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Endogenous enzyme-triggered DNA walker and its application for detecting UDG

An endogenous enzyme, walker technology, applied in DNA preparation, recombinant DNA technology, microbial assay/inspection, etc., can solve problems such as limited sensitivity, and achieve the effect of improving sensitivity and good specificity

Active Publication Date: 2019-08-06
SHANDONG UNIV
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Problems solved by technology

However, the inventors of the present disclosure found that in order to maintain the operation of the intracellular DNA walker, exogenous metal ions (Mn 2+ , Zn 2+ ) or fuel oligonucleotides, which lead to changes in cell state or degradation of intact fuel DNA, ultimately limiting the operation of DNA walkers in cells due to the resistance of DNA strands to degradation by nonspecific nucleases and the unique amplification of walkers ability and strong DNA enrichment ability on the 3-D surface of nanoparticles, DNA walkers run in living cells to image UDG activity, while the imaging of UDG activity is caused by the presence of unprotected DNA probes and difficulties in intracellular amplification before The problem of limited sensitivity of the

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Embodiment

[0050] 1. Experimental part

[0051] 1.1 Reagents and raw materials

[0052] TE buffer solution (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) was prepared for diluting the synthesized DNA powder, and then stored at -20°C. 13nm gold nanoparticles are prepared from chloroauric acid trihydrate and sodium citrate dihydrate. Uracil-DNA glycosylase (UDG), uracil glycosylase inhibitor (UGI), 8-oxidized guanine DNA glycosylase (hOGG1), human alkyladenine DNA glycosylase (hAGGA ), endonuclease IV (endo IV) and abasic / purinase 1 (APE1) were purchased from New England Biological Co., Ltd. (Beijing, China). Tween 20 was purchased from Beijing Suolaibao Technology Co., Ltd. Phosphate buffered saline (8mM Na 2 HPO 4 ,2mM KH 2 PO 4 , 136mM NaCl, 2.6mM KCl, pH 7.4) were purchased from Cromwell Bioindustries, USA. Some chemical reagents of analytical grade do not require purification. All solutions were prepared with ultrapure water (resistivity >18.25 MΩcm) produced by a Millipore Milli-Q wate...

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Abstract

The present disclosure provides an endogenous enzyme-triggered DNA walker and its application for detecting UDG, The DNA walker comprises gold nanoparticles, at least one walking chain and a pluralityof track chains, wherein the walking chain and the tracks are single-stranded DNA, one end of the walking chain is connected with the gold nanoparticles, one end of each track chain is connected to the gold nanoparticles, the other end of each track chain is connected to fluorophore, the walking chain contains a first sequence, the track chain contains a second sequence, the first sequence is complementary to the second sequence, and one base in the second sequence is a uracil base. The constructed DNA walker is capable of sensitive imaging of UDG activity in cells.

Description

technical field [0001] The present disclosure relates to an endogenous enzyme-triggered DNA walker and its application for detecting UDG. Background technique [0002] The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art. [0003] Uracil-DNA glycosylase (UDG) is a DNA damage repair protein whose main function is to remove uracil by catalyzing the hydrolysis of the N-glycosidic bond between uracil and deoxyribose. The function of UDG is the first step of uracil base excision repair (UBER), followed by the action of apurinic / apyrimidinic (AP) endonuclease, ligase and polymerase to complete the UBER pathway and maintain the integrity of the genome. However, abnormal activity of UDG in cells can lead to misintroduction of uracil during DNA replication, resulting in incorrect base pairing and gene mutations, ultimately increasing the incidence of diseases such as immunodeficiency, Bloom syndrom...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/34G01N21/64
CPCC12N15/10C12Q1/34G01N21/6428G01N2021/6432
Inventor 姜玮徐晓文张苹苹张芮源
Owner SHANDONG UNIV
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