CN102226185A - RNA aptamer of targeting hepatocyte and nucleotide sequence thereof - Google Patents
RNA aptamer of targeting hepatocyte and nucleotide sequence thereof Download PDFInfo
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- CN102226185A CN102226185A CN 201110117632 CN201110117632A CN102226185A CN 102226185 A CN102226185 A CN 102226185A CN 201110117632 CN201110117632 CN 201110117632 CN 201110117632 A CN201110117632 A CN 201110117632A CN 102226185 A CN102226185 A CN 102226185A
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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
The invention belongs to the Clinics and Practices field of hepatic diseases.The present invention relates to utilize the adaptive son of the hepatocellular RNA of a kind of target of SELEX technology screening acquisition, and the nucleotide sequence of this adaptive son.
Background technology
Chronic hepatic diseases is a kind of important and disease of refractory.Usually need the long-term prescription treatment for diseases such as viral hepatitis, liver cirrhosis and liver cancer, but most drug and nonspecific action are in liver.Medicine is combined with the liver target carrier, make its selectivity be targeted to specific liver cell, thereby make medicine be delivered to liver more efficiently, the therapeutic action of bringing into play medicine better is the treatment plan that has prospect.But, still lack the hepatocellular carrier of specific target at present.
(asialoglycoprotein receptor is that liver target is treated the maximum and most promising target spot of research in numerous action target spots ASGPR) to liver cell specificity asialoglycoprotein receptor.ASGPR is the endocytosis acceptor of the abundant a kind of allos oligopolymer of quantity, mainly is present in the surface of cell membrane of liver parenchyma towards sinusoid one side, is called glycogen aggegation albumen or hepatic lectin again.Aspects such as ASGPR sends at gene target, the research of targeted drug, clinical detection possess very high using value.
SELEX technology (systematic evolution of ligands by exponential enrichment) be the early 1990s-a kind of new combinatorial chemistry technique that grows up.It utilizes jumbo random oligonucleotide storehouse and target molecule to interact, therefrom filter out oligonucleotide with the target molecule specific combination, and in conjunction with PCR amplification in vitro technology, make it obtain exponential enrichment, through multi-turns screen, finally obtain the oligonucleotide aptamer of high-affinity, high specific.With respect to traditional protein antibody molecule, the oligonucleotide aptamer molecule that obtains through SELEX screening is littler, and immunogenicity is low, can faster infiltration cell, and be the novel agent of the prevention, diagnosis and the treatment disease that have potentiality.
Summary of the invention
The purpose of this invention is to provide the adaptive son of a kind of RNA, realize liver cell targetedly by specificity in conjunction with the liver asialoglycoprotein receptor, can be used for the Clinics and Practices of hepatic diseases.
The present invention is achieved through the following technical solutions:
External synthetic single-stranded DNA banks that contains 25 stochastic sequences that length is 115 Nucleotide constructs the adaptive sub-random library of single stranded RNA by in-vitro transcription; Adopt the method for affinity column chromatography, purifying liver ASGPR from human liver tissue as target protein, adopts the SELEX technology screening to have the adaptive son of ASGPR specific RNA of high-affinity with its big subunit H1; Secondary structure by the adaptive son of structure prediction software RNA Structure Program analyses and prediction; In conjunction with determination experiment, the adaptive son of gel retardation assasy evaluation and screening specificity and avidity, obtained an adaptive son with H1 protein affinity height, high specificity, called after H1-A25 by film to target protein; The RNA sequence of the adaptive son of this RNA is: 5 '-GUUGAUUGCGUGUCAAUCAUGGCGUAGUAAAAGACAAGUAGCUACGAGGUCAUGUG UAUGUUGGGGAUUAGGACCUGAUUGAGUUCAGCCCACAUAC-3 ' can form loop-stem structure.This adaptive son can be bonded to liver cell in conjunction with liver asialoglycoprotein receptor big subunit H1 albumen or energy specificity high-affinity target by the specificity high-affinity.The fluorine for preparing anti-RNA enzyme liberating is modified H1-A25, it is carried out the green fluorescence mark, identify that it combines and endocytosis with hepatocellular specificity, found that this adaptive son can be bonded to liver cell and by cell endocytic by ASGPR is selectively targeted, and the non-liver cell of debond.
Advantage of the present invention is:
1. obtained can target to be bonded to liver cell and by the adaptive son of endocytosis, this adaptive son is artificial preparation in a large number, method is simple, and is with low cost.
2. lay a good foundation based on the diagnosis for liver disease and the medicine of the adaptive son of RNA for further screening.
Description of drawings
Fig. 1: be the schema of SELEX screening H1 protein-specific in conjunction with the adaptive son of RNA.
Fig. 2: the secondary structure analysis figure that is the adaptive son of RNA of screening acquisition.Be the stochastic sequence zone of adaptive son in the red square frame of Fig. 2 wherein, this zone has formed a special loop-stem structure.
Fig. 3: illustrated adaptive sub-H1-A25 can the specificity high-affinity in conjunction with the proteic figure as a result of the big subunit H1 of ASGPR.
