ANALYST
·
2024
·Vol. 149
·期 4
·1271-1279
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IF 3.6
Aiwen Su,Dan Luo,Shixuan Li,Yanli Zhang,Hongbin Wang,Lijuan Yang,Wenrong Yang,Pengfei Pang
T4 polynucleotide kinase helps with DNA recombination and repair. In this study, an electrochemical biosensor was developed for a T4 polynucleotide kinase activity assay and inhibitor screening based on phosphate pillar[5]arene and multi-walled carbon nanotube nanocomposites. The water-soluble pillar[5]arene was employed as the host to complex thionine guest molecules. The substrate DNA with a 5′-hydroxyl group initially self-assembled on the gold electrode surface through chemical adsorption of the thiol group, which was phosphorylated in the presence of T4 polynucleotide kinase. Titanium dioxide nanoparticles served as a bridge to link phosphorylated DNA and phosphate pillar[5]arene and multi-walled carbon nanotube composite due to strong phosphate-Ti4+-phosphate chemistry. Through supramolecular host–guest recognition, thionine molecules were able to penetrate the pillar[5]arene cavity, resulting in an enhanced electrochemical response signal. The electrochemical signal is proportional to the T4 polynucleotide kinase concentration in the range of 10−5 to 15 U mL−1 with a detection limit of 5 × 10−6 U mL−1. It was also effective in measuring HeLa cell lysate-related T4 polynucleotide kinase activity and inhibitor screening. The proposed method offers a unique sensing platform for kinase activity measurement, holding great potential in nucleotide kinase-target drug development, clinical diagnostics, and inhibitor screening.
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
JOURNAL OF THE ELECTROCHEMICAL SOCIETY
·
2022
·Vol. 169
·期 2
·027504
·
IF 4.386
Zaiqiong Liu,Jinpeng Tao,Zhenyu Zhu,Yanli Zhang,Hongbin Wang,Pengfei Pang,Haibo Wang,Wenrong Yang
An ultrasensitive electrochemical assay for T4 polynucleotide kinase (T4 PNK) activity and inhibition was developed based on magnetic Fe3O4@TiO2 core–shell nanoparticles and gold nanoparticles hybrid probe (Fe3O4@TiO2-dsDNA-AuNPs) modified magnetic gold electrode (MGE). Fe3O4@TiO2-dsDNA-AuNPs hybrid probe was formed via DNA hybridization between phosphorylated DNA S1 modified Fe3O4@TiO2 and complementary DNA S2 assembled AuNPs. [Ru(NH3)6]3+ was used as an electrochemically active indicator to enhance the electrochemical signal. The proposed method is sensitive for T4 PNK activity assay with a linear range of 0.0001 to 10 U ml−1 and low detection limit of 0.00003 U ml−1. This strategy can be used to screen the T4 PNK inhibitors, holding a great potential for discovery of nucleotide kinase-target drug and early clinical diagnosis of cancer.
研究领域:
生物化学
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电化学
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纳米技术
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分子生物学
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癌症诊断
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DOI
高影响因子
ENVIRONMENTAL POLLUTION
·
2023
·
IF 8.9
Xinyi Li,Kaiyue Li,Jiayi Li,Zhiqin Zhang,Hongyuan Wang
Endocrine disruptors have been demonstrated to exert adverse effects on growth and development of amphibians by disrupting hormone levels. Tail resorption, which is one of the most remarkable events during amphibian metamorphosis, is closely associated with thyroid hormones levels. However, limited research has been conducted on the effects of endocrine disruptors on tail resorption in amphibians. This study explored the effects of NaClO 4 and T4 on the growth, development and tail resorption during the metamorphosis of Rana Chensinensis. The results demonstrated that exposure to NaClO 4 led to an increase in body size and a delay in metamorphosis of R. Chensinensis tadpoles. Histological analysis revealed that both NaClO 4 and exogenous T4 exposure resulted in thyroid gland injury, and NaClO 4 treatment delayed the degradation of notochord and muscles, thereby delaying tail resorption. Moreover, transcriptome sequencing results showed that apoptosis-related genes ( APAF1 , BAX and CASP6 ) and cell component degradation-related genes ( MMP9 and MMP13 ) were highly expressed in the T4 exposure group, and the expression of oxidative stress-related genes ( SOD and CAT ) was higher in the NaClO 4 exposure group. Taken together, both NaClO 4 and exogenous T4 affect tail resorption in R. Chensinensis , thereby affecting their adaptation to terrestrial life. The present study will not only provide a reference for future experimental research on the effects of other endocrine disruptors on the growth, development and tail resorption of amphibians but will also provide insights into environmental protection and ecological risk assessment.
