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CN-121294448-B - Aptamer specifically binding to DR5, derivative and application thereof

CN121294448BCN 121294448 BCN121294448 BCN 121294448BCN-121294448-B

Abstract

The invention relates to the technical field of biomedicine, in particular to a nucleic acid aptamer specifically combined with DR5, a derivative and application thereof. The nucleic acid aptamer is Apt-DR5 single-stranded DNA, the nucleotide sequence of the nucleic acid aptamer is shown as SEQ ID NO. 1, the nucleic acid aptamer comprises a modification body, the modification body is obtained by modification on the basis of the DNA sequence, and the modification adopts at least one of phosphorylation, methylation, amination, sulfhydrylation, oxygen substitution by sulfur, oxygen substitution by selenium and isotopicization. Compared with the DR5 antibody, the nucleic acid aptamer combined with DR5 has small molecular weight, stability, easy preservation and marking, can be singly or combined for detecting tumor cells with high expression of DR5, such as ovarian cancer cells, and can inhibit proliferation of tumor cells with high expression of DR5, such as ovarian cancer cells.

Inventors

  • HU KONGWANG
  • WANG JING
  • FANG XIAONA
  • LI PING
  • ZHANG MEI
  • YE WEI
  • WANG MINGQING
  • ZHONG YAN

Assignees

  • 安徽安龙基因科技有限公司
  • 安徽医科大学第一附属医院

Dates

Publication Date
20260505
Application Date
20251208

Claims (7)

  1. 1. The nucleic acid aptamer specifically binding to DR5 is characterized in that the nucleic acid aptamer is Apt-DR5 single-stranded DNA, and the nucleotide sequence of the nucleic acid aptamer is shown as SEQ ID NO. 1: the sequence is as follows: 5’-CGAGCATCGTGGAGGTTGTCAATGGTGTCCAGGTAGGCGTAAGGGTTTTGAGTGTTTTCCACGATGCTGC-3’。
  2. 2. The aptamer of claim 1, wherein the aptamer is a modification of single-stranded DNA of Apt-DR5, said modification being by at least one of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, and isotopicization.
  3. 3. The derivative of a nucleic acid aptamer that specifically binds DR5 according to claim 2, wherein 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, digoxin, a nano luminescent material, and an enzyme label, and the nucleic acid aptamer is one of Apt-DR5 single-stranded DNA or a modified form.
  4. 4. The derivative of a nucleic acid aptamer that specifically binds to DR5 according to claim 2, wherein the derivative comprises a phosphorothioate backbone prepared by engineering a backbone of a nucleotide sequence of at least one of Apt-DR5 single-stranded DNA, a modified form, to specifically bind to DR 5.
  5. 5. The derivative of a nucleic acid aptamer that specifically binds DR5 of claim 2, wherein the derivative comprises a peptide nucleic acid prepared from at least one of Apt-DR5 single-stranded DNA, a modification engineered to specifically bind DR 5.
  6. 6. Use of a nucleic acid aptamer specifically binding to DR5 as defined in any one of claims 2-4 in the preparation of an anti-ovarian and/or liver cancer formulation.
  7. 7. Use of a nucleic acid aptamer specifically binding to DR5 as defined in any one of claims 2-4 for the preparation of an anti-ovarian and/or liver cancer drug or for the preparation of a diagnostic reagent for detecting DR5 expression.

Description

Aptamer specifically binding to DR5, derivative and application thereof Technical Field The invention relates to the technical field of biology and medicine, in particular to a nucleic acid aptamer specifically binding ovarian cancer and liver cancer cells and application thereof. Background Death receptor 5 (DR 5, TNFRSF 10B) is a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) and binds TNF-related apoptosis-inducing ligand (TRAIL), activating an exogenous apoptosis pathway, inducing apoptosis of tumor cells. DR5 is highly expressed in various tumor cells such as ovarian cancer, has low expression level in normal cells, has selective tumor killing potential, and is an anti-tumor drug target with great prospect. Currently, therapies directed against DR5 mainly include recombinant TRAIL proteins and anti-DR 5 agonistic antibodies. However, recombinant TRAIL protein has short half-life (0.56-1.02 hours) in vivo, limited ability of inducing DR5 trimerization, weak apoptosis signal, insufficient receptor aggregation and insufficient activation of antibody drugs, long antibody preparation period, large batch difference, poor tissue penetrability and high immunogenicity. Small molecule drugs face the problems of lack of action pockets, low specificity, short half-life and the like. The nucleic acid aptamer is an oligonucleotide sequence obtained by in vitro screening, and can be combined with a target with high affinity and specificity. Compared with the antibody, the antibody has the advantages of simple preparation, good chemical stability, easy modification, small molecular weight, strong tissue penetrating capacity, low immunogenicity and the like, and has wide application prospect in the fields of biological analysis, diagnosis and treatment. However, no published nucleic acid aptamer targeting DR5 exists at home and abroad at present. Disclosure of Invention In order to overcome the defects in the prior art, the invention aims to provide a nucleic acid aptamer which has high specificity, stable chemical property and easy preservation and marking and can be combined with DR5 protein and a derivative thereof, and also provides a screening method and application of the nucleic acid aptamer. In order to solve the technical problems, an in vitro index enriched ligand system evolution (SELEX) technology is used for screening to obtain the nucleic acid aptamer specifically binding to DR 5. Specifically, the present inventors designed and synthesized a random single-stranded DNA library and corresponding primers for screening nucleic acid aptamers capable of binding DR5 with high specificity, stable chemical properties, easy preservation and labeling, thereby screening nucleic acid aptamers specifically binding DR5 and detecting affinity of the screened nucleic acid aptamers with DR5 protein. On this basis, the present inventors have completed the present invention. In a first aspect, the invention provides a nucleic acid aptamer that specifically binds DR5, said nucleic acid aptamer sequence comprising or consisting of: (1) The nucleotide sequence of the Apt-DR5 single-stranded DNA is 5'-CGAGCATCGTGGAGGTTGTCAATGGTGTCCAGGTAGGCGTAAGGGTTTTGAGTGTTTTCCACGATGCTGC-3'. Or (b) (2) A nucleotide sequence having at least 60% homologous DNA to the Apt-DR5 single-stranded DNA sequence and specifically binds DR 5. (3) An RNA sequence transcribed from the nucleotide sequence of (1) or (2) and specifically binding to DR 5. In another aspect, the invention also provides derivatives of the nucleic acid aptamer. It will be appreciated by those skilled in the art that, as an improvement to the above-described technical scheme, a fluorescent substance, a radioactive substance, a therapeutic substance, biotin, digoxin, a nano-luminescent material, a small peptide, an siRNA or an enzyme label, etc. may be attached to the nucleotide sequence of the above-described nucleic acid aptamer, provided that the nucleic acid aptamer sequence thus modified has desirable properties, for example, may have an affinity for binding DR5 equal to or higher than that of the parent nucleic acid aptamer sequence before modification, or has higher stability although the affinity is not significantly improved. Furthermore, as a general technical idea, the present invention provides a nucleic acid aptamer derivative which is a phosphorothioate backbone derived from the backbone of the nucleotide sequence of the nucleic acid aptamer in all the above-mentioned technical schemes, or a corresponding peptide nucleic acid modified from the nucleic acid aptamer in all the above-mentioned technical schemes. Provided that the derivatives all have substantially the same or similar molecular structure, physicochemical properties and functions as the original aptamer and all bind to DR 5. A biologic that specifically binds DR5, said biologic comprising at least any one of Apt-DR5 single-stranded DNA, homologous DNA, transcribed RNA, a modification, a der