Wherein Fig. 3 A partly is adaptive sub-H1-A25 and the proteic binding curve of H1, has shown bonded high-affinity between the two; B and C two portions are respectively adaptive sub-H1-A25 and combine experiment and gel retardation assasy with the protein bound film of H1, have shown bonded high specific between the two.
Fig. 4: illustrated that adaptive sub-H1-A25 can be bonded to liver cell and by the figure as a result of cell endocytic by ASGPR is selectively targeted.Wherein Fig. 4 A, B and C three parts are respectively the experiment that combines that the adaptive sub-H1-A25 of fluorescent mark and HepG2, Huh7 and HeLa cell carry out, the result show H1-A25 can specificity in conjunction with liver cell; D and E partly are respectively with ASGPR antibody and excessive unmarked adaptive son and block combining between adaptive son and the HepG2 cell, and the result shows that combining by ASGPR between H1-A25 and the liver cell mediates; F partly tests the endocytosis of H1-A25 for the HepG2 cell of living, and the result has shown that H1-A25 can be by the liver cell endocytosis.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and embodiment, but be not restriction the present invention.
Embodiment 1:H1 protein-specific is in conjunction with the SELEX screening of the adaptive son of RNA
The SELEX screening process as shown in Figure 1, initial oligonucleotide random library of chemosynthesis and primer, sequence is as follows: 5 '-TTAATACGACTCACTATAGTTGATTGCGTGTCAATCATGG-25N-GGTCATGTGTA TGTTGGGGATTAGGACCTGATTGAGTTCAGCCCACATAC-3 ' (the T7 promoter sequence is indicated that by underscore 25N represents 25 random nucleotides); Use
Primer 1:5 '-TTAATACGACTCACTATAGTTGATTGCGTGTCAATC-3 ',
Primer 2: 5 '-GTATGTGGGCTGAACTCAAT-3 '.The single-stranded DNA banks amplification is double-stranded DNA, and product is through 2% agarose gel electrophoresis and cut glue recovery purifying; Double-stranded DNA with recovery is a template, and in-vitro transcription goes out the single stranded RNA random library, and transcription product is through the PAGE purifying.Remove and membrane-bound RNA molecule through anti-sieve of nitrocellulose filter in 68 μ gRNA libraries, hatches 40min with 2 μ gH137 ℃ then, and reaction solution filters through nitrocellulose filter, the washing filter membrane; Then filter membrane is shredded, (1mmol/L EDTA boils 5min in 0.2%SDS), and is centrifugal, gets supernatant for 7mol/L urea, 0.5mol/L ammonium acetate, dehydrated alcohol precipitated rna, and being dissolved in again in the 20 μ l DEPCR water to place elution buffer; With RNA is template RT-PCR amplifying doulbe-chain DNA, and in-vitro transcription goes out the RNA library and is used for the next round screening; Every RT-PCR that takes turns in the screening process obtains size and is 115bp double-stranded DNA library, is the adaptive word bank of RNA that the template in-vitro transcription goes out 97nt with this double-stranded DNA, and screening is carried out 12 altogether and taken turns.
The secondary structure analysis of the adaptive son of embodiment 2:RNA
Take turns the library clone that obtains of screening to the pMD19-T carrier with the 12nd, be converted into bacillus coli DH 5 alpha, 48 clones of random choose, order-checking.Obtain the sequence information of the adaptive son that screens, predict all order-checking clones' RNA secondary structure by structure prediction software RNA Structure Program, it can form a special loop-stem structure at stochastic sequence zone (23-47nt) to obtain a kind of adaptive son, as shown in Figure 2.
Embodiment 3: the adaptive son of H1 protein binding that obtains the specificity high-affinity
The adaptive son of RNA that will have above-mentioned secondary structure is got 1 μ g respectively, and with 37 ℃ of digestion of calf intestinal alkaline phosphatase (CIP) 1h, purifying reclaims dephosphorylized RNA; By T4 polynucleotide kinase mark [γ-
32P] ATP is in dephosphorylized RNA molecular end.The adaptive son of the radiolabeled RNA of 10nmol ℃ is hatched 40min with the H1 protein 37 of different concns (10-200nM) respectively, each group reaction liquid filters through nitrocellulose filter, washing filter membrane, dry filter membrane, liquid scintillation counter is measured residual exit dose on the filter membrane, the parallel mensuration of doing twice of same sample.Calculate each adaptive son and the proteic dissociation constant of H1, obtain the highest adaptive son of avidity, called after H1-A25.
32The H1-A25 of P mark adds H1 albumen with H1 albumen, irrelevant albumen HBsAg and excessive unlabelled H1-A25 respectively and hatches 40min at 37 ℃, each group reaction liquid filters through nitrocellulose filter, the washing filter membrane, dry filter membrane, liquid scintillation counter is measured residual exit dose on the filter membrane, the parallel mensuration of doing twice of same sample.Same grouping hatch the non-sex change PAGE of sample to 6% gel on the afterreaction liquid, 120V electrophoresis 2h takes out gel, the imaging of phosphorus screen, the result is as shown in Figure 3.