研究领域:
生态毒理学
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发育生物学
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内分泌学
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分子生物学
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DOI
PubMed
高影响因子
TALANTA
·
2023
·
IF 6.1
Dan Luo,Zaiqiong Liu,Aiwen Su,Yanli Zhang,Hongbin Wang,Lijuan Yang,Wenrong Yang,Pengfei Pang
T4 polynucleotide kinase (T4 PNK) is an important DNA repair-related enzyme that plays a crucial role in DNA recombination, replication and damage repair. Herein, an electrochemical biosensor was developed for detection of T4 PNK activity and inhibitor screening based on supramolecular host–guest recognition between phosphate pillar (Dumitrache and McKinnon, 2017) [5] arene (PP5) and methylene blue (MB). The water-soluble PP5 employed as the host for complexation of MB guest molecules. The substrate DNA with 5′-hydroxyl group was first self-assembled on the gold electrode surface through the chemical adsorption of the thiol group , which was phosphorylated in the presence of T4 PNK and adenosine triphosphate (ATP). TiO 2 served as a bridge to link phosphorylated DNA and PP5 via the robust phosphate-Ti 4+ -phosphate chemistry . The immobilized PP5 captured the MB on electrode surface via the supramolecular host–guest recognition interaction, resulting in an enhanced electrochemical response signal. The electrochemical signal is proportional to the T4 PNK concentration in the range of 2 × 10 −4 to 5 U mL −1 with a detection limit of 1 × 10 −4 U mL −1 . It was also successfully used for PNK inhibitor screening and PNK activity assay in HeLa cell lysates sample. The proposed strategy provides a novel sensing platform for kinase activity assay and inhibitor screening, holding a great potential in clinical diagnostics, inhibitor screening, and nucleotide kinase-target drug discovery.
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
·
2019
·
IF 6.393
Zhen Song,Yang Li,He Teng,Caifeng Ding,Guiyun Xu,Xiliang Luo
Herein, an antifouling and sensitive electrochemical enzyme sensing platform was developed based on a self-designed zwitterionic peptide and sacrificial iron metal-organic framework (Fe-MOF). The designed zwitterionic peptide was hydrophilic and neutral in charge, and it was used to improve the antifouling capability of the sensing system. Fe-MOF with amine groups was loaded with Au nanoparticles and further functionalized with a specific DNA sequence, and it was used as a sacrificial label. The sensing target, T4 polynucleotide kinase (PNK), can catalyze the 5′-hydroxyl termini of a unique hairpin DNA to phosphate group, and the hairpin DNA can then be hydrolyzed by lambda exonuclease (λ-Exo) and release a single nucleotide. In the presence of the released DNA sequence, a sandwich-type hybridization reaction was triggered, and the DNA linked Fe-MOF was selectively attached to the electrode surface, which can react with K 4 Fe(CN) 6 to form the electroactive Prussian blue and generate significantly enlarged current response. Based on the antifouling peptide and the sacrificial Fe-MOF, the biosensor exhibited satisfying analytical performance for the detection of T4 PNK, with a wide linear range from 1.0×10 −3 to 10 U·mL -1 and a low detection limit of 3.5×10 -4 U·mL -1 . Moreover, the biosensor was capable of effectively eliminating nonspecific protein adsorption and assaying target in human serum, indicating its promising potential for practical application.
