Nucleic acid aptamers, derivatives and uses thereof specifically binding dr5

Abstract

This invention relates to the field of biomedical technology, specifically to a nucleic acid aptamer that specifically binds to DR5, its derivatives, and their applications. The nucleic acid aptamer is an Apt-DR5 single-stranded DNA, the nucleotide sequence of which is shown in SEQ ID NO:1. The nucleic acid aptamer includes a modifier, which is obtained by modifying the DNA sequence. The modification employs at least one of phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium, and isotopization. Compared with DR5 antibodies, the nucleic acid aptamer binding to DR5 of this invention has a smaller molecular weight, is more stable, easier to store and label, and can be used alone or in combination for the detection of tumor cells that highly express DR5, such as ovarian cancer cells; and for inhibiting the proliferation of tumor cells that highly express DR5, such as ovarian cancer cells.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medical technology, and in particular to nucleic acid aptamers that specifically bind to ovarian cancer and liver cancer cells and their applications. Background Technology

[0002] Death receptor 5 (DR5, TNFRSF10B) is a member of the tumor necrosis factor receptor superfamily (TNFRSF). It can bind to TNF-associated apoptosis-inducing ligand (TRAIL), activate the extrinsic apoptosis pathway, and induce programmed cell death in tumor cells. DR5 is highly expressed in various tumor cells, including ovarian cancer, but its expression level is low in normal cells. It has the potential for selective tumor killing and is a promising target for anti-tumor drugs.

[0003] Currently, treatments targeting DR5 mainly include recombinant TRAIL protein and anti-DR5 agonist antibodies. However, recombinant TRAIL protein has a short half-life in vivo (0.56-1.02 hours), limited ability to induce DR5 trimerization, and weak apoptosis signals. Antibody drugs suffer from insufficient receptor aggregation and inadequate activation, and also have long preparation cycles, large batch-to-batch variations, poor tissue penetration, and high immunogenicity. Small molecule drugs face challenges such as a lack of effective pockets, low specificity, and short half-life.

[0004] Nucleic acid aptamers are oligonucleotide sequences obtained through in vitro screening that can bind to targets with high affinity and specificity. Compared with antibodies, they have advantages such as simple preparation, good chemical stability, ease of modification, small molecular weight, strong tissue penetration, and low immunogenicity, and have broad application prospects in the fields of bioanalysis, diagnostics, and therapy. However, there are currently no publicly published nucleic acid aptamers targeting DR5, either domestically or internationally. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention aims to provide a nucleic acid aptamer and its derivatives that are highly specific, chemically stable, easy to preserve and label, and capable of binding to the DR5 protein. The invention also provides a screening method and application for the nucleic acid aptamer.

[0006] To address the aforementioned technical problems, the in vitro exponential enrichment of ligands using systematic evolutionary phylogenetic analysis (SELEX) was employed to screen for nucleic acid aptamers that specifically bind to DR5. Specifically, the inventors designed and synthesized a random single-stranded DNA library and corresponding primers to screen for nucleic acid aptamers that are highly specific, chemically stable, easy to store and label, and capable of binding to DR5. The specific binding affinity of these aptamers to the DR5 protein was then tested. Based on this, the inventors completed this invention.

[0007] In a first aspect, the present invention provides a nucleic acid aptamer that specifically binds to DR5, wherein the nucleic acid aptamer sequence comprises or consists of the following:

[0008] (1) Apt-DR5 single-stranded DNA, with the nucleotide sequence: 5'-CGAGCATCGTGGAGGTTGTCAATGGTGTCCAGGTAGGCGTAAGGGTTTTGAGTGTTTTCCACGATGCTGC-3'. Or

[0009] (2) A nucleotide sequence that has at least 60% homology with the Apt-DR5 single-stranded DNA sequence and specifically binds to DR5.

[0010] (3) An RNA sequence transcribed from the nucleotide sequence of (1) or (2) that specifically binds to DR5.

