CN115960884B - Screening method for nucleic acid aptamer APT-Tan targeting activated hepatic stellate cells - Google Patents

Abstract

The invention provides a screening method for the nucleic acid aptamer APT-Tan targeting activated hepatic stellate cells. The aptamer is a single-stranded DNA structure, and the nucleotide sequence of the nucleic acid aptamer is SEQ ID NO. : The nucleotide sequence of any DNA fragment shown in 1-7, the nucleic acid aptamer can specifically enter activated hepatic stellate cells (HSC). The present invention can be used for the diagnosis of liver fibrosis, and can be used as an intracellular drug transport carrier by carrying bioactive molecules such as microRNA and siRNA.

Description

Screening method for nucleic acid aptamer APT-Tan targeting activated hepatic stellate cells

Technical Field

The present invention relates to the field of biomedicine, and in particular to a nucleic acid aptamer and a use thereof.

Background Art

Nucleic acid aptamers are single-stranded oligonucleotides that can specifically recognize target substances, obtained through multiple rounds of screening in vitro using the Systematic Evolution of Ligands by Exponential Enrichment technology (SELEX). Nucleic acid aptamers have many advantages, such as high specificity, high affinity, strong stability, and easy chemical synthesis and chemical modification. At present, as a new type of widely-watched detection and treatment tool, nucleic acid aptamers have shown broad application prospects in human medical research, disease diagnosis, and virus infection mechanism research. In the past 20 years, a large number of aptamers for important molecules related to tumors and other diseases have been screened and applied in basic biomedical research, disease diagnosis and treatment, and drug development. Pegaptanib (trade name Macugen) is the first aptamer drug approved by the US FDA for the treatment of age-related macular degeneration. There is also the nucleic acid aptamer drug AS1411 developed by Aptamera. Clinical trials have shown that AS1411 has a strong inhibitory effect on tumor cells such as breast cancer and cervical cancer. All these indicate that nucleic acid aptamers have a good prospect for clinical application. Cell-SELEX technology, developed on the basis of tradition, is a technology that uses whole living cells as targets to obtain aptamers that can bind to target cells. This technology can make cells closer to their natural state, not only can it obtain aptamers with unknown target information, but also can distinguish certain states of cells, such as differentiated or undifferentiated cells, normal cells, cancer cells, etc. Therefore, this technology has great application potential in tumor diagnosis, treatment and the discovery of tumor markers.

Liver fibrosis is characterized by abnormal proliferation and deposition of connective tissue in the liver and is a common pathological change in chronic liver diseases caused by various causes. The incidence of liver disease is increasing worldwide, and liver fibrosis and its end-stage cirrhosis constitute a huge health care burden worldwide. In 2015, approximately 1.3 million people died from chronic liver disease (CLD) and cirrhosis worldwide. The causes of CLD include chronic viral hepatitis, alcohol, nonalcoholic fatty liver disease (NAFLD), hemochromatosis, α-1-antitrypsin deficiency, cholestasis, and autoimmune diseases. Regardless of the cause, the final result of untreated CLD is inflammation, loss of liver parenchyma, fibrosis, and regenerative healing. Hepatic stellate cells (HSCs) are non-parenchymal cells located in the perisinusoidal space of the liver, also known as Ito cells, fat storage cells, and vitamin A storage cells. In the normal liver, HSCs are in a quiescent state, characterized by the ability to store retinol esters in lipid droplets in the cytoplasm and having ultrastructural characteristics of perivascular cells. In the process of liver injury healing, although other cells also make important contributions, the key fibrotic effector cell type is activated HSC. Liver injury caused by multiple causes activates quiescent HSC, causing them to lose stored vitamin A and transform into myofibroblasts that express α-SMA positively, which in turn secrete fibrillar collagen, elastin and matrix proteins, secrete profibrotic mediators, and lead to liver fibrosis. Therefore, taking practical measures to inhibit the activation of HSC is considered to be the key to preventing or even reversing the process of liver fibrosis and preventing and treating the occurrence of cirrhosis.

Summary of the invention

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular biology, nucleic acid chemistry and other operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

As used herein, the term "biomolecule" refers to a general term for molecules present in organisms, including but not limited to nucleic acids, oligopeptides, polypeptides, nanoparticles, carbohydrates, lipids, and small molecule compounds and any complexes thereof.

The term "aptamer" usually refers to a short RNA or DNA fragment that can bind specifically and efficiently to ligands such as proteins or metabolites obtained through in vitro screening. The term "liver fibrosis" usually refers to a pathophysiological process, which refers to the abnormal proliferation of connective tissue in the liver caused by various pathogenic factors.

The purpose of the present invention is to provide a new nucleic acid aptamer, which is an exogenously synthesized nucleotide chain, which has the ability to specifically bind to activated HSC and enter such cells, and the cell sources are hepatic stellate cells, normal liver cells, and other normal tissue cells. The nucleotide sequence of the nucleic acid aptamer is the nucleotide sequence of any DNA fragment shown in SEQ ID NO: 1-7, and the nucleic acid aptamer can specifically enter activated HSC cells.

The fluorescent marker is FAM.

As a preferred embodiment, the nucleotide sequence of the present invention is the sequence shown in SEQ ID NO: 2, i.e., GGTTTGCTGT ATGGTGGGCG TTGAAAGAGG GGTGGACACG GTGG (SEQ ID NO: 2), named 2 (31-74), which is truncated from SEQ ID NO: 1.

The aptamer includes at least one chemical modification: modification of the phosphodiester bond in the aptamer nucleic acid sequence, including thiolation modification; modification of the ribose in the aptamer nucleic acid sequence, including replacement of 2'-H with F, _NH2 , or OMe; modification of any base in the aptamer nucleic acid sequence with an amino group, a carboxyl group, a thiol group, a biotin group, a cholesterol group, or a polyethylene glycol group; modification of the 5' end of the nucleotide sequence of the aptamer with polyethylene glycol (PEG) and the 3' end with deoxyuracil (dU), deoxythymine (dT), and deoxyhypoxanthine (dL); and at least one nucleotide in the nucleotide sequence of the aptamer is a locked nucleic acid.

The application of the nucleic acid aptamer in the preparation of drugs for biomolecule transport carriers, the biomolecules include one or any complex of nucleic acids, oligopeptides, polypeptides, carbohydrates, lipids, nanoparticles, nanoblocks or small molecule compounds, and the nucleic acid is selected from si-RNA or Micro-RNA for anti-liver fibrosis.

The present invention also provides a method for screening the nucleic acid aptamer, which comprises: selection of a screening library, negative and positive screening, PCR amplification and high-throughput analysis, flow cytometry detection to obtain an aptamer with the strongest fluorescence, and then truncation based on the aptamer to finally obtain the optimal aptamer, comprising the following steps:

(1) providing a random single-stranded DNA library, the single-stranded DNA library comprising a single-stranded sequence represented by the following formula: 5'ATCCAGAGTGACGCAGCA(45N)TGGACACGGTGGCTTAGT3', wherein N45 represents a random sequence of 45 bp in the middle; preparing a secondary library using a primer pair of the nucleotide sequence shown in SEQ ID NO: 8-9;

(2) After denaturation, the single-stranded DNA library was subjected to negative screening by other normal liver cells (hepatocytes, hepatic sinusoidal endothelial cells, HSCs, and Kupffer cells) and positive screening by activated HSC cells to obtain the first round of screening products;

(3) PCR amplification of the first round screening products using primers with nucleotide sequences shown by upstream primer-FAM and downstream primer-Biotin to obtain the second round screening products;

(4) Similarly, the aptamer library of the previous round is denatured, negatively screened with other normal liver cells, and positively screened with HSC to obtain the screening products of the previous round, and then the aptamer library of the next round is obtained by PCR amplification with the primers described in step (1), and then the next round of screening is performed. A total of 11 rounds of screening are performed to finally obtain the aptamer, and the nucleotide sequence of the aptamer is the nucleotide sequence of the DNA fragment shown in the high-throughput sequencing results of the first 18 sequences in Table 2.