研究领域:
生物化学
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分析化学
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生物传感器
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分子诊断
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产品关联
DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2018
·
IF 8.173
Chengji Sui,Tingting Wang,Yunlei Zhou,Huanshun Yin,Xiangjian Meng,Shenran Zhang,Geoffrey I.N. Waterhouse,Qin Xu,Yuping Zhuge,Shiyun Ai
5-Hydroxymethylcytosine (5hmC) plays an important role in switching genes on and off in mammals, and it is implicated in both embryonic development and cancer progression. Herein, a novel photoelectrochemical (PEC) biosensor was developed for 5hmC detection based on WS 2 nanosheets as the photoactive material and boronic acid functionalized carbon dots (B-CDs) for signal amplification unit. This biosensor can also be used for T4-β-glucosyltransferase (β-GT) activity assessment. Firstly, WS 2 nanosheets and gold nanoparticles (AuNPs) were immobilized on an ITO electrode surface . Then probe DNA was immobilized on this electrode surface via Au-S bond. Afterwards, the complementary DNA containing 5hmC was then captured on the modified electrode surface by hybridization. Subsequently, β-GT transferred glucose from uridine diphosphoglucose to the hydroxyl groups of the 5hmC residues. After glycosylation , B-CDs could further be immobilized on the modified electrode surface resulting in a strong photocurrent . The PEC biosensor afforded high selectivity , excellent sensitivity and good reproducibility, with detection limits of 0.0034 nM and 0.028 unit/mL for 5hmC and β-GT, respectively. Results demonstrate that the photoelectrochemical strategy introduced here based on WS 2 nanosheets and B-CDs offers a versatile platform for hydroxymethylated DNA detection, β-GT activity assessment and β-GT inhibitor screening.
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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分析化学
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Jing Zhu,Liutao Chen
Fluorescent dye DITO-1 has almost no fluorescence in the absence of nucleic acid . G bases in single strand DNA can induce maximum fluorescent enhancement followed by the A bases when it binds the DITO-1. However, the incorporation efficiency of the dATP was higher than dGTP in terminal transferase (TdT) polymerization. As a consequence, ploy (A) n , rather than ploy (G) n via TdT polymerization had the superior photoluminance when it binded DITO-1 fluorescent dye. Here, we developed a high selective and sensitive sensing strategy for assaying TdT and T4 polynucleotide kinase activity (T4 PNK) based on the ploy (A) n -DITO-1 fluorescent probe. An increasing amounts of TdT enzyme could promote the distinct incorporation of dATP on the DNA primer and form poly (A) n ssDNA with a difference in length. A good linear relationship between the ΔF and the concentrations of TdT in a range of 0.2–50 U/mL was obtained and the detection limit was 0.05 U/mL. Based on the experimental results for TdT, we further expanded the application of this method for detection of a series of concentrations of T4 PNK. The ΔF and the logarithm concentrations of T4 PNK in the range of 0.1–10 U/mL showed a good linear response and the detection limit of 0.02 U/mL was obtained. In addition, the detection of T4 PNK in Hela cell lysate was achieved, demonstrating that the proposed method had the potential application in complex system. The ploy (A) n -DITO-1 fluorescent probe had the excellent properties of one-step readout, robustness for target detection in complex system, and easiness operation, and showed the great potential in clinical diagnostics, inhibitor screening, and drug discovery.