[0011] On the other hand, the present invention also provides derivatives of nucleic acid aptamers. Those skilled in the art should understand that, as an improvement to the above technical solution, fluorescent substances, radioactive substances, therapeutic substances, biotin, digoxigenin, luminescent nanomaterials, small peptides, siRNA, or enzyme labeling can be attached to the nucleotide sequence of the above-mentioned nucleic acid aptamers, provided that the nucleic acid aptamer sequence obtained after such modification has the desired properties. For example, it can have an affinity for DR5 equal to or higher than that of the parent nucleic acid aptamer sequence before modification, or although the affinity is not significantly improved, it has higher stability.

[0012] Furthermore, as a general technical concept, this invention also provides nucleic acid aptamer derivatives, which are phosphate thioester backbones derived from the nucleotide sequences of the nucleic acid aptamers described in all the foregoing technical solutions, or corresponding peptide nucleic acids modified from the nucleic acid aptamers described in all the foregoing technical solutions. The condition is that all derivatives have essentially the same or similar molecular structure, physicochemical properties, and functions as the original nucleic acid aptamers, and all bind to DR5.

[0013] A biological product that specifically binds to DR5, wherein the biological product comprises at least one of Apt-DR5 single-stranded DNA, homologous DNA, transcribed RNA, modified form, or derivative.

[0014] As a further improvement to the above scheme, the biological products include: reagents, reagent kits, and chips.

[0015] As a further improvement to the above solution, the biological product has at least one of the following uses:

[0016] Used for the preparation of anti-tumor drugs or for the preparation of diagnostic reagents for detecting DR5 expression.

[0017] The beneficial effects of this invention are: compared with DR5 antibodies, the nucleic acid aptamers of this invention have smaller molecular weights, are more stable, easier to preserve and label, and can be used alone or in combination for the detection of tumor cells that highly express DR5, such as ovarian cancer cells; and to inhibit the proliferation of tumor cells that highly express DR5, such as ovarian cancer cells. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the screening method for nucleic acid aptamers according to the present invention;

[0019] Figure 2 These are the secondary library affinity test results obtained through screening in this invention;

[0020] Figure 3 The results of SPR detection of the affinity between Apt-DR5 and DR5 protein in this invention;

[0021] Figure 4 The inhibitory effect of the nucleic acid aptamer Apt-DR5 obtained in this invention on the ovarian cancer cell line A2780 was detected by CCK-8 assay.

[0022] Figure 5 The inhibitory effect of the nucleic acid aptamer Apt-DR5 screened in this invention on the cisplatin-resistant ovarian cancer cell line A2780-DDP (A2780 cisplatin-resistant strain) was detected by CCK-8 assay.

[0023] Figure 6 The inhibitory effect of the nucleic acid aptamer Apt-DR5 obtained in this invention on the Li-7 liver cancer cell line was detected by CCK-8 assay. Detailed Implementation

[0024] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0025] Example 1: Screening of ssDNA aptamers that specifically bind to DR5 protein

[0026] Synthesize the random single-stranded DNA library and primers shown in the following sequences.

[0027] Please see Figures 1-6 As shown, the random single-stranded DNA library information is: 5'-cgagcaTCGTGGAGGTTGT(36N)TCCACGATGCTGCTTCAcc-3', where "36N" represents a sequence composed of 36 arbitrary nucleotide bases. This library was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0028] Primer information is shown in Table 1, and the primers were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0029] Table 1 shows the primer sequence information.

[0030]

[0031] In this primer name, FP represents the forward primer, RP represents the reverse primer, 19A in the sequence represents the polyA tail composed of 19 adenosine nucleotides (A), and "Spacer18" represents the 18-atom hexaethylene glycol intermediate arm.

[0032] Primers were prepared into 100 μM stock solutions using DPBS buffer (CaCl2: 0.1 g / L, KCl: 0.2 g / L, KH2PO4: 0.2 g / L, MgCl2·6H2O: 0.1 g / L, NaCl: 8 g / L, Na2HPO4: 1.15 g / L; pH 7.4, 25℃) and stored at -20℃ for later use.

[0033] 2. Nucleic acid aptamer screening using DR5 protein as a target.

