BIOCHEMISTRY
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2018
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IF 2.997
Qianwei Zhao,Wen Yang,Tong Qin,Zhen Huang
RNA is a key player in the cellular central dogma, including RNA transcription and protein synthesis. However, it is unknown whether RNA can directly interfere with DNA synthesis. Recently, we have found in vitro that while binding to DNA polymerase nonspecifically, RNA can transform DNA polymerase to display a moonlighting activity, dNTP phosphatase, in turn interfering with DNA synthesis. This phosphatase activity removes the γ-phosphate from dNTPs (generating dNDPs) and subsequently removes the β-phosphate from the formed dNDPs (generating dNMPs), confirmed by the noncleavable α,β-CH2-dGTP and β,γ-CH2-dGTP analogues. We also found that dGTP is the best substrate for the phosphatase, and the dNTP phosphatase activity is sensitive to the reaction medium. In addition, we have revealed that RNA can tune the activity of closely related proteins and give rise to new catalytic functions with subtle differences. Moreover, we have demonstrated in vitro that at the lower dNTP level, this phosphatase can directly inhibit DNA synthesis by dNTP depletion, though the phosphatase activity is 690-fold slower than the polymerase activity. Our observation in vitro suggests a plausible strategy for RNA to directly interfere with DNA polymerase and DNA synthesis in vivo.
研究领域:
分子生物学
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生物化学
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DOI
PubMed
高影响因子
FOOD CHEMISTRY
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2019
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IF 5.399
Bin Wang,Jiushang Zheng,Ailing Ding,Liqun Xu,Jiuncun Chen,Chang Ming Li
A novel aptasensor, based on a perylene probe (PAPDI; N,N′-bis(propylenetrimethylammonium)-3,4,9,10-perylenediimide), was developed for the detection of aflatoxin B1 (AFB1) in maize samples. AuNPs/DNA composites were synthesized and integrated with aptamers-modified magnetic nanoparticles (MNPs) via DNA hybridization. For AFB1 determination, AuNPs/DNA composites were released from MNPs through specific binding of AFB1 with the aptamer and used for assembly of the PAPDI probe. To enhance the method sensitivity, terminal deoxynucleotidyl transferase (TdT)-catalyzed DNA polymerization was performed to elongate DNA on AuNPs/DNA composites. As a result, more PAPDI probes were assembled on the AuNPs/DNA composites. Through a multiple signal amplification strategy, the proposed method exhibited high sensitivity towards AFB1, with a detection limit of 0.01 nM (3.1 pg/mL). In summary, the proposed method has great potential to be a universal strategy for monitoring AFB1 in food samples.
研究领域:
食品科学
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分子生物学
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DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2021
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IF 10.618
Ying Peng,Yanhong Pan,Zhaowei Sun,Jinlong Li,Yongxiang Yi,Jie Yang,Genxi Li
The pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is continuously worsening globally, herein we have proposed an electrochemical biosensor for the sensitive monitoring of SARS-CoV-2 RNA. The presence of target RNA firstly triggers the catalytic hairpin assembly circuit and then initiates terminal deoxynucleotidyl transferase-mediated DNA polymerization. Consequently, a large number of long single-stranded DNA products can be produced, and these negatively charged DNA products will bind a massive of positively charged electroactive molecular of Ru(NH 3 ) 6 3+ due to the electrostatic adsorption. Therefore, significantly amplified electrochemical signals can be generated for sensitive analysis of SARS-CoV-2 RNA in the range of 0.1–1000 pM with the detection limit as low as 26 fM. Besides the excellent distinguishing ability for SARS-CoV-2 RNA against single-base mismatched RNA, the proposed biosensor can also be successfully applied to complex matrices, as well as clinical patient samples with high stability, which shows great prospects of clinical application.