研究领域:
分子生物学
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生物化学
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分析化学
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生物医学工程
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DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2023
·Vol. 190
·期 10
·1-10
·
IF 5.7
Wu Yongju,Yi Jinfei,Su Aiwen,Zhang Yanli,Wang Hongbin,Yang Lijuan,Yang Wenrong,Pang Pengfei
T4 polynucleotide kinase (T4 PNK) helps with DNA recombination and repair. In this work, a phosphate pillar[5]arene@palladium nanoparticles@reduced graphene oxide nanocomposite (PP5@PdNPs@rGO)–based electrochemical biosensor was created to identify T4 PNK activities. The PP5 used to complex toluidine blue (TB) guest molecules is water-soluble. With T4 PNK and ATP, the substrate DNA, which included a 5ʹ-hydroxyl group, initially self-assembled over the gold electrode surface by chemical adsorption of the thiol units. Strong phosphate-Zr 4+ -phosphate chemistry allowed Zr 4+ to act as a bridge between phosphorylated DNA and PP5@PdNPs@rGO. Through a supramolecular host–guest recognition connection, TB molecules were able to penetrate the PP5 cavity, where they produced a stronger electrochemical response. With a 5 × 10 −7 U mL −1 detection limit, the electrochemical signal is linear in the 10 −6 to 1 U mL −1 T4 PNK concentration range. It was also effective in measuring HeLa cell lysate–related PNK activities and screening PNK inhibitors. Nucleotide kinase–target drug development, clinical diagnostics, and screening for inhibitors all stand to benefit greatly from the suggested technology, which offers a unique sensing mechanism for kinase activity measurement. Graphical Abstract
研究领域:
生物化学
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医学诊断
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药物开发
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DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2020
·Vol. 187
·期 3
·1-8
·
IF 6.232
Chen Yan,Yin Huanshun,Sui Chengji,Fu Weizhang,Yang Yue,Ai Shiyun
A photoelectrochemical method was proposed for DNA hydroxymethylation determination using black TiO 2 (B-TiO 2 ) nanosphere as photoactive material and ZnO as photoactivity inhibitor. After hydroxymethylated DNA (5hmC-DNA) was captured on the probe modified B-TiO 2 /ITO electrode surface through hybridization, a glycosyl can be then transferred from uridine diphosphoglucose to 5hmC-DNA and formed a covalent structure with –CH 2 OH in the presence of T4 β-glucosyltransferase (β-GT). Afterwards, based on a series of covalent reaction, amino functionalized ZnO nanoparticles are further immobilized to the surface of the electrode. Due to the capacity to expend the irradiation light and the photogenerated electron of electron donor, the modified ZnO nanoparticles can result in a decreased photocurrent. The developed method shows wide linear ranges from 0.05-200 nM for hydroxymethylated DNA and 1-220 unit·mL −1 for T4-β-glucosyltransferase. The corresponding determination limits were 0.013 nM and 0.24 unit·mL −1 , respectively. The enzyme activity inhibited by 4-phenylimidazole was evaluated. This photoelectrochemical method shows high specificity for 5hmC-DNA (compared to 5fC, 5mC, m6A, control) and β-GT (compared to β-AGT, UGT2B7), and shows excellent stability for testing 5hmC (RSD = 2.75%).
Schematic representation of photoelectrochemical method for DNA hydroxymethylation and β-glucosyltransferase detection based on the glycosylation reaction of –CH 2 OH in 5-hydroxymethylcytosine and the inhibition activity of ZnO to the photoactivity of black TiO 2 nanospheres.
