shown in SEQ ID NO.3; a fluorescent group is modified between the 10th and 11th bases.

2. A method for preparing a palindrome-mediated self-assembled DNA molecule as described in claim 1, characterized in that, The A1 chain, L1 chain, and C1 chain are mixed in a molar ratio of 1:1:0.7 and reacted at 90-98℃ for 4-6 min to obtain the palindrome-mediated self-assembled DNA molecule.

3. The application of the palindrome-mediated self-assembled DNA molecule as described in claim 1 in the preparation of products for detecting miR-21, characterized in that, The products include reagents, reagent kits, or chips.

4. A product for detecting miR-21, characterized in that, The product contains the palindromic self-assembling DNA molecule as described in claim 1.

5. The application of the palindromic self-assembly DNA molecule as described in claim 1 in the preparation of a tumor-targeted drug delivery system.

6. A method for preparing a tumor-targeting drug delivery system, characterized in that, The palindromic self-assembled DNA molecule of claim 1 is mixed with a drug and incubated to obtain the tumor-targeting drug delivery system.

7. The preparation method according to claim 6, characterized in that, The incubation temperature is 20-25℃, and the time is 10-14h.

8. A tumor-targeting drug delivery system prepared by the method described in claim 6 or 7.

9. The use of the tumor-targeting drug delivery system as described in claim 8 in the preparation of a medicament for treating tumors.

10. The use of a palindromic self-assembled DNA molecule as described in claim 1 in the preparation of products for tumor imaging.

Images