研究领域:
分子生物学
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分析化学
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医学诊断
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产品关联
DOI
PubMed
高影响因子
Food Bioscience
·
2021
·
IF 4.24
Jing Li,Yong-Wei Feng,Li-Jun Huang,Rui Jiang,Xiao-Fang Shen
Proper labeling of meat products is critical for consumers who want to avoid specific meat species for cultural or religious reasons. Herein, we developed a convenient strand-displacement DNA polymerase induced isothermal circular amplification (SDPICA) fluorescence sensor for detection of undeclared pork component in meat products. The proposed assay was performed at 37 °C for 20 min with no cross-reactivity, and could detect 1 pg of pork DNA per reaction. The limit of detection of the SDPICA assay was as low as 0.1% pork in 30 mg multi-component meat mixtures. This assay would contribute to the quality control of meat products in food industry.
研究领域:
食品科学
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分子生物学
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分析化学
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产品关联
DOI
高影响因子
MICROCHEMICAL JOURNAL
·
2023
·
IF 4.8
Lilin Qin,Fangxu Lou,Yan Wang,Yinhao Zhang,Shishi Liu,Xu Hun
Molecular switches formed by enzyme-DNA hybrid compounds combined with CRISPR-Cas12a cis -cleavage used for photoelectrochemical nucleic acid assay was fabricated. A compound, molecular switch, consisting of Taq DNA polymerase, aptamer, block DNA and methylene blue labeled primer (primer-Mb) is formed. Block DNA is assembled on the surface of MBs (block DNA-MBs), and hybridized with the primer-Mb. In the presence of Human Immunodeficiency Virus (HIV) target, molecular switches are turned on by hybridization of the target with block DNA. The Taq DNA polymerase is released and activated. The activated Taq DNA polymerase elongates the primer via the template, block DNA, which attached onto Magnetic beads (MBs). A dsDNA with Mb (dsDNA-Mb) was formed. With the assistance of Cas12a, a amplified photoelectrochemical signal was obtained and the HIV target DNA was qualified. Under the best conditions, the limit of detection (LOD) can be as low as 0.3 fM. This photoelectrochemical assay extends the tool kit of molecular nanotechnology.
研究领域:
分子生物学
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生物化学
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纳米技术
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病毒学
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产品关联
DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
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2016
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IF 7.476
Kai Shi,Baoting Dou,Jianmei Yang,Ruo Yuan,Yun Xiang
Specific and sensitive detection of protein biomarkers is of great importance in biomedical and bioanalytical applications. In this work, a dual amplified signal enhancement approach based on the integration of catalytic hairpin assembly (CHA) and terminal deoxynucleotidyl transferase (TdT)-mediated in situ DNA polymerization has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. The presence of the target thrombin leads to the unfolding and capture of a significant number of hairpin signal probes with free 3′-OH termini on the sensor electrode . Subsequently, TdT can catalyze the elongation of the signal probes and formation of many G-quadruplex sequence replicates with the presence of dGTP and dATP at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a completely label-free fashion. The sensor shows a linear range of 0.5 pM–10.0 nM and a detection limit of 0.12 pM for thrombin. Moreover, the developed sensor can selectively discriminate the target thrombin against other non-target proteins and can be employed to monitor thrombin in human serum samples.