The nucleic acid aptamer is further truncated to obtain 2(31-74).

During the negative screening process, the cells were incubated at 37°C in a constant temperature shaker at 120-150 rpm for 30-60 min.

During the positive screening process, the activated HSC cells were washed, added with binding buffer, and incubated at 37°C in a constant temperature shaker at 120-150 rpm for 60-30 min.

The PCR amplification conditions are: 95°C for 5 min; 94°C for 30 s, 56-66°C for 30 s, 72°C for 30 s, 72°C for 5 min; the PCR cycle conditions are: 95°C for 5 min; 94°C for 30 s, 55-66°C for 30 s, 72°C for 30 s, extension after 21-35 cycles; 72°C for 5 min.

Another technical solution of the present invention is to use the nucleic acid aptamer in the preparation of drugs for biomolecule transport carriers, wherein the biomolecules include nucleic acids, oligopeptides, polypeptides, carbohydrates, lipids, nanoparticles, nanoblocks or small molecule compounds or any complexes thereof.

The nucleic acid is selected from si-RNA or Micro-RNA against liver fibrosis.

The complex comprises a drug transport carrier and a molecule that can be used as a drug, wherein the drug transport carrier is directly or indirectly connected to the molecule that can be used as a drug through a linker;

Wherein, the drug transport carrier is the nucleic acid aptamer; the molecules that can be used as drugs include one or any complex of nucleic acids, oligopeptides, polypeptides, sugars, lipids, nanoparticles, nanoblocks or small molecule compounds.

The complex is used in preparing medicine for treating liver fibrosis cancer disease or diagnosing liver fibrosis cancer disease.

The technical solution of the present invention discovered for the first time that the screened nucleic acid sequence has the function of specifically binding to activated HSC and can carry biological molecules such as RNA across the membrane into the cell. It is a transmembrane transport carrier of biologically active molecules such as nucleic acids and targeted drugs with great development prospects.

The advantages of the present invention are that, compared with other transmembrane bioactive substances, it is highly efficient, targeted, has no toxic side effects on cells, and has relatively few potential unsafe factors. Therefore, it has a broader application prospect as a drug molecule carrier for clinical application.

The present invention aims to use Cell-SELEX technology to use activated HSC as positive screening cells, and normal hepatocytes, liver sinusoidal endothelial cells, HSCs and Kupffer cells as negative screening cells. First, the random library and negative cells are incubated, and after collecting the unbound library, they are incubated with target cells, and finally the sequence bound to the target cells is eluted and amplified as a template for PCR, and used as a secondary library for the next round of screening. After multiple rounds of cyclic screening, the sequence bound to negative cells is discarded, and finally the aptamer (Figure 1) that specifically recognizes activated HSC is screened out. Then the obtained nucleic acid aptamer is deduced from its secondary structure according to Mfold, and the structure with the lowest Gibbs free energy ΔG is selected. The G-quadruplex structure of the nucleic acid aptamer is calculated by QGRS Mapper. Under the condition that the G-quadruplex maintains a stable structure and does not change the inherent stem-loop structure, the free end and the stem-loop structure are truncated one by one, and the possible binding domain is found by molecular simulation docking reference. The Vienna format of the secondary structure of the truncated aptamer was used as a template for constructing the tertiary structure, and the tertiary structure was generated on the RNAComposer website. The RNA tertiary structure was converted into a DNA sequence in Discovery Studio, and the predicted tertiary structure of the nucleic acid aptamer was obtained, which was named APT-Tan. The affinity of APT-Tan to cultured activated HSC, HSC, some other normal cells and tumor cells was observed, and the specificity, concentration and time gradient and other related properties of APT-Tan were further detected, providing a scientific basis for further using it as a drug delivery carrier for the treatment of liver fibrosis and targeted intervention in the process of liver fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the cell-SELEX screening process.

FIG. 2 is a gel electrophoresis diagram showing the screening annealing temperature and cycle number.

Figure 3 Cell affinity and flow cytometry analysis of lanes 1-18, A: affinity fluorescence graph of lanes 1-9 in activated HSC-T6 and LX-2 cells; B: affinity fluorescence graph of lanes 10-18 in activated HSC-T6 and LX-2 cells; C: fluorescence quantification of flow cytometry analysis of lanes 1-18 in activated HSC-T6.

FIG. 4 is a concentration gradient analysis of nucleic acid aptamer 2.

FIG5 is the tertiary structure of nucleic acid aptamer 2 and its truncated sequence.

FIG. 6 is an affinity analysis of the original length and truncated sequences of nucleic acid aptamer 2 in activated HSC-T6.

Figure 7 is a flow cytometric graph of the original length and truncated sequence of the second lane, A: affinity comparison between aptamer 1 and the truncated sequence; B: affinity comparison between aptamer 2 and the truncated sequence. ***p<0.005.

FIG8 is the affinity curve of APT-Tan at different concentration gradients.

FIG. 9 shows the stability of APT-Tan in HSC-T6 cells and serum, A: the stability time of APT-Tan in HSC-T6 cells; B: the stability time of APT-Tan in serum.

Figure 10 is a diagram of the cell specificity experiment of the aptamer APT-Tan, A: flow cytometry peak diagram of APT-Tan in 293FT, H9C2, LSEC and RAW264.7 cells; B: flow cytometry statistical results of APT-Tan in 293FT, H9C2, LSEC and RAW264.7 cells. nsp>0.05.

FIG. 11 is a graph comparing the temperature and incubation buffer of APT-Tan, ns p>0.05, **p<0.01.

Figure 12 APT-Tan incubation temperature, *p<0.05, ***p<0.005.

FIG. 13 APT-Tan cell entry inhibitor experiment, ***p<0.005.

Figure 14 Trypsin digestion target experiment. A: Flow cytometry peak diagram of APT-Tan and 2 treated with trypsin and untreated cells; B: PAGE electrophoresis gel diagram of APT-Tan and 2 target proteins. *p<0.05, ***p<0.005.

FIG. 15 is an affinity graph obtained by incubating APT-Tan-miR-23b-5p with activated and non-activated HSC-T6 cells, respectively, and observing the binding affinity under a fluorescence microscope.

FIG. 16 is a diagram showing Western-blot detection of harvested cells.

DETAILED DESCRIPTION

The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative and qualitative tests in the following examples are repeated three times or more, and the results are averaged.

Main reagents:

(1) DMEM culture medium: Gibco, USA.

(2) Pancreatic enzyme: Haoyang Biological Products (Tianjin) Technology Co., Ltd.

(3) Streptomycin/penicillin (P/S) reagent: Haoyang Biological Products (Tianjin) Technology Co., Ltd.

(4) PBS powder: Wuhan Biotech Biotechnology Co., Ltd.

(5) Fetal bovine serum: Gibco Company, USA.

(6) Newborn calf serum: Sijiqing Bioengineering Materials Co., Ltd. (Hangzhou).

(7) Turbofect transfection reagent: Thermo Fisher Scientific, USA.

(8) Aptamer library and primers: Sangon Biotech (Shanghai) Co., Ltd.

(9) Streptavidin Separopore 4B: Murray (Shanghai) Biotechnology Co., Ltd.

(10) Bovine serum albumin: Shanghai Sangon Biotechnology Co., Ltd.

(11) Small-scale plasmid extraction and DNA purification and recovery kit: Nanjing Novozyme Biotechnology Co., Ltd.

(12) Competent cell preparation kit: Takara Biotechnology Co., Ltd.

