ANALYTICA CHIMICA ACTA
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2022
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IF 6.911
Jingjing Li,Zhixue Zhu,Manru Zhang,Qingxin Zhang,Wenyu Sun,Yeru Wang,Long Jiang,Zhiqiang Guo,Yu Wang,Su Liu,Jiadong Huang,Jinghua Yu
Uracil-DNA glycosylase (UDG) is a common glycosylase that can expressly recognize and remove damaged uracil bases, and the ultrasensitive detection of which is significant to maintain genomic stability and early clinical diagnosis of disease. Herein, we proposed a sensitive colorimetric sensing platform to detect UDG. Combined with target-manipulated drawstring DNAzyme and [email protected] nanorods ( [email protected] NRs) indicator, we achieved in naked-eyes observation and ultrasensitive detection of UDG. Briefly, when the UDG exists, the dynamic reaction of rope pulling will occur generating the active conformation of DNAzyme. The cutting effect will be further produced when we add Mg 2+ , thus the generated trigger chain can mediate the occurrence of CHA reaction, followed by generating amount of ·OH which can etch [email protected] NRs causing the shifted of localized surface plasmon resonance (LSPR) peak. By contrast, there is no obvious shift of LSPR peak. This strategy shows extraordinary specificity and sensitivity toward UDG providing a detection limit of 4.6 × 10 −5 U mL −1 . By using of this method, we detected UDG specifically in complex samples, proving that it’s potential applications in biomedical research and clinical diagnosis are fantastic.
研究领域:
生物化学
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纳米技术
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生物医学工程
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临床诊断
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产品关联
DOI
PubMed
高影响因子
TALANTA
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2018
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IF 4.244
Guirong Liu,Wenli He,Chenghui Liu
As an important base excision repair (BER) enzyme, uracil-DNA glycosylase (UDG) can repair the uracil-induced DNA lesion and maintain the genomic integrity. Herein, we have proposed a sensitive UDG assay based on the terminal deoxynucleotidyl transferase (TdT)-assisted formation of fluorescent copper nanoclusters (CuNCs). In this study, a uracil-containing stem-loop DNA substrate is rationally designed with its 3′-end blocked with 2′, 3′-dideoxycytosine (ddC). UDG can remove the uracil in the DNA substrate to generate an apurinic/apyrimidinic (AP) site, which can then be specifically cleaved by endonuclease IV (Endo IV) to expose a 3′-OH terminus. TdT will initiate the template-free DNA extension along the exposed 3′-OH terminus to produce a quite long poly(T) tail, which will perfectly template the production of fluorescent CuNCs. By recording the fluorescence of the CuNCs, the UDG activity can be faithfully detected. In contrast, if UDG is absent, the 3′-ddC terminus of the DNA substrate cannot be recognized by TdT and thus no TdT-based extension and formation of CuNCs will occur. The use of ddC as a 3′-end blocker can greatly decrease the nonspecific DNA extension and improve the signal-to-noise ratio. Furthermore, TdT is a template-free and sequence-independent DNA polymerase, which can effectively catalyze the tailing process up to a maximum of thousands of thymines , and each tail can form many fluorescent CuNCs. Therefore, an ultrahigh sensitivity is achieved and as low as 0.00005 U/mL of UDG can be clearly detected. Moreover, with an additional AP site-contained poly(A) oligonucleotide during TdT-mediated extension, a branched amplification mechanism is also preliminarily devised, which can further push the detection limit of UDG to an extremely low level of 0.000002 U/mL.
研究领域:
生物化学
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分子生物学
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分析化学
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DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2019
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IF 6.393
Yu Ling,Jiao Zhou,Xiao Fang Zhang,Xiao Hu Wang,Nian Bing Li,Hong Qun Luo
As one of critical base excision repair enzymes, uracil-DNA glycosylase (UDG) can specifically repair uracil-induced DNA damage to maintain the genome integrity of organisms. Moreover, abnormal expression of UDG is related to various cancers and other serious diseases. Therefore, it is essential to accurately and sensitively monitor UDG activity. In this work, a ratiometric fluorescence method using poly-(thymine) DNA template copper nanoclusters (Cu NCs) and 4′,6-diamidino-2-phenylindole (DAPI) as the output signals is developed for simple, selective, and sensitive detection of UDG. A double-stranded DNA (dsDNA) consisting of a uracil-containing single chain and a trigger sequence is designed as a substrate dsDNA. Meanwhile, another dsDNA comprising 30 pairs of A-T bases ((AT) 30 dsDNA) is designed to be partially complementary to the trigger chain. The (AT) 30 dsDNA itself has no blunt or recessed 3′ end and cannot be cut by exonuclease III (Exo III). With the Exo III-assisted amplification, adding UDG results in the success synthesis of red-emissive Cu NCs, while the dsDNAs in the system are hydrolyzed and the fluorescence signal of DAPI is weak. In contrast, without addition of UDG, the fluorescence intensity of Cu NCs is low, while the DAPI emits significantly enhanced fluorescence signal. The proposed method presents a detection limit of 5.0 × 10 −5 U mL −1 and can be applied to the HeLa cell lysate sample with satisfactory results. Additionally, the kinetic parameter is studied and the UDG inhibition effect is evaluated. In summary, this work provides a potential platform for DNA repair enzyme-related analysis and clinical diagnosis.
研究领域:
生物化学
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分子生物学
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癌症研究
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分析化学
✓ 已验证
产品关联
DOI
高影响因子
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