研究领域:
生物化学
·
分子生物学
·
分析化学
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2013
·
IF 5.695
Yang Tang,Jie-Mei Chu,Wei Huang,Jun Xiong,Xi-Wen Xing,Xiang Zhou,Yu-Qi Feng,Bi-Feng Yuan
5-Methylcytosine (5-mC), an important epigenetic modification involved in development, can be converted enzymatically to 5-hydroxymethylcytosine (5-hmC). 5-hmC is considered an intermediate of active DNA cytosine demethylation and makes itself serve as an epigenetic mark. 5-hmC content in most mammalian cells is low and the quantification of 5-hmC by liquid chromatography–mass spectrometry (LC–MS) frequently suffers from ion suppression by the presence of unmodified nucleosides. To circumvent this problem, we developed a method to selectively transfer a glucosyl group to the hydroxymethyl moiety of 5-hmC and form a more hydrophilic residue (β-glucosyl-5-hydroxymethyl-2′-deoxycytidine, 5-gmdC) by using T4 β-glucosyltransferase. The more hydrophilic 5-gmdC can be selectively enriched by using NH2-silica via hydrophilic interaction prior to liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis, which eliminates the ion suppression and significantly improves the detection sensitivity and accuracy. Using this method, we successfully quantified 5-hmC content in genomic DNA of three human cell lines and seven yeast strains. To the best of our knowledge, this is the first report about the existence of 5-hmC in the model organism of yeast. In addition, the contents of 5-hmC in two yeast strains of Schizosaccharomyces pombe are even higher than those of 5-mC, indicating that 5-hmC may play important roles on the physiological functions of yeast.
研究领域:
表观遗传学
·
分析化学
·
生物化学
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DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2021
·Vol. 188
·期 8
·1-10
·
IF 5.833
Yang Hongmei,Wang Yafen,Tang Jing,Wang Fang,Chen Zilin
A promising electrochemical strategy for assay of N6-methyladenosine (m6A)/N6-methyladenine (6mA) in RNA/DNA is proposed. The key of this strategy is the end-labeling of nucleic acid, which makes it possible to detect methylation level in unknown sequence. Firstly, the end of m6A-RNA or 6mA-DNA was labeled with sulfhydryl group through T4 polynucleotide kinase (T4 PNK) and then directly assembled on a gold nanoparticle–modified glassy carbon electrode (AuNPs/GCE). Secondly, methylation sites in RNA/DNA were specifically recognized by anti-m6A-antibody, and then, horseradish peroxidase–labeled goat anti-rabbit IgG (HRP-IgG) was further conjugated on the antibody. Thirdly, HRP-IgG catalyzed the hydroquinone oxidation reaction to generate amplified current signal which correlates with the amount of m6A/6mA in nucleic acid. This method showed a wide linear range from 0.0001 to 10 nM for m6A-RNA, 0.001 to 100 nM for 6mA-dsDNA, and 0.0001 to 10 nM for 6mA-ssDNA. The method was successfully applied to detection of m6A/6mA in RNA/DNA from HeLa cells and E. coli cells and validation of the decrease of m6A-RNA in HeLa cells after treatment with FTO protein. Graphical abstract
研究领域:
电化学
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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
BIOMACROMOLECULES
·
2025
·
IF 5.4
Lin Zhang,Jiaxing Zhang,Lvping Wu,Shengping You,Rongxin Su,Wei Qi
Single-stranded DNA-binding protein gp32 is essential for DNA replication and widely used in isothermal amplification techniques such as recombinase polymerase amplification (RPA). However, its inherent thermal instability and reaction condition sensitivity restrict its broader applications. Here we develop the Fermentation Integrated Rational Engineering (FIRE-3S) strategy to enhance the functional properties of gp32 through molecular dynamics (MD) simulations, structure-guided fermentation tuning and stability-focused rational design. Starting from a disulfide-bonded mutant (V62C/T80C) that showed enhanced RPA performance but reduced thermostability, MD simulations revealed local structural disruption near the Zn2+-binding motif. Then, structure-guided fermentation tuning was applied, which restored structural integrity and enhanced DNA-binding affinity. Building on 2S-step, a computationally guided clique-based design pipeline rapidly identified a multisite mutant, gp32-M1, with elevated thermostability (ΔTm = +6.03 °C), robust ssDNA-binding, and strong inhibitor tolerance─maintaining >65% activity under 4% isopropanol or ethanol. This work establishes a systematic framework for engineering ssDNA-binding proteins with enhanced biostability and environmental resilience for molecular diagnostics.
研究领域:
分子诊断
·
蛋白质工程
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生物技术
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核酸扩增
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结构生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）