(13) Transfer RNA (yeast): Murray (Shanghai) Biotechnology Co., Ltd.

(14) Ethidium bromide (EB), dimethyl sulfoxide (DMSO): Sigma-Aldrich, USA.

(15) UNIQ-10 oligonucleotide purification kit: Sangon Biotech (Shanghai) Co., Ltd.

(16) AGAROSE (agarose): BioFroxx (Germany).

(17) TRYPTONE (tryptone) and YEAST EXTRACT (yeast extract): OXOID LTD (UK).

(18) Affinity chromatography column: Sangon Biotech (Shanghai) Co., Ltd.

(19) 100 bp DNA Ladder and DL5000 DNA Marker: Nanjing Novogene Biotechnology Co., Ltd.

(20) Pronase, DNase and collagenase type IV: Sigma-Aldrich, USA.

(21) Tris-base and glycine: Shanghai MacLean Biochemical Technology Co., Ltd.

(22) Sodium dodecyl sulfate (SDS): Henan Huamei Bioengineering Co., Ltd.

(23) Taq PCR master mix: Nanjing Novozyme Biotechnology Co., Ltd.

(24) Sodium bicarbonate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium chloride and calcium chloride: Shanghai Biotechnology Co., Ltd.

(25) DNase/RNase-Free Water: Beijing Solarbio Technology Co., Ltd.

(26) Ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (EGTA): Amresco Corporation (USA).

(27) 4-Hydroxyethylpiperazineethanesulfonic acid (Hepes): Sigma-Aldrich, USA.

(28) Isopropyl alcohol, glacial acetic acid, sodium hydroxide, magnesium chloride: Sinopharm Chemical Reagent Co., Ltd. (Shanghai).

(29) Agar A: Shanghai Sangon Biotechnology Co., Ltd.

(30) XhoΙ, SalΙ, EcoRΙ: Thermo Fisher Scientific, USA.

(31) 75% alcohol: Wuhan Feiyang Reagent Co., Ltd.

(32) Chloral hydrate: Sinopharm Chemical Reagent Co., Ltd.

(33) OptiPrep density gradient separation solution: Axis-shield, Norway

(34) 4% paraformaldehyde, DAPI staining solution: Wuhan Sewell Biotechnology Co., Ltd.

(35) High-throughput sequencing: Shanghai Bioengineering Co., Ltd.

(36) Streptavidin-labeled magnetic beads: MedChemExpress, USA

(37) EIPA: MedChemExpress, USA

(38) Dynasore: Abcam Plc, USA

(39) Filipin III: GLP Bio, USA

Cell lines:

(1) Rat hepatic stellate cell line (HSC-T6): donated by Professor Song Yuhu of Tongji Hospital, Huazhong University of Science and Technology.

(2) Hepatocytes, sinusoidal endothelial cells, and Kupffer cells: Our research group isolated and extracted hepatocytes from the livers of adult male SD rats.

(3) Rat cardiomyocyte cell line (H9C2): donated by Professor Zhang Shizhong of the Cardiovascular Pharmacology Laboratory of China Three Gorges University.

(4) Mouse mononuclear macrophages (RAW264.7): donated by Professor Wang Decheng from the Institute of Infection and Inflammatory Injury, Three Gorges University.

(5) Human renal epithelial cells (293T) and human embryonic kidney cells (293FT): maintained by our research group.

(6) Human hepatoma cells (HepG2): cultured and maintained by the Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, Three Gorges University.

(7) Human breast cancer cells (MCF7): cultured and maintained by the Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, Three Gorges University.

(8) Human lung adenocarcinoma cells (A549): cultured and maintained by the Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, Three Gorges University.

(9) Cervical cancer cells (HeLa): cultured and preserved by the Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, Three Gorges University.

(10) Human hepatic stellate cells (LX-2): preserved by our research group.

Strains and plasmids:

(1) Escherichia coli XL-Blue strain: donated by Dr. Zha Yunhong from the Institute of Cell Therapy, First People's Hospital, Three Gorges University.

(2) Plasmids PCDNA3.1(+), PCDNA3.1-ALK3, and PCDNA3.1-ALK5 were donated by Professor Liu Changbai of the Key Laboratory of Tumor Microenvironment and Immunotherapy, Three Gorges University.

Experimental Animals:

SD male rats: weighing 450-550 g, provided by the Experimental Animal Center of Three Gorges University.

Preparation of bacterial culture reagents:

(1) CaCl ₂ solution: 11.1 g of CaCl ₂ powder was added to 80 mL of ddH ₂ O to fully dissolve, and the volume was finally adjusted to 100 mL to prepare a 1 M CaCl ₂ solution. After high-pressure sterilization, it was stored at 4°C for later use.

(2) Ampicillin solution (Amp, 100 μg/μL): 1 g of Amp powder was added to 7 mL of ddH ₂ O and fully dissolved. The volume was finally adjusted to 10 mL. After dialyzing through a 0.22 μm filter membrane, the solution was stored at -20°C for later use.

(3) Liquid culture medium (LB(-), without Amp): 4 g sodium chloride, 2 g yeast extract, 4 g tryptone, add 300 mL ddH ₂ O to fully dissolve, and finally make up to 400 mL. After high-pressure sterilization, store at 4°C for future use.

(4) Liquid culture medium (LB(+), containing Amp): Add 400 μl of 100 μg/μL Amp to 400 mL of LB(-) liquid culture medium, mix thoroughly, and store at 4°C until use.

(5) Solid culture medium (without Amp): weigh 4g sodium chloride, 2g yeast extract, 4g tryptone, 6g agar powder (1.5%), add 300mL ddH ₂ O to fully dissolve, and finally make up to 400mL. Autoclave, when the temperature drops to 50℃, quickly and evenly pour into a sterile culture dish, and wait until the temperature cools to

After the culture medium solidifies at room temperature, seal it and store it at 4°C for later use.

(6) Solid culture medium (containing Amp): Prepare 400 mL of solid culture medium without Amp, sterilize under high pressure, and when the temperature drops to 50°C, add 400 μL of 100 μg/μL Amp, mix thoroughly, and quickly and evenly pour into a sterile culture dish. After the temperature cools to room temperature and the culture medium solidifies, seal it and store it at 4°C for later use.

Plasmid extraction related reagents:

(1) 1×TE Buffer: 0.121 g Tris-Base powder, 0.037 g EDTANa ₂ .2H ₂ O powder, add 70 mL ddH ₂ O to fully dissolve, and finally adjust the volume to 100 mL. After high-pressure sterilization, cool to room temperature, store in separate devices at -20°C for later use.

(2) Lithium chloride: 21.2 g lithium chloride powder was added to 70 mL ddH ₂ O to fully dissolve, and the volume was finally adjusted to 100 mL and stored at 4°C for later use.

(3) 10% SDS: 10 g of SDS powder was added with 60 mL of ddH ₂ O to fully dissolve, and the volume was finally adjusted to 100 mL and stored at room temperature for later use.

(4) Chloroform-phenol: Mix 100 mL of chloroform and 100 mL of Tris-saturated phenol thoroughly, allow to stand to separate into layers, and store at 4°C in the dark for later use.

(5) Solution I: Take 10 mL of 1M Tris-HCl solution, 25 mL of 1M glucose solution, and 10 mL of 0.5M EDTA solution, add 300 mL of ddH ₂ O, fully dissolve and mix, and finally make up to 500 mL. After high-pressure sterilization, store at 4°C for future use.

(6) Solution II: Take 50 mL of 10% SDS solution and 50 mL of 2M NaOH solution, add 300 mL of ddH ₂ O and mix thoroughly, finally make up to 500 mL and store at room temperature for future use.

(7) Solution III: Take 330 mL of 5M potassium acetate solution and 57.5 mL of glacial acetic acid, add 300 mL of ddH ₂ O and mix thoroughly, and finally make up to 500 mL. After autoclaving, store at 4°C for future use.