[0034] 2.1 Library renaturation: Dilute the library to the working concentration with DPBS buffer and aliquot into PCR tubes for renaturation. The process is as follows: Set the PCR instrument program to 95°C for 10 minutes to unfold the strands, then incubate at 4°C for 5 minutes and store at room temperature for later use.

[0035] 2.2 Immobilization of DR5 protein with carboxylic magnetic beads: Take 50 μL of carboxylic magnetic beads (Invitrogen, Dynabeads™ MyOne™ Carboxylic Acid, #65012), wash four times with 200 μL of ultrapure water, magnetize the beads, and discard the supernatant. Take equal volumes of prepared NHS (N-hydroxysuccinimide; 0.1M aqueous solution) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution), add to the magnetic beads, and incubate at 25°C for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads. Wash the magnetic beads twice with pure water and set aside. Take 20 μL of DR5 protein (concentration of 0.5 mg / ml), add 180 μL of pH 4.0 NaAC solution, mix well, and add to the magnetic beads from the previous step. Incubate on a shaker at room temperature for 40 minutes. Magnetize the beads, and discard the supernatant. Add 200 μL of 1M ethanolamine (pH 8.5) to the magnetic beads from the previous step. Incubate on a shaker at room temperature for 10 min. Discard the supernatant and wash four times with 200 μL of LPBS. Label the beads as MB-DR5 for later use. Use the same method to conjugate BSA protein to the magnetic beads as reverse screening beads.

[0036] 2.3 Screening: The library treated in 2.1 was added to 50 μL of MB-BSA magnetic beads and incubated at room temperature for 30 minutes. The mixture was placed on a magnetic rack, and the supernatant was collected and labeled "pool-". The reverse-screened library was added to 50 μL of MB-DR5 magnetic beads for positive screening. The mixture was placed on a magnetic rack, the supernatant was discarded, and the magnetic beads were retained. The magnetic beads were washed four times with 200 μL of LPBS. After washing, the magnetic beads were added to 200 μL of ultrapure water and boiled for 10 minutes. The supernatant was collected and labeled "elution". The elution was used as a template for the next step of PCR amplification.

[0037] 2.4 Amplification and Single-Strand Preparation: Using the nucleic acid molecules obtained above as templates, amplification was performed using emulsion PCR. The method is as follows: All template was added to 2 mL of PCR mix and mixed well. Four volumes of ePCR microdroplet generating oil were added, and the mixture was vortexed to prepare an emulsion. The emulsion was divided into 100 μL portions and added to PCR tubes. The amplification conditions were as follows: 95℃ pre-denaturation for 2 minutes, 95℃ denaturation for 60 seconds, 60℃ annealing for 60 seconds, and 72℃ extension for 60 seconds, for a total of 35 cycles. The ePCR microdroplet generating oil was purchased from Anhui Aptamy Biotechnology Co., Ltd. (Catalog No.: EPO100). The formulation of the PCR mix is ​​shown in Table 2.

[0038] Table 2 shows the PCRmix formulations.

[0039]

[0040] Purification of amplification products with n-butanol: Collect all ePCR products in 15ml conical centrifuge tubes, add 2 volumes of n-butanol, and vortex to mix thoroughly; centrifuge at 9000 rpm for 10 minutes using a benchtop centrifuge; discard the supernatant to obtain concentrated PCR amplification products, add TBE / urea denaturing buffer at a 1:1 volume ratio, and boil for denaturation for 10 minutes. Perform urea-denaturing polyacrylamide gel electrophoresis on all samples at 400V until bromophenol blue reaches the bottom of the gel, separating the elongated antisense strand from the FAM-labeled strand. Gel extraction and recovery of FAM-labeled strands: Remove the gel and place it on a plastic membrane. Detect the desired FAM-labeled ssDNA at Ex: 495 nm, Em: 517 nm. Use a clean blade to cut the target band directly. Transfer the gel strip to a 1.5 mL EP tube and break it up. Add 1 mL ddH2O and boil for 10 minutes to transfer the ssDNA from the gel to a solution. Centrifuge to remove gel fragments, retaining the supernatant. Add another 1 mL ddH2O to the gel fragments and boil for 10 minutes. Centrifuge again to recover the supernatant. Combine the two supernatants and transfer them to a 15 mL centrifuge tube. Add 11.5 mL n-butanol and mix by inversion. Centrifuge at 9000 rpm for 5 minutes. Dialyze the obtained DNA single strands overnight using a 3.5 kDa dialysis bag. This can then be used as the library for the next round of screening.