研究领域:
生物医学
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生物分析学
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产品关联
DOI
PubMed
高影响因子
BIOORGANIC CHEMISTRY
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2022
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IF 5.307
Junjie Wu,Mengmeng Zhang,Lulu Song,Yahong Tan,Yosuke Taniguchi,Christopher John Hipolito,Youming Zhang,Yizhen Yin
Oxidative lesions, such as 8-oxo-dG and 8-oxo-dA, are continuously generated from exposure to reactive oxygen species. While 8-oxo-dG has been extensively studied, 8-oxo-dA has not received as much attention until recently. Herein, we report the synthesis of duplex DNAs incorporating dA, 8-oxo-dA, 7-deaza-dA, 8-Br-dA, and 8-Br-7-deaza-dA, which have different substitutions at 7- and 8-position, for the investigation into the implications of N7-hydrogen and C8-keto on the base pairing preference, mutagenic potential and repair of 8-oxo-dA. Base pairing study suggested that the polar N7-hydrogen and C8-keto of 8-oxo-dA, rather than the syn -preference, might be essential for 8-oxo-dA to form a stable base pair with dG. Insertion and extension studies using KF-exo − and human DNA polymerase β indicated that the efficient dGTP insertion opposite 8-oxo-dA and extension past 8-oxo-dA:dG are contingent upon not only the stable base pair with dG, but also the flexibility of the active site in polymerase. The N7-hydrogen in 8-oxo-dA or C7-hydrogen in 7-deaza-dA and 8-Br-7-deaza-dA was suggested to be important for the recognition by hOGG1, although the excision efficiencies of 7-deaza-dA and 8-Br-7-deaza-dA were much lower than 8-oxo-dA. This study provides an insight into the structure-function relationship of 8-oxo-dA by nucleotide analogues.
研究领域:
分子生物学
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生物化学
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遗传学
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DNA修复
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DOI
PubMed
高影响因子
FOOD CHEMISTRY
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2019
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IF 5.399
Xuetong Li,Yuan Su,Huashuo Chu,Shuxia Lyu,Jingjing Tian,Wentao Xu
Vibrio parahaemolyticus is a major hidden danger of food safety. To develop a rapid, sensitive and on-site detecting method of Vibrio parahaemolyticus ( V. parahaemolyticus ), a strand replacement primer thermostat phosphate (SRPP) visual sensor was proposed, based on Bst DNA polymerase and pyrophosphatase. The novel strand replacement primer (SRP) facilitates chain substitution and to open a self-folding hairpin by adding region at its 3′ end. Under the action of the SRP, a pair of external primers and two inner primers, target DNA is specifically amplified at 63 °C relies mainly on the hairpin. Many pyrophosphates (PPi) are simultaneously generated as by-products, which can be converted into phosphates (Pi) by pyrophosphatase for phosphomolybdate blue visual detection within 5 min. The proposed biosensor can detect 1.29 × 10 3 copies of V. parahaemolyticus within 35 min.
研究领域:
微生物学
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食品科学
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生物技术
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL BIOCHEMISTRY
·
2019
·
IF 2.507
Shuyan Niu,Cheng Bi,Weiling Song
A sensitive and selective fluorescence assay for DNA methyltransferase (MTase) activity detection was designed based on aggregation-induced emission (AIE) and target initiated template-free DNA polymerization. Quaternized tetraphenylethene salt was synthesized as the AIE probe, which binds to single-stranded DNA by electrostatic interaction . A hairpin probe was designed with a specific sequence for DNA MTase. In the presence of DNA MTase, the methylation reaction initiated DNA polymerization with terminal deoxynucleotidyl transferase (TdT), which activated the fluorescence intensity through AIE. The designed DNA sensor displayed a linear response to concentrations of DNA adenine methyltransferase (Dam) MTase from 0.5 U·mL −1 to 100 U mL −1 , with a limit of detection of 0.16 U mL −1 . The assay was also effective for detection of DNA MTase activity in human serum and for showing the inhibitory effect of 5-fluorouracil on Dam MTase.