Agarose gel electrophoresis related reagent formula:

(1) Ethidium bromide solution (EB, 10 mg/mL): 1 g EB powder was added to 70 mL ddH ₂ O to fully dissolve, and the volume was finally adjusted to 100 mL. The solution was divided into small portions and stored at 4°C in a dark place for later use.

(2) 50×TAE stock solution: 242 g Tris-Base powder, 37.2 g EDTANa ₂ ·2H ₂ O powder and 57.1 mL glacial acetic acid, add 600 mL ddH ₂ O to fully dissolve, finally make up to 1000 mL, adjust pH to 8.5, store at room temperature for later use, and dilute 50 times before use.

(3) 2% agarose gel: 0.8 g agarose powder and 40 mL 50-fold diluted TAE were placed in a conical flask and heated in a microwave oven until the powder was completely dissolved. When the solution temperature dropped to 50°C, 1.8 μL EB was quickly added and mixed evenly. The mixture was immediately poured into the mold groove and an 18-hole comb was inserted. The solution could be used only after it cooled and solidified.

Reagent formula for nucleic acid aptamer screening:

(1) Washing buffer (WB): 1.237 g glucose, 0.254 g MgCl.6H ₂ O, dissolved in 250 mL sterile PBS (pH=7.4), cooled to room temperature, dialyzed through a 0.22 μm filter membrane, and stored in separate devices at 4°C for later use.

(2) Binding buffer (BB): 0.025 g tRNA, 0.25 g BSA, dissolved in 250 mL WB solution, dialyzed through a 0.22 μm filter membrane, and stored in separate devices at 4°C for later use.

(3) 0.2 M sodium hydroxide solution (NaOH): 0.8 g NaOH powder was placed in a beaker, and 80 mL ddH ₂ O was added to the beaker to fully dissolve. Finally, the volume was adjusted to 100 mL and transferred to a glass container. It was stored at room temperature for later use.

Cell culture related reagent formula:

(1) PBS phosphate buffer solution: 0.2g KCl, 0.2g KH ₂ PO ₄ , 8g NaCl, 2.886g _{Na 2} HPO ₄ , add 700mL ddH ₂ O to fully dissolve, and finally make up to 1000mL. Adjust pH to 7.4, sterilize by high pressure, and store at 4°C for future use.

(2) DMEM(+): Take 3 mL of 1% P/S dual antibody solution and 30 mL of 10% fetal bovine serum, add them to 267 mL of DMEM(-) and mix well to obtain 300 mL of 10% DMEM(+) culture medium, which is stored at 4°C for later use.

(3) Cell freezing solution (containing serum): prepare 10 mL of freezing solution according to the solution volume of FBS:DMSO = 9:1, mix the three ingredients thoroughly and store at 4°C in the dark for later use.

Reagent formula for separation and extraction of rat hepatocytes, hepatic sinusoidal endothelial cells, and Kupffer cells:

(1) Enzyme preparation solution: weigh 0.9 g glucose, 8 g NaCl, 0.566 g CaCl ₂ , 0.4 g KCl, 0.4 g NaHCO ₃ , 0.06 g Na ₂ HPO ₄ , 0.19 g EGTA and 2.38 g Hepes, add 600 mL ddH ₂ O to fully dissolve, and make up to 1000 mL. After autoclaving, adjust the pH to 7.4 and store at 4°C for future use.

(2) D-Hanks balanced salt solution: weigh 0.4 g NaHCO ₃ , 0.053 g Na ₂ HPO ₄ , 0.4 g KCl, 0.06 g KH ₂ PO ₄ , 0.19 g EGTA, 2.38 g Hepes and 8 g NaCl, add 700 mL ddH ₂ O to fully dissolve, and make up to 1000 mL. After autoclaving, adjust the pH to 7.4 and store at 4°C for later use.

(3) GBSS: Weigh 1.0 g glucose, 0.21 g MgCl ₂ .6H ₂ O, 4.8 g Hepes, 0.07 g MgSO ₄ ·7H ₂ O, 0.133 g Na ₂ HPO ₄ ·12H ₂ O, 0.227 g NaHCO ₃ , 0.37 g KCl and 0.03 g KH ₂ PO ₄ , add 600 mL ddH ₂ O to fully dissolve, and finally make up to 1000 mL. Autoclave, adjust pH to 7.4, and store at 4°C for later use.

(4) High concentration pronase solution: Weigh 150 mg of pronase powder and place it in 150 mL of enzyme preparation solution to fully dissolve it. Use it after dialyzing through a 0.22 μm filter membrane.

(5) Low concentration pronase solution: Weigh 10 mg of pronase powder and place it in 50 mL of enzyme preparation solution to fully dissolve it. Use it after dialyzing through a 0.22 μm filter membrane.

(6) Type IV collagenase solution: Weigh 18 mg of type IV collagenase powder and place it in 150 mL of enzyme preparation solution to fully dissolve it. Use it after dialyzing through a 0.22 μm filter membrane.

(7) Enzyme digestion solution: Weigh 2.5 mg of DNase powder and place it in a mixture of 50 mL of low-concentration Pronase solution and 50 mL of type IV collagenase solution to fully dissolve it. Use it after dialyzing through a 0.22 μm filter membrane.

(8) Red blood cell lysis solution: Weigh 1 g KHCO ₃ , 0.037 g EDTA and 8.29 g NH ₄ Cl, add 600 mL ddH ₂ O to fully dissolve, and finally make up to 1000 mL. After dialyzing through a 0.22 μm filter membrane, store at 4°C for later use.

Example 1

Cell-SELEX technology to screen nucleic acid aptamers targeting HSC activation

1. Synthesis of nucleic acid library: Design and synthesize single-stranded DNA library (ssDNA library, 81 bases in total) and upstream and downstream primers. The library consists of a random sequence in the middle and constant sequences at both ends, which can be chemically synthesized at the laboratory level (Table 1).

Table 1 Library and primer sequence names

name Base sequence (5'→3') ssDNA library ATCCAGAGTGACGCAGCA(45N)TGGACACGGTGGCTTAGT Upstream primer-FAM ATCCAGAGTGACGCAGCA Downstream primer-Biotin ACTAAGCCACCGTGTCCA

2. Screening of specific nucleic acid aptamers

2.1 The above single-stranded DNA library (ssDNA library) and upstream and downstream primers were constructed by in vitro chemical synthesis method, and activated HSC-T6 was used as positive screening cells, and normal rat hepatocytes, liver sinusoidal endothelial cells, HSCs and Kupffer cells were used as negative screening cells.

2.2 Before dissolving the library and primers, centrifuge at 4000g for 1 min at 4°C (primers and random library are in dry powder form and will settle to the bottom of the tube after centrifugation). Prepare a stock solution (i.e. add 13ul _ddH2O per OD of the library) and freeze at -20°C for later use.

2.3 Extract cells from normal rat liver by lavage and digestion.

2.4 Take out the stock solution of the library from step 2.2 and dilute it 10 times with double distilled water to obtain the original solution (i.e., take 1 μL of the stock solution from step 2 and dilute it 10 times).

2.5 Pre-denaturation: Take all the original library solution, add 500 μL binding buffer, blow evenly, place the ssDNA library solution in a 95°C metal bath for 5 minutes (to denature the DNA), then immediately take it out and place it on ice for 10 minutes.

