RNA aptamer of targeting hepatocyte and nucleotide sequence thereof

A nucleotide sequence and aptamer technology, applied in the field of diagnosis and treatment of liver diseases, can solve problems such as lack of specific targeting of liver cells, and achieve the effect of simple method and low cost

Inactive Publication Date: 2011-10-26
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of specific vectors targeting hepatocytes

Method used

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  • RNA aptamer of targeting hepatocyte and nucleotide sequence thereof
  • RNA aptamer of targeting hepatocyte and nucleotide sequence thereof
  • RNA aptamer of targeting hepatocyte and nucleotide sequence thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: SELEX screening of H1 protein specifically binding to RNA aptamer

[0018] SELEX screening process such as figure 1 As shown, chemically synthesized initial oligonucleotide random library and primers, the sequence is as follows: 5'-TTAATACGACTCACTATAGTTGATTGCGTGTCAATCATGG-25N-GGTCATGTGTATGTTGGGGATTAGGACCTGATTGAGTTCAGCCCACATAC-3' (T7 promoter sequence is underlined, 25N represents 25 random nucleotides);

[0019] Primer 1: 5′-TTAATACGACTCACTATAGTTGATTGCGTGTCAATC-3′,

[0020] Primer 2: 5'-GTATGTGGGCTGAACTCAAT-3'. The single-stranded DNA library is amplified into double-stranded DNA, and the product is subjected to 2% agarose gel electrophoresis and then gel-cut to recover and purify; the recovered double-stranded DNA is used as a template to transcribe a single-stranded RNA random library in vitro, and the transcript is purified by PAGE . 68 μg RNA library was screened through nitrocellulose membrane to remove membrane-bound RNA molecules, then incubated wit...

Embodiment 2

[0021] Example 2: Secondary structure analysis of RNA aptamers

[0022] The library obtained in the 12th round of screening was cloned into the pMD19-T vector, transformed into Escherichia coli DH5α, and 48 clones were randomly selected and sequenced. Obtain the sequence information of the screened aptamers, predict the RNA secondary structure of all sequencing clones through the structure prediction software RNA Structure Program, and obtain an aptamer that can form a special stem-loop in the random sequence region (23-47nt) structure, such as figure 2 shown.

Embodiment 3

[0023] Example 3: Obtaining specific high-affinity H1 protein binding to suitable gametes

[0024] Take 1 μg of the RNA aptamers with the above secondary structures, digest them with calf intestinal alkaline phosphatase (CIP) at 37°C for 1 h, purify and recover the dephosphorylated RNA; label [γ- 32 P]ATP at the end of dephosphorylated RNA molecules. 10nmol of radioactively labeled RNA aptamers were incubated with different concentrations (10-200nM) of H1 protein at 37°C for 40min. The reaction solutions of each group were filtered through a nitrocellulose membrane, washed, dried, and measured by a liquid scintillation counter. The amount of radioactivity remaining on the filter membrane was measured twice in parallel on the same sample. The dissociation constant between each aptamer and H1 protein was calculated, and the aptamer with the highest affinity was obtained, which was named H1-A25. 32 P-labeled H1-A25 was incubated with H1 protein, irrelevant protein HBsAg, and ex...

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PUM

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Abstract

The invention discloses a RNA aptamer of a targeting hepatocyte and a nucleotide sequence thereof, and relates to the field of hepatic disease diagnosis and treatment. The RNA aptamer provides a novel specific and efficient molecule of a targeting hepatocyte for the field of hepatic disease diagnosis and treatment. In the invention, through using the new combinatorial chemical technology SELEX, and taking hepatic ASGPR (asialoglycoprotein receptor) large subunits as target proteins, an RNA aptamer which can specify the ASGPR is screened from a single-stranded RNA random library. The aptamer can be formed into a special stemloop structure in a random sequence region thereof, combines with big liver ASGP receptor subunit H1 protein with high specific affinity and can combine to liver cells in a targeted mode by high specific affinity. The RNA aptamer provides a new choice for developing a liver disease targeted diagnostic reagent and treatment drug.

Description

technical field [0001] The invention belongs to the field of diagnosis and treatment of liver diseases. The invention relates to an RNA aptamer targeting hepatic cells obtained by screening with SELEX technology, and the nucleotide sequence of the aptamer. Background technique [0002] Chronic liver disease is an important and intractable disease. Diseases such as viral hepatitis, liver cirrhosis, and liver cancer usually require long-term drug treatment, but most drugs do not specifically act on the liver. Combining drugs with liver-targeting carriers to selectively target specific liver cells, so that drugs can be delivered to the liver more efficiently and the therapeutic effect of drugs can be better exerted is a promising treatment plan. However, there is still a lack of specific vectors targeting hepatocytes. [0003] Hepatocyte-specific asialoglycoprotein receptor (ASGPR) is the most studied and most promising target among many targets of liver-targeted therapy. A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115
Inventor 杨东亮杨燕刘嘉
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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