Embodiment 4: verify that adaptive sub-H1-A25's is liver cell targeted
The fluorine of in-vitro transcription green fluorescence mark is modified adaptive sub-H1-A25, and the system of transcribing is as follows: 1 μ gDNA template, 5 μ l 10 * transcribe damping fluid, 400 μ g/ μ l PEG, 0.5M MgCl2,100U/ml IPP, 50U RNasin, 2.5mM 2F '-CTP, 2.5mM2F '-UTP, 2.5mM ATP, 2.5mM GTP, 10mM FITC mark UTP and 0.36 μ g/ μ l T7RNA polymerase, moisturizing is transcribed for 37 ℃ and is spent the night to final volume 50 μ l.Transcription product is through the PAGE purifying.HepG2, HuH-7 and HeLa cell inoculation 24 orifice plates, 50% methyl alcohol is fixed 15 minutes for 4 ℃, the adaptive sub-H1-A25 of FITC mark that adds 50nM then respectively, add excessive unmarked adaptive sub-H1-A25 in the section H epG2 cell hole simultaneously or add ASGPR polyclonal antibody, incubated at room 20 minutes; Discard Incubating Solution; The PBS washing; The imaging of fluorescence inverted microscope, the result as shown in Figure 4.
Claims (2)
1. adaptive son of the hepatocellular RNA of target and nucleotide sequence thereof, it is characterized in that: the RNA sequence of the adaptive son of this RNA is: 5 '-GUUGAUUGCGUGUCAAUCAUGGCGUAGUAAAAGACAAGUAGCUACGAGGUCAUGUG UAUGUUGGGGAUUAGGACCUGAUUGAGUUCAGCCCACAUAC-3 ' can form loop-stem structure.
2. adaptive son of the hepatocellular RNA of the described target of claim 1 and nucleotide sequence thereof is characterized in that: the adaptive son of this RNA can the specificity high-affinity in conjunction with liver asialoglycoprotein receptor big subunit H1 albumen or can be bonded to liver cell by specificity high-affinity target.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392024A (en) * | 2011-12-02 | 2012-03-28 | 华中科技大学同济医学院附属同济医院 | Nucleic acid aptamer combining hepatitis B virus surface antigen and sequence thereof |
| US20150275212A1 (en) * | 2012-11-15 | 2015-10-01 | Roche Innovation Center Copenhagen A/S | Oligonucleotide Conjugates |
| CN106032534A (en) * | 2015-02-02 | 2016-10-19 | 苏州方舟基因药业有限公司 | Ribonucleic acid aptamer capable of specifically being combined with human non-small cell lung cancer cells and screening method thereof |
| CN110408620A (en) * | 2019-08-16 | 2019-11-05 | 中山大学附属第三医院 | A kind of nucleic acid aptamer, its preparation method, its derivative and its application |
| CN113563414A (en) * | 2020-04-29 | 2021-10-29 | 泰比棣医药科技(石家庄)有限公司 | Tissue-targeted protein targeted degradation compound and application thereof |
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| CN101148667A (en) * | 2006-09-22 | 2008-03-26 | 国家纳米技术与工程研究院 | Preparation and use for affinity human albumin nucleic acid aptamer |
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| CN101148667A (en) * | 2006-09-22 | 2008-03-26 | 国家纳米技术与工程研究院 | Preparation and use for affinity human albumin nucleic acid aptamer |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392024A (en) * | 2011-12-02 | 2012-03-28 | 华中科技大学同济医学院附属同济医院 | Nucleic acid aptamer combining hepatitis B virus surface antigen and sequence thereof |
| US20150275212A1 (en) * | 2012-11-15 | 2015-10-01 | Roche Innovation Center Copenhagen A/S | Oligonucleotide Conjugates |
| US10077443B2 (en) * | 2012-11-15 | 2018-09-18 | Roche Innovation Center Copenhagen A/S | Oligonucleotide conjugates |
| US11155816B2 (en) | 2012-11-15 | 2021-10-26 | Roche Innovation Center Copenhagen A/S | Oligonucleotide conjugates |
| CN106032534A (en) * | 2015-02-02 | 2016-10-19 | 苏州方舟基因药业有限公司 | Ribonucleic acid aptamer capable of specifically being combined with human non-small cell lung cancer cells and screening method thereof |
| CN106032534B (en) * | 2015-02-02 | 2019-08-23 | 苏州方舟生物医药有限公司 | A kind of rna aptamer and its screening technique in conjunction with Non-small cell lung carcinoma cell-specific |
| CN110408620A (en) * | 2019-08-16 | 2019-11-05 | 中山大学附属第三医院 | A kind of nucleic acid aptamer, its preparation method, its derivative and its application |
| CN110408620B (en) * | 2019-08-16 | 2023-03-21 | 中山大学附属第三医院 | Nucleic acid aptamer, obtaining method thereof, derivative thereof and application thereof |
| CN113563414A (en) * | 2020-04-29 | 2021-10-29 | 泰比棣医药科技(石家庄)有限公司 | Tissue-targeted protein targeted degradation compound and application thereof |
| CN113563414B (en) * | 2020-04-29 | 2022-08-12 | 泰比棣医药科技(石家庄)有限公司 | Tissue-targeted protein targeted degradation compound and application thereof |
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