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Nucleic acid aptamers targeting and antagonizing HMGB1 molecules

A nucleic acid aptamer and antagonism technology, which is applied in the field of HMGB1 nucleic acid aptamer antagonists, targeting and antagonizing HMGB1 molecular nucleic acid aptamers, can solve the problems of low immunogenicity and achieve low immunogenicity, small molecular weight, good affinity effect

Active Publication Date: 2022-07-29
中国科学院基础医学与肿瘤研究所(筹)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aptamers have the ability to specifically bind to targets similar to antibodies, but exhibit very low immunogenicity compared to antibodies

Method used

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  • Nucleic acid aptamers targeting and antagonizing HMGB1 molecules
  • Nucleic acid aptamers targeting and antagonizing HMGB1 molecules
  • Nucleic acid aptamers targeting and antagonizing HMGB1 molecules

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Preparation of nucleic acid aptamers

[0097] 1. Construction of screening library

[0098] The ssDNA library used in this study has a full length of 70 nt, consisting of 20 nt primer fragments fixed at both ends and a random fragment of 30 nt in the middle; the 5'-modified FITC fluorophore of the forward primer is fixed to the 5' end of the initial library The sequence is the same; the 5' end of the reverse primer is labeled with Biotin, and the sequence is reverse complementary to the fixed sequence at the 3' end of the original library;

[0099] The sequence of the screening library targeting HMGB1 is:

[0100] SEQ ID NO. 3: AAGGAGCAGCGTGGAGGATA (N) 30 TTAGGGTGTGTCGTCGTGGT.

[0101] The primers used to construct the library are:

[0102] Forward primer: SEQ ID NO. 4: AAGGAGCAGCGTGGAGGATA;

[0103] Reverse primer: SEQ ID NO. 5: ACCACGACGACACACCCTAA.

[0104] 2. Conjugation of protein and magnetic beads

[0105] Take the carboxyl magnetic beads (MB), wash them w...

Embodiment 2

[0115] Validation of nucleic acid aptamer binding to HMGB1 protein

[0116] The protein of SKOV3 cells was extracted and divided into 6 equal parts. The biotin-labeled control sequence and candidate chain were added to the protein, mixed well, incubated at 4°C for 45 min, and then 50 μl of streptavidin-modified magnetic beads were added to it. , after mixing, continue to incubate at 4 °C for 1 h, after the incubation, remove the supernatant for washing 3 times, then add 2× protein loading buffer to the tube, mix well, denature at 100 °C for 10 min, and separate the protein , and finally take the supernatant and load the sample, and use SDS-PAGE for electrophoresis. After the electrophoresis is completed, the gel is stained with Coomassie brilliant blue to identify the different bands.

[0117] Test results such as image 3 As shown, according to the test results, it can be seen that the nucleic acid aptamer ZH-1 can specifically recognize and bind the HMGB1 protein in the cel...

Embodiment 3

[0119] Optimization of nucleic acid aptamer ZH-1 sequence

[0120] Through the sequence truncation strategy, ZH-1 was truncated into 6 optimized sequences, and the affinity of the 6 sequences was determined by WesternBlot, and the internal reference was tublin; the detection results are as follows Figure 4 shown, by Figure 4 It can be seen that ZH-1a (SEQ ID NO. 2) has the strongest affinity, so ZH-1a is determined to be the optimal nucleic acid aptamer for subsequent experiments.

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Abstract

The invention discloses a nucleic acid aptamer for targeting and antagonizing HMGB1 molecules, and belongs to the technical field of biology. The aptamer can be efficiently combined with HMGB1, has the advantages of good affinity, low immunogenicity, small molecular weight, low synthesis cost, easiness in storage, convenience in modification and substitution and the like, and has important application value in the fields of nucleic acid antagonists of HMGB1, detection and the like. Besides, the aptamer can be used for preparing medicines for treating inflammations such as acute pneumonia and the like, and experiments find that the aptamer can effectively reduce significant down-regulation of IL-6 and TNF-alpha in blood of mice with acute inflammations, which indicates that the aptamer ZH-1a can inhibit release of inflammation-related factors in inflammatory diseases.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to the development and application of nucleic acid aptamers for targeting and antagonizing HMGB1 molecules and HMGB1 nucleic acid aptamer antagonists. Background technique [0002] High mobility group box-I protein (HMGB1) is a ubiquitous and highly conserved chromatin-binding protein that is widely present in all mammalian nucleated cells and platelets. HMGB1 protein is a multifunctional protein molecule, which has a variety of different biological functions in different locations of cells and can regulate various signaling pathways. The regulation and detection of HMGB1 has shown an important role in various diseases, but the development of HMGB1 antagonists is very slow, and the protein antibody of HMGB1 has been mainly developed. [0003] In recent years, nucleic acid aptamers (aptamers) called chemical antibodies have shown broad application prospects in the field of disea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68A61K31/711A61K47/26A61K47/54A61K49/00A61K51/04A61P11/00A61P29/00A61P31/14A61P35/00
CPCC12N15/115G01N33/68A61K31/711A61P29/00A61P11/00A61P31/14A61P35/00A61K47/549A61K47/26A61K49/0052A61K51/0491C12N2310/16C12N2310/35C12N2310/3521C12N2310/3533C12N2310/315C12N2310/3181
Inventor 谭蔚泓张慧甘绍举
Owner 中国科学院基础医学与肿瘤研究所(筹)
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