2.6 Negative screening (added in the third round): Add the treated normal rat liver mixed cell fluid to a 15mL tube, centrifuge at 120g for 3min, discard the supernatant, repeat washing several times, add 500uL binding buffer to resuspend, and then add to the denatured ssDNA library, blow evenly, place on a desktop constant temperature shaker at 37℃, 120rpm, 30min (as the number of screening rounds increases, the screening pressure increases, and the negative incubation time increases). To prevent the cells from sinking, shake the EP tube every 10min to keep the cells in a suspended state. After incubating with the cell suspension for 30-60min, place in a centrifuge at 4000g, 4℃, 4min, discard the precipitate, and take the supernatant for positive screening (ssDNA that is not bound to normal liver cells)

2.7 Positive screening:

(1) Take out the HSC-T6 cells passaged the day before from a 37°C constant temperature incubator, observe the cell morphology and cell number under a microscope, and take out a culture dish with a cell density of about 80%-90%.

(2) HSC-T6 cells were transfected with pcDNA3.1-ALK5 plasmid for 24h-36h.

(3) Discard the culture medium, wash the cells three times with washing buffer, add binding buffer (in the first and second rounds, the cell culture dish is 100×20 mm ² and the binding buffer is 5 mL. After the third round, it is 60×15 mm ² and the binding buffer is 2 mL), add the negative screening supernatant, and incubate in a 37°C constant temperature incubator for 60-30 min.

2.8 Washing:

(1) Collect the supernatant in an EP tube, wash three times with washing buffer, add 200uL of trypsin to digest for 5min, add complete medium preheated at 37℃ to stop digestion, and collect the cell suspension in a 1.5mL EP tube.

(2) Centrifuge the cell suspension at 120 g for 3 min, discard the supernatant, add 1 mL of washing buffer, resuspend and wash at 120 g for 3 min, discard the supernatant, and repeat this step 5 times.

2.9 Elution

(1) Add 500uL of binding buffer (add _ddH2O in the first round) to the cells, heat in a 95℃ metal bath for 15min (DNA denaturation, cell lysis), centrifuge at high speed (12000g, 4℃, 10min) in a refrigerated centrifuge, discard the precipitate, and use the supernatant as a template for PCR amplification.

2.10 Primers

Primers were prepared as 100uM stock solution according to the instructions, diluted to 10μM as required, and stored at -20°C. The working concentration during PCR was 1μM.

2.11 PCR amplification system and procedure

(1) Annealing temperature optimization

The reaction system includes 1 μL of template (screening fragment), 0.5 μL of upstream primer, 0.5 μL of downstream primer, 12.5 μL of 2×powerTaq PCR mastermix, 10.5 μL of water, a total of 25 μL. The PCR amplification conditions are: 95°C for 5 min pre-denaturation; then 94°C for 30 s, 56-66°C (55.0°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, 66°C) for 30 s, 72°C for 30 s for 35 cycles; and finally 72°C for 5 min extension.

After the PCR reaction program is completed, run 3% agarose gel at 120V for 55min and observe whether the bands are in the correct position on a gel imager. Select the temperature with the highest brightness and the least nonspecific amplification as the PCR annealing temperature for this round of screening.

(2) Cycle number optimization

The reaction system included 1 μL of template (screening fragment), 0.5 μL of upstream primer, 0.5 μL of downstream primer, 12.5 μL of 2×powerTaq PCR mastermix, and 10.5 μL of water, for a total of 25 μL. The PCR cycle conditions were: 95°C for 5 min pre-denaturation; then 94°C for 30 s, 55-66°C (55.0°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, 66°C) for 30 s, and 72°C for 30 s for 19-35 cycles; and finally 72°C for 5 min extension.

Perform PCR according to the optimized annealing temperature, and set the number of cycles to 19, 21, 23, 25, 27, 29, 31, 33, and 35.

After PCR, run electrophoresis on 3% agarose gel at 100V for 55min and observe the optimal cycle number on a gel imager. Select the cycle number with the brightest band and the least nonspecific amplification.

2.12 After determining the optimal temperature and cycle, amplify the screening fragments according to the previous reaction system. There are 40 tubes in total, 25uL in each tube, and a total of 1000μL of PCR products are obtained, which are divided into 2 large EP tubes, 500uL in each tube (all the first round of screening templates are used for PCR).

2.13 PCR product purification

(1) Add 500 μL of binding buffer to each EP tube, then add 500 μL of isopropanol and let stand for 5 min.

(2) Add the mixture in (1) to the purification column several times, let it stand for 5 minutes, centrifuge at 12000g for 1 minute, and discard the waste liquid at the bottom.

(3) Add 700 μL of wash buffer (PW) to each purification column, let stand for 2 min, centrifuge at 12,000 g for 2 min, and discard the waste liquid at the bottom.

(4) Centrifuge again for 2 minutes

(5) Move the column to a new EP tube and air-dry it in a clean bench away from light for 5 min.

(6) Add 50uL RNAsee free _ddH2O to each column and let stand for 5min.

(7) Centrifuge at 12000 g for 1 min and collect the liquid, a total of 100 uL.

2.14 Alkaline denaturing affinity column chromatography to separate double-stranded DNA and convert it into single-stranded DNA

(1) Add 200uL of strepeividin sepharose to the affinity chromatography column.

(2) After equilibration of the column with DPBS (or PBS), add the double-stranded DNA amplified by PCR.

(3) Washing the chromatography column with DPBS

(4) Separate and elute the positive strand ssDNA with 500uL 0.2M NaOH

2.15 Desalination

The secondary library (FAM-ssDNA) was obtained by UNIQ-10 column purification (according to the instructions). The first round of screening was completed.

Thus, the first round of screening is completed. Repeat the above steps to complete the second, third, fourth, and fourth rounds of negative and positive screening. The number of PCR cycles must be optimized before each round of screening (Figure 1-2).

Figure 2 shows the results of 11 rounds of temperature and cycle screening of nucleic acid aptamers, in which the first round of temperature and cycle screening, the temperature was between 55-66°C, the annealing temperatures were 55°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, 66°C from left to right, and the cycles were 19, 21, 23, 25, 27, 29, 31, 33, 35 cycles from left to right;

In the second round of temperature and cycle screening, the temperature was between 55-66°C, the annealing temperatures from left to right were 55°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, 66°C, and the cycles from left to right were 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 cycles;

In the third round of temperature and cycle screening, the temperature was between 55-66°C, the annealing temperatures from left to right were 55°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, 66°C, and the cycles from left to right were 19, 21, 23, 25, 27, 29, 31, 33, 35 cycles;

In the fourth round of temperature and cycle screening, the temperature was between 55-66°C, the annealing temperatures from left to right were 55.0°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, 66°C, and the cycles from left to right were 19, 21, 23, 25, 27, 29, 31, 33, 35 cycles;

For the fifth round of temperature and cycle screening, the temperature was between 55-66°C, the annealing temperatures from left to right were 55°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, 66°C, and the cycles from left to right were 19, 21, 23, 25, 27, 29, 31, 33, and 35 cycles.

In the sixth round of temperature and cycle screening, the temperature was between 55-66°C, the annealing temperatures from left to right were 55°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, 66°C, and the cycles from left to right were 23, 25, 27, 29, 31, 33, and 35 cycles.

In the seventh round of temperature and cycle screening, the temperature was between 55-66°C, the annealing temperatures from left to right were 55°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, and 66°C, and the cycles from left to right were 23, 25, 27, 29, 31, 33, and 35 cycles.

In the eighth round of temperature and cycle screening, the temperature was between 55-66°C, the annealing temperatures from left to right were 55°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, 66°C, and the cycles from left to right were 23, 25, 27, 29, 31, 33, and 35 cycles.

In the ninth round of temperature and cycle screening, the temperature was between 55-66°C, the annealing temperatures from left to right were 55°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, 66°C, and the cycles from left to right were 23, 25, 27, 29, 31, 33, and 35 cycles.

In the tenth round of temperature and cycle screening, the temperature was between 56°C and 66°C, the annealing temperatures from left to right were 55.0°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, and 66°C, and the cycles from left to right were 23, 25, 27, 29, 31, 33, and 35 cycles.