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 Physical Chemistry Letters
·
2023
·
IF 5.7
Tianyu Cai,Jiaqing Zhang,Jingjing Deng,Qinman Tian,Ruoqi Zhang,Haokai Geng,Yuedong Liu,Keding Zhong,Liting Wen,Chunxiang Liu,Xun Hu,Yuehui Li,Zhipeng Li,Shipeng Hao,Kai Yang,Huizhong Xu
DNA alkylating agents are widely used in anticancer pharmacology. Although shown to induce cross-linking and/or methylation of DNA, how they affect the mechanical properties of DNA and activity of DNA enzymes remains to be elucidated. Here, we perform single-molecule optical tweezer experiments on DNA treated with alkylating agents, including melphalan, cisplatin, and dacarbazine. While all three drugs induce a significant increase of overstretching force and a reduction of hysteresis, suggesting stabilization of DNA against shearing forces, their effects on elasticity of DNA were quite different, with the largest change in persistence length induced by cisplatin. Furthermore, we find that these alkylating-agent-induced changes on DNA have different effects on processivity of DNA polymerase, with melphalan and cisplatin showing significantly reduced activity and dacarbazine showing little effect. Overall, our results provide new insights into the effects for these alkylating agents, which could potentially facilitate a better design of related drugs.
研究领域:
生物化学
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生物物理
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分子生物学
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药理学
·
癌症研究
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DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2022
·
IF 8.008
Lancheng Wang,Mingjie Xu,Huimin Zhou,Kun Yan,Shiqi Duan,Dandan Xue,Youmei Wang,Bin Di,Chi Hu
The target of typical PCR analysis is restricted to nucleic acids. To this end, we report here a novel strategy to simultaneously detect genetic and metabolic markers using commercial PCR kits with cucurbit[8]urils (CB[8]) implemented to manipulate the activity of Taq DNA polymerase. CB[8] binds with the nonionic surfactants and displaces them from the polymerase surface, resulting in decreased enzyme activity. Meanwhile, the inhibited enzyme can be reversibly activated when spermine, the downstream metabolite of ornithine decarboxylase (ODC), is present in the sample, which competitively binds to CB[8] and recovers polymerase activity. CB[8] was implemented in conventional PCR kits not only to reduce false-positive results but also to extend the detection range of PCR technology. With this novel method to detect ODC in cell lysates containing both the nucleotides and intracellular metabolites, positive results were only observed in highly active HEK 293T cells, whereas silent cells treated with ODC inhibitor showed negative readouts, therefore providing a simple but elegant dual-modality PCR method for precision diagnosis.
研究领域:
分子生物学
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生物技术
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分析化学
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产品关联
DOI
PubMed
高影响因子
FEBS Journal
·
2024
·
IF 5.5
Wanyi Chen,Binbin Chen,Xinjia Li,Gang Xu,Lirong Yang,Jianping Wu,Haoran Yu
Responsible for synthesizing the complementary strand of the DNA template, DNA polymerase is a crucial enzyme in DNA replication, recombination and repair. A highly conserved tyrosine (Tyr), located at the C-terminus of the O-helix in family A DNA polymerases, plays a critical role in enzyme activity and fidelity. Here, we combined the technology of genetic code extension to incorporate non-canonical amino acids and molecular dynamics (MD) simulations to uncover the mechanisms by which Tyr671 impacts substrate binding and conformation transitions in a DNA polymerase from Thermus aquaticus . Five non-canonical amino acids, namely l -3,4-dihydroxyphenylalanine ( l -DOPA), p -aminophenylalanine ( p AF), p -acetylphenylalanine ( p AcF), p -cyanophenylalanine ( p CNF) and p -nitrophenylalanine ( p NTF), were individually incorporated at position 671. Strikingly, Y671 p AF and Y671DOPA were active, but with lower activity compared to Y671F and wild-type. Y671 p AF showed a higher fidelity than the Y671F, despite both possessing lower fidelity than the wild-type. Metadynamics and long-timescale MD simulations were carried out to probe the role of mutations in affecting protein structure, including open conformation, open-to-closed conformation transition, closed conformation, and closed-to-open conformation transition. The MD simulations clearly revealed that the size of the 671 amino acid residue and interactions with substrate or nearby residues were critical for Tyr671 to determine enzyme activity and fidelity.
研究领域:
生物化学
·
分子生物学
·
结构生物学
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产品关联
DOI
PubMed
高影响因子
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