[0041] 2.5 The screening was repeated 7 times, with each operation using the secondary library obtained from the previous operation as the starting nucleic acid library. Figure 1 The obtained enriched library was analyzed for affinity for the target protein DR5 using SPR assay, and the results are as follows: Figure 2 As shown, the affinity between the library and the target gradually increased as the screening process progressed. The library was then sent for high-throughput sequencing. After analyzing the sequencing results, several sequences were selected and synthesized by Shanghai Sangon Biotech. Affinity was then tested, and in subsequent tests, one sequence was identified as having a very strong binding ability and named Apt-DR5.

[0042] Example 2: Surface plasmon resonance (SPR) detection of the affinity between Apt-DR5 and DR5 protein

[0043] 1. Dilute the synthesized nucleic acid aptamer Apt-DR5 with DPBS buffer to: 6.25, 12.5, 25, 50, 100, 200 nM;

[0044] 2. DR5 protein conjugation to the carboxyl chip surface: Equal volumes of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution) and NHS (N-hydroxysuccinimide; 0.1M aqueous solution) were mixed and injected into the chip to activate it. The DR5 protein was diluted with 10mM sodium acetate at pH 4.0 to a final concentration of 50 μg / mL and then injected in a volume of 50 μL. The DR5 protein conjugation amount was 5000 Ru. After injection, ethanolamine was injected to block the chip at a flow rate of 5 μL / min in a volume of 50 μL.

[0045] 3. Detection: Using a surface plasmon resonance spectrometer (GE Healthcare, model: Biacore8K), set the kinetic detection parameters as follows: injection 30 μL / min * 2 min, dissociation 30 μL / min * 3 min, regeneration 1M NaCl 30 μL / min * 0.5 min, and then inject the diluted nucleic acid aptamers of each concentration sequentially.

[0046] Affinity test data can be found Figure 3 These data indicate that Apt-DR5 has a strong binding to the DR5 protein, as detected by the SPR instrument, with a KD value of 47.7 nM.

[0047] Example 3: CCK-8 assay to detect the inhibitory effect of aptamers on the proliferation of ovarian cancer cell lines A2780 and A2780-DDP (cisplatin-resistant ovarian cancer cell line).

[0048] Seed cells in 96-well plates, with 1,000 cells per well. Add 5 auxiliary wells per group. Add PBS to a ring of wells around the perimeter of the 96-well plate to prevent evaporation. Incubate for 24 hours before use.

[0049] The nucleic acid aptamer Apt-DR5 was diluted with DPBS containing a final concentration of 2 mM magnesium chloride to prepare solutions with concentrations of 0, 100, 1000, and 2000 nM, respectively. The solutions were added to 96-well plates, and the aptamers and cells were incubated at 37°C for 1 hour. The supernatant was discarded, and the cells were cultured in 10% ordinary medium containing fetal bovine serum for about 48 hours.

[0050] Cell proliferation was assessed using the CCK-8 (CellCounting Kit-8) assay. Following the kit instructions, CCK-8 was added to serum-free DMEM at a 9:1 ratio. 120 μL of the prepared CCK-8 solution was added to each well of the control and experimental groups. After incubation at 37°C in the dark for 2 hours, the 96-well plates were placed on a microplate reader, and the absorbance at 450 nm was measured. Data were collected, analyzed, and plotted.

[0051] The CCK-8 results show that, Figure 4 and Figure 5 As shown, the proliferation ability of ovarian cancer cell line A2780 decreased with increasing Apt-DR5 concentration, indicating that Apt-DR5 can inhibit the proliferation of ovarian cancer cell line A2780. After different concentrations of Apt-DR5 were bound to A2780-DDP (cisplatin-resistant ovarian cancer cell line), 2000 nM of Apt-DR5 significantly inhibited the cell proliferation rate.