In the eleventh round of temperature and cycle screening, the temperature was between 55-66°C, the annealing temperatures from left to right were 55°C, 55.2°C, 55.8°C, 56.6°C, 57.9°C, 59.5°C, 61.5°C, 63.1°C, 64.4°C, 65.2°C, 65.8°C, 66°C, and the cycles from left to right were 23, 25, 27, 29, 31, 33, and 35 cycles.

2.16 High-throughput sequencing

According to the conditions of the 11th round of screening, amplification was performed in large quantities, the PCR target bands were recovered by gel excision, and sent for high-throughput sequencing, and the top 18 bands with the highest enrichment content were screened for subsequent experiments.

Table 2 High-throughput sequencing results of the first 18 sequences

The activated hepatic stellate cell aptamers were screened twice, for a total of 12 rounds. Negative screening was added in the third round.

Preprocessing

Take the DNA library, denature it at 95°C for 5 minutes, and then renature it on ice for 10 minutes.

Negative screening

(1) Primary hepatocytes, hepatic stellate cells, hepatic sinusoidal endothelial cells, and Kupffer cells were placed in 1.5 ml EP tubes, and the pretreated DNA library was added thereto. After thorough mixing, the cells were incubated in a 37°C constant temperature metal bath, and the mixture was mixed every 5 minutes. The incubation time was 30 minutes to 60 minutes. As the number of screening rounds increased, the negative incubation time gradually increased, and the number of negative screening cells gradually increased.

(2) After incubation, centrifuge at 4°C, 110 g for 3 min, take the supernatant into another clean EP tube, and centrifuge several times to completely remove residual cells and tissue fragments.

(3) Take the supernatant left at the end as the library for positive screening. The screening is performed twice in total, and negative screening is added in the third round.

Positive screening

(1) Positive screening: activated HSC-T6 cells were used as target cells and stimulated with pcDNA3.1-ALK5 for 24-36 hours.

(2) Discard the original culture medium of HSC-T6 cells after stimulation and activation, wash with WB three times, add BB, add the pre-treated negative screening supernatant, mix thoroughly, and incubate in a 37°C incubator for 30-60 minutes, shaking gently every 5 minutes. As the number of screening rounds increases, the positive incubation time gradually decreases, and the number of positive screening cells gradually decreases.

(3) Discard the incubation supernatant, add WB to wash three times, digest with trypsin for 5 min, stop digestion with cell culture medium, and collect the cell suspension in a 1.5 ml EP tube.

(4) Centrifuge at 110 g for 3 min at 4°C. Discard the supernatant and resuspend in 1 ml of WB. Repeat 5 times.

(5) After discarding the supernatant, add 500ul BB to resuspend (add 500ul distilled water for the first round of screening), incubate at 95°C for 10 min to fully lyse the cells, centrifuge at 4°C, 12,000g for 15 min, and use the supernatant as the PCR template.

PCR amplification

(1)

PCR amplification system

(2) Selection of PCR amplification annealing temperature: According to the PCR amplification system, the amplification conditions are: 95℃5min pre-denaturation; then 94℃30s, 55-66℃30s, 72℃30s for 35 cycles; finally 72℃5min extension. Among them, the annealing temperatures are 55.0℃, 55.8℃, 56.6℃, 57.9℃, 59.5℃, 61.5℃, 63.1℃, 64.4℃, 65.2℃, 65.8℃, 66.0℃. After the PCR reaction is completed, 3% agarose gel is electrophoresed at 120v for 55min, and the bands are observed on the gel imager to see if they are in the correct position. The temperature with the highest brightness and the least non-specific amplification is selected as the PCR annealing temperature for this round of screening.

(3) Selection of PCR amplification cycle number: According to the PCR amplification system, the annealing temperature is determined in step (2). The amplification conditions are: pre-denaturation at 95°C for 5 min; then cycle 19-35 times at 94°C for 30 s, 55-66°C for 30 s, and 72°C for 30 s; and finally extend at 72°C for 5 min. The cycle numbers are set to 19, 21, 23, 25, 27, 29, 31, 33, and 35. After the PCR program is completed, 3% agarose gel is electrophoresed at 120v for 55min. The bands are observed on the gel imager to see if they are in the correct position. The cycle with the highest brightness and the least nonspecific amplification is selected as the cycle for large-scale PCR amplification in this round of screening.

(4) After selecting the optimal annealing temperature and number of cycles for screening, amplify the screening fragments according to the reaction system of step (1). Amplify 40 tubes per round (all the templates for the first round of screening are subjected to PCR).

PCR double-stranded DNA recovery

(1) Take a clean 1.5 ml EP tube, add 500 μl of PCR product, then add 500 μl of BB and 500 μl of isopropanol, and let it stand on ice for 5 min.

(2) The mixed solution in step (1) was added to the purification column CP2 in several portions, allowed to stand for 5 min, centrifuged at 12000 g for 1 min, and the waste liquid at the bottom was discarded.

(3) Add 700 μl of wash buffer (PW) to each purification column, let stand for 2 min, centrifuge at 12,000 g for 2 min, and discard the waste liquid at the bottom.

(4) Centrifuge again for 2 min to completely remove any remaining liquid.

(5) Transfer the purification column to a new 1.5 ml EP tube and air-dry at room temperature in the dark for 5 min.

(6) Add 50 μl of enzyme-free distilled water to the middle of the purification column filter membrane and let it stand at room temperature for 5 minutes.

(7) After centrifugation at 12000g for 1 min, collect the liquid and repeat step (6) once to increase the DNA recovery efficiency.

(8) The obtained DNA was sent for high-throughput sequencing at round 11 and round 12 respectively.

Separation of single-stranded DNA by alkaline denaturation of affinity chromatography column

(1) Pre-pack the affinity chromatography column and add 200 ul of streptavidin-labeled agarose.

(2) Equilibrate the affinity chromatography column with DPBS for more than 6 hours to ensure that the liquid level is above the agar and that the agar remains moist.

(3) Slowly add PCR recovery product along the tube wall, allowing the PCR product to slowly pass through the affinity chromatography column, and repeat several times to allow biotin and streptavidin to bind as much as possible. The whole process takes about 30 minutes.

(4) Wash the affinity chromatography column several times with DPBS at a volume 5 times that of agar.

(5) Add 500ul of 0.2M NaOH to the cleaned affinity chromatography column to denature the double-stranded DNA, collect the outflowing liquid, and then pass the collected liquid through the affinity chromatography column several times. The whole process takes about 30 minutes.

(6) Collect the liquid that flows out, which is the single-stranded DNA.

Single-stranded DNA desalting and recovery

(1) Use UNIQ-10 column kit for recovery.

(2) Take four 1.5 ml sterilized EP tubes, add 100 ul of ssDNA supernatant to each tube, and then add 1 ml of Binding Buffer I to mix well.

(3) Transfer the mixed solution to the adsorption column several times, let it stand at room temperature for 2 minutes, and centrifuge it at 8000 rpm for 2 minutes. Pour away the waste liquid in the collection tube and put the adsorption column back into the collection tube.

(4) Add 500 μl of Wash Solution (add the corresponding volume of anhydrous ethanol according to the label on the bottle before use) to each adsorption column and centrifuge at 10,000 rpm for 1 min.

(5) Repeat step (4) once and discard the waste liquid at the bottom.

(6) Place the adsorption column back into the collection tube and centrifuge at 10,000 rpm for 2 min to completely dry the remaining liquid (remove residual ethanol, otherwise ethanol will affect the ssDNA recovery efficiency and subsequent experiments).

(7) Place the adsorption column into a new clean 1.5 ml EP tube and leave it uncovered at room temperature for 5 min.

(8) Add 50 μl of enzyme-free ddH ₂ O to the middle of the adsorption column membrane, let stand at room temperature for 5 min, and centrifuge at 12,000 rpm for 2 min.

(9) Repeat step (8) once to improve the recovery efficiency.

(10) Collect the resulting ssDNA liquid, which is the next round of screening library.

HSC-T6 cells in the logarithmic growth phase were seeded in a 24-well plate, and plasmid pcDNA3.1-ALK5 stimulated HSC-T6 activation. When the cell density was about 70%-80%, the cells were treated with pcDNA3.1-ALK5 for 24h-36h, and the 18 sequences were co-incubated with the cells at a final concentration of 100nM, respectively, as a control for stimulation and activation, and the cell fluorescence was observed under an inverted fluorescence microscope after incubation for 1h. The results showed that the 1st, 2nd, and 14th aptamers entered the largest number of activated HSC-T6 cells (pcDNA3.1-ALK5 stimulation group) (Figure 3). The flow cytometry results were consistent with the fluorescence results, and the average fluorescence intensity of the 1st, 2nd, and 14th aptamer groups was the strongest (Figure 3). The present invention named the second aptamer as nucleic acid aptamer 2, and selected it for subsequent experiments.

2. The activated HSC-T6 cells were incubated with aptamer 2 at concentrations of 50, 100, 150, 200, 250, and 300 nM in binding buffer for 1 h, and the fluorescence intensity of aptamer 2 at different concentrations was detected by flow cytometry. The results showed that the fluorescence intensity of aptamer 2 reached a maximum at 100 nM (Figure 4).

3. Use Discovery Studio software to simulate the tertiary structure of nucleic acid aptamer 2 according to the nucleotide sequence for further analysis.

The aptamer was truncated to effectively improve its cell entry efficiency (Figure 5).

Example 2: Experimental analysis of affinity optimization of nucleic acid aptamer 2

1. Structural prediction of the secondary and tertiary structures of aptamer 2 and the possible G-quadruplex structure. The aptamer 2 was truncated in sequence without changing the secondary, tertiary and G-quadruplex structures.

Table 3 2 Truncated aptamer sequences

Note: Black sequences represent retained sequences, and grey sequences represent truncated sequences.

2. The spatial conformation of nucleic acid aptamer 2 and its truncated aptamer was simulated using Discovery Studio, as shown in FIG5 .

3. Qualitative and quantitative experiments on truncated sequences.

3.1 The truncated sequence was labeled with FAM fluorescent group. HSC-T6 cells in the logarithmic growth phase were seeded in 24-well plates, activated for 24 hours by plasmid pcDNA3.1-ALK5 treatment, and the truncated sequence was added at a final concentration of 50uM. After incubation for 1 hour, the cells were observed under an inverted fluorescence microscope. The results showed that the original length of aptamer 2, 2(31-74), 2(1-51+38-80), 2(10-15+38-52), 2(59-71), 2(59-74), and 2(61-74) had the strongest fluorescence (Figure 6).

3.2 1×10 ⁵ HSC-T6 cells in the logarithmic growth phase were inoculated in a 24-well plate and treated in the same manner as 3.1. The six sequences with the strongest fluorescence were added and incubated for 1 hour, and the cells were collected. The average fluorescence intensity was detected by flow cytometry. The average fluorescence intensity of 2 (31-74) was the strongest (Figure 7), indicating that it entered the activated HSC-T6 in large quantities, and the other bands were less. 2 (31-74) was selected for further study and named APT-Tan.

Example 3

Study on concentration dependence, stability, specificity, incubation system and temperature of APT-Tan

1. Saturation experiment of APT-Tan.

1.1 1×10 ⁵ HSC-T6 cells were seeded into 24-well plates and transfected with 1 μg pcDNA3.1-ALK5 for 24-36 hours.

1.2 After heating APT-Tan at concentrations of 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, and 700nM at 95°C for 5min, the cells were immediately placed on ice for 10min, added to the corresponding 24-well plates in the dark, incubated with binding buffer for 1h, and then the mean fluorescence intensity of the cells was detected by flow cytometry. The results are shown in Figure 8. The mean fluorescence intensity of APT-Tan continued to increase at a concentration of 0-200nM and basically reached saturation at a concentration of 200nM.

2. Stability

2.1 APT-Tan cell stability

APT-Tan at a concentration of 200 nM was incubated with activated HSC-T6 in the same manner as above. After incubation in 10% FBS serum for 0.5h, 1h, 2h, 4h, 6h, 8h, and 10h, the cells were collected and the affinity of APT-Tan to HSC-T6 was analyzed by flow cytometry. The fluorescence of APT-Tan remained unchanged at 0.5h-4h, and then continued to increase at 6-10h. At 24h, the fluorescence intensity was still similar to that at 0.5h, indicating that APT-Tan can be stably present in cells for more than 24h (Figure 9A).

2.2 Serum stability

Blood was collected from the eyeballs of c57BL mice, and the serum was separated. APT-Tan at a concentration of 10uM was co-incubated with the same volume of serum. The samples were taken after incubation for 0.5h, 1h, 2h, 4h, 6h, 8h, and 10h, and electrophoresed on 10% native-PAGE for 4h. The gel was imaged after staining with gelred dye at room temperature for 30min. APT-Tan began to degrade at 0.5h and was almost completely degraded after 2h (Figure 9B).

3. Cell specificity of APT-Tan The APT-Tan obtained by screening in Example 2 was incubated with rat cardiomyocytes (H9C2), mouse mononuclear macrophages (RAW264.7), human embryonic kidney cells (293FT), rat liver sinusoidal endothelial cells (LSEC) and other cell lines to observe the affinity of APT-Tan to these cells. The results are shown in Figure 10. APT-Tan has poor binding ability with these cells.

4. Selection of APT-Tan incubation system and temperature

4.1 APT-Tan incubation system

After incubating APT-Tan at a concentration of 200 nM with activated HSC-T6 in the buffer systems of DMEM, DMEM+10% FBS, BB and PBS for 1 hour, the cells were collected for flow cytometry detection. The results are shown in Figure 11. The fluorescence intensities of BB and DMEM+10% FBS are close, while those of DMEM and PBS are weaker, indicating that different incubation systems have a greater impact on the entry of APT-Tan into activated HSC-T6.

4.2 APT-Tan incubation temperature

APT-Tan at a concentration of 200 nM was incubated with activated HSC-T6 at 4°C and 37°C for 1 h, and the cells were collected for flow cytometry detection. The results are shown in Figure 12. Different incubation temperatures have a great influence on the entry of APT-Tan into activated HSC-T6, and low temperature conditions will reduce the transmembrane efficiency.

5. APT-Tan cell endocytosis inhibition experiment

1×10 ⁵ HSC-T6 cells were seeded into 24-well plates and transfected with 1μg pcDNA3.1-ALK5 for 24-36h. Endocytosis inhibitors dynasore (80uM), EIPA (50uM), filipinⅢ (2ug/ml), NaN ₃ (40uM), NH4Cl (50uM), Heparin (50ug/ml), wartmannin (5uM) and Chlorpromazine (30uM) were added for pretreatment for 30min, and then APT-Tan at a concentration of 200nM was added for incubation for 1h and the cells were collected for flow cytometry detection. The results are shown in Figure 13. The results show that dynasore, filipinⅢ and Heparin all have inhibitory effects, indicating that APT-Tan may enter the cells through receptor protein-mediated endocytosis and macropinocytosis, and receptor protein-mediated endocytosis plays a major role.