[0052] Example 4: CCK-8 assay to detect the inhibitory effect of aptamers on the proliferation of hepatocellular carcinoma cell line Li-7.

[0053] 1. Seed cells in 1.96-well plates, with 1,000 cells per well. Add 5 accessory wells per group. Place PBS around the outer edge of the 96-well plate to prevent evaporation. Incubate for 24 hours before use.

[0054] 2. Dilute the nucleic acid aptamer Apt-DR5 with DPBS containing a final concentration of 2mM magnesium chloride to prepare solutions with concentrations of 0, 100, 1000, and 2000 nM, respectively; add the solutions to the wells, incubate for about 72 hours, and then remove the supernatant.

[0055] 3. CCK-8 (CellCounting Kit-8) assay for cell proliferation. Following the kit instructions, CCK-8 was added to serum-free DMEM at a 9:1 ratio. 120 μL of the prepared CCK-8 solution was added to each well of the control and experimental groups. After incubation at 37°C in the dark for 2 hours, the 96-well plate was placed on a microplate reader, and the absorbance at 450 nm was measured. Data were collected, analyzed, and plotted.

[0056] The CCK-8 results show that, Figure 6As shown, the proliferation ability of cancer cells decreased with increasing Apt-DR5 concentration, indicating that Apt-DR5 can inhibit the proliferation of the liver cancer cell line Li-7.

[0057] The nucleic acid aptamers, their derivatives, and their derivatives provided by this invention bind to DR5 with high specificity, and have small molecular weight, stable chemical properties, and are easy to store and label. They can be used alone or in combination for the detection of ovarian cancer cells and liver cancer cells, as well as for inhibiting the proliferation of ovarian cancer cells or liver cancer cells.

[0058] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A nucleic acid aptamer that specifically binds to DR5, characterized in that, The nucleic acid aptamer is Apt-DR5 single-stranded DNA, and its nucleotide sequence is shown in SEQ ID NO: 1: The sequence is: 5'-CGAGCATCGTGGAGGTTGTCAATGGTGTCCAGGTAGGCGTAAGGGTTTTGAGTGTTTTCCACGATGCTGC-3'.

2. The nucleic acid aptamer that specifically binds to DR5 as described in claim 1, characterized in that, The modified form is obtained by modifying Apt-DR5 single-stranded DNA, wherein the modification is at least one of phosphorylation, methylation, aminoation, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium, and isotopization.

3. The derivative of the nucleic acid aptamer that specifically binds to DR5 as described in claim 2, characterized in that, The derivative comprises a nucleic acid aptamer and a substance attached to the nucleic acid aptamer, wherein the substance is at least one of a fluorescent label, a radioactive substance, a therapeutic substance, biotin, digoxigenin, a nanoluminescent material, and an enzyme label, and the nucleic acid aptamer is one of Apt-DR5 single-stranded DNA or a modified version.

4. The derivative of the nucleic acid aptamer that specifically binds to DR5 as described in claim 2, characterized in that, The derivative comprises a thiophosphate backbone, which is prepared by modifying the backbone of at least one of the nucleotide sequences of Apt-DR5 single-stranded DNA and a modifier to specifically bind to DR5.

5. The derivative of the nucleic acid aptamer that specifically binds to DR5 as described in claim 2, characterized in that, The derivative comprises a peptide nucleic acid, which is prepared by modifying at least one of Apt-DR5 single-stranded DNA and a modifier to specifically bind to DR5.

6. The use of a nucleic acid aptamer that specifically binds to DR5 as described in any one of claims 2-4 in the preparation of an anti-ovarian cancer and / or liver cancer agent.

7. The use of a nucleic acid aptamer that specifically binds to DR5 as described in any one of claims 2-4 in the preparation of drugs for treating ovarian cancer and / or liver cancer or in the preparation of diagnostic reagents for detecting DR5 expression.

Images