Example 4 Optimization of the stability and effectiveness of the nucleic acid aptamer of the present invention Since nucleic acid aptamers are sensitive to temperature, ions and pH values, these will affect the hydrophobic effect and hydrogen bond distortion in their structure, resulting in changes in the nucleic acid molecular skeleton and loss of binding ability. At the same time, the flexible nucleotide conformation will expose their single-stranded binding regions to nucleases and degrade. Therefore, the serum stability time of unmodified nucleic acid aptamers is very short, and after modification, its degradation time can be significantly extended. Currently, the commonly used modifications of nucleic acid aptamers include modifications of phosphodiester bonds, such as thiolation modifications, etc.; modifications of ribose in the nucleic acid sequence of the aptamer, such as replacement of 2'-H by F, OMe, etc.; modifications of amino, carboxyl, thiol, etc. to any base in the nucleic acid sequence of the aptamer; modifications such as polyethylene glycol (PEG) at the 5' end of the nucleotide sequence of the aptamer and modifications such as deoxyuracil (dU) at the 3' end.

Modification of ribose in the aptamer nucleic acid sequence, such as replacement of 2'-H with F, NH, OMe, etc.; modification of any base in the aptamer nucleic acid sequence, such as modification of amino, carboxyl, sulfhydryl, biotin, cholesterol, polyethylene glycol group, etc.; modification of deoxyuracil (dU), deoxythymine (dT) and deoxyhypoxanthine (dL) at the 3' end of the aptamer nucleic acid sequence (see Eckstein et al., International Publication PCT No 92/07065; Usman et al., International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711; Beigelman et al., International PCT Publication WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No.5,627.053; Woolf et al., International Publication WO 98/13526; J.Org.Chem.47(1982),3623-3628; Verheyden et al.(1971),supra; J.Org.Chem.40(1975),1659). Deoxyfluorination reaction of nucleic acid aptamers: (R)-N-Cbz-3-hydroxypyrrolidine (221 mg, 1.0 mmol, ee>99.9%) dissolved in dichloromethane (3.0 mL) was cooled to -78°C, and DBU (224 μL, 1.5 mmol) and XtalFluor-E (344 mg, 1.5 mmol) were added in sequence. After stirring for 30 minutes under nitrogen, the reaction mixture was allowed to warm to room temperature and stirred for 24 hours. The reaction mixture was quenched with 5% aqueous sodium bicarbonate solution and stirred for 15 minutes. The resulting mixture was extracted twice with dichloromethane, the organic groups were combined, dried over magnesium sulfate, and filtered through a filter pad to evaporate the solvent to give a crude product. The crude product was then purified by silica gel flash chromatography using hexanes/EtOAc (3/1) to give the title compound (192 mg, 86%) mixed with N-Cbz-2,5-dihydropyrrole (6.9:1 ratio respectively). The 3' end of the nucleic acid aptamer is covalently linked to a C7 indirect arm (-(CH2)7-), and then the end of the C7 indirect arm is covalently modified with an amino group to obtain an amino-modified aptamer; Polyethylene glycol (PEG) modification of the 5' end of the nucleotide sequence: (1) PEG5000, 0.4 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 0.1 M N-hydroxysuccinimide (NHS) are mixed in a molar ratio of 6:6:1 (50 μL each) and the mixture is stirred at 400 °C for 2 h. The mixture was shaken at 80 rpm for 25 min, (2) the aptamer with a volume ratio of 1:100 to PEG was added to 50 μL of 2-(N-morpholino)ethanesulfonic acid buffer (MES buffer) with a pH of 6, and then added to the mixed solution prepared in step 1, and shaken at a speed of 80 rpm for 5 h. After the reaction, gel electrophoresis was performed to obtain a 5'-end polyethylene glycol (PEG)-modified aptamer; 2'-H was replaced by OMe: 2'-O-methyl RNA was connected to the 5' end of the aptamer by a standard phosphoramidite chemical synthesis method, and purified by reverse phase high performance liquid chromatography; thiomonomers dATP, dTTP, dGTP and dNTP were subjected to PCR reaction to obtain a thiophosphate-modified aptamer; the nucleic acid molecule of the present invention also includes at least one LNA (locked nucleic acid) nucleotide, such as 2',4'-C methylene bicyclic nucleotide (see Wengel et al., International PCT Publications WO 00/66604 and WO 99/14226). The average fluorescence intensity of the modified aptamers was not reduced compared with APT-Tan and was generally distributed between 450-550.

Example 5

Specificity, concentration dependence, time dependence and functional detection of APT-Tan drug delivery

The 3' end of APT-Tan was linked to miR-23b-5p through C6 to synthesize APT-Tan-miR-23b-5p: GGTTTGCTGTATGGTGGGCGTTGAAAGAGGGGTGGACACGGTGG/C6/GGGUUCCUGGC AUGCUGAUUU

1. APT-Tan-miR-23b-5p was incubated with activated and non-activated HSC-T6, respectively, and its affinity was observed under a fluorescence microscope. The results are shown in FIG15 . APT-Tan can still enter the activated HSC-T6 to a large extent after carrying miR-23b-5p.

2. Functional detection of APT-Tan-miR-23b-5p

2.1 4×10 ⁵ HSC-T6 cells were seeded into 6-well plates and transfected with 3 μg PCDNA3.1-ALK5 for 24 h.

2.2 Add 200 nM APT-Tan-miR-23b-5p to the corresponding 6-well plate and incubate for one hour in the dark. After replacing the culture medium, continue culturing for 24 hours.

4.3 The cells were collected for Western-blot detection. The results are shown in Figure 16. APT-Tan-miR-23b-5p has the effect of targeted downregulation of fibrosis-related proteins Itga5, Tgfb2, and α-SMA.

Under the same configuration as the APT-Tan aptamer of the present application, the 3' end of the SEQ ID NO: 1-6 sequence is connected to miR-23b-5p through C6 to synthesize the -miR-23b-5p of the SEQ ID NO: 1-6 sequence, which also has the effect of targeted downregulation of fibrosis-related proteins Itga5, Tgfb2, and α-SMA.

Under the same configuration as the APT-Tan aptamer of the present application, the 3' end of the thiophosphate-modified APT-Tan nucleic acid aptamer sequence prepared in Example 2-1 was connected to miR-23b-5p through C6 to synthesize thiophosphate-modified APT-Tan-miR-23b-5p, which also has the effect of targeted downregulation of fibrosis-related proteins Itga5, Tgfb2, and α-SMA.

The present invention uses Cell-SELEX technology to first screen out an aptamer APT-Tan that can enter activated HSC-T6 cells with high affinity and high specificity. The present invention uses Discovery Studio software to simulate the spatial conformation of aptamer 2, and continuously optimizes and shortens aptamer 2 without changing the spatial structure to obtain the shortest aptamer APT-Tan, which lays a good experimental foundation for subsequent targeted drug delivery. At the same time, the present invention detects the concentration dependence, stability, specificity, incubation system and temperature, and membrane penetration mechanism of APT-Tan, and the results show that APT-Tan has good affinity for HSC-T6, but is not strong in binding to some other normal cells. The aptamer is basically saturated at a concentration of 200nM, and at the same time, its average fluorescence intensity reaches the maximum at 0.5h, indicating that it can quickly enter the cell and reach a saturated state. These results show that APT-Tan, as a new tool for targeted activation of HSC-T6 cells, can specifically enter activated HSC-T6 cells. The development of APT-Tan will provide another targeted and efficient transport tool for scientific research (targeted drug delivery to in vitro cultured cells), diagnosis and treatment of clinical diseases (carrying bioactive molecules such as microRNA, siRNA and drug delivery carriers).

Claims (2)

1. The application of nucleic acid aptamers in the preparation of drugs for treating or diagnosing liver fibrosis diseases. The nucleic acid aptamers are the nucleotide sequences of the DNA fragments shown in SEQ ID NO: 2. The nucleic acid aptamers can Specific entry into activated HSC cells. 2. The application according to claim 1, characterized in that the cells are mammalian cells.