BIOCHEMISTRY
·
2018
·
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
·
生物化学
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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
·
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
·
生物化学
·
纳米技术
·
病毒学
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产品关联
DOI
高影响因子
BIOORGANIC CHEMISTRY
·
2022
·
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
·
生物化学
·
遗传学
·
DNA修复
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DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2019
·
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
·
生物技术
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产品关联
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
·
生物物理
·
分子生物学
·
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
·
生物技术
·
分析化学
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产品关联
DOI
PubMed
高影响因子
Biosensors-Basel
·
2024
·Vol. 14
·期 12
·618
·
IF 4.9
Shurui Tao,Yi Long,Guozhen Liu
The Phi29 DNA polymerase is renowned for its processivity in synthesizing single-stranded DNA amplicons by rolling around a circularized DNA template. However, DNA synthesis rolling circle amplification (RCA) is significantly hindered by the secondary structure in the circular template. To overcome this limitation, an engineered circular template without secondary structure could be utilized to improve the sensitivity of RCA-based assays without increasing its complexity. We herein proposed an entropy-driven special RCA technology for the detection of HPV16 E7 gene at room temperature. The strategy is composed of a molecular beacon containing a loop region for nucleic acid target recognition and a stem region to initiate RCA. With the target analyte, the stem region of the molecular beacon will be exposed and then hybridized with a special circular template to initiate the DNA amplification. We tested different designs of the molecular beacon sequence and optimized the assay’s working conditions. The assay achieved a sensitivity of 1 pM in 40 min at room temperature. The sensitivity of this assay, at 1 pm, is about a hundred-fold greater than that of conventional linear RCA performed in solution. Our proposed sensor can be easily reprogrammed for detecting various nucleic acid markers by altering the molecular beacon’s loop. Its simplicity, rapid assay time, and low cost make it superior to RCA sensors that utilize similar strategies.
研究领域:
分子生物学
·
生物技术
·
纳米技术
·
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
CHEMICAL ENGINEERING JOURNAL
·
2022
·
IF 16.744
Xinglu Jiang,Kangjing Li,Beibei Xie,Jun Zhou
Taq DNA polymerase is the most widely used enzyme for PCR amplification and it contains 832 amino acids but no cysteines. Bovine serum albumin (BSA) is an 832-amino acid protein containing 35 cysteines, and at high temperature BSA can produce protein hydrogels through recombined disulfides and noncovalent bonds. When hydrogels are prepared using a mixture of Taq DNA polymerase and BSA, no disulfide bonds occur between them, and noncovalent bonds are the main forces holding together the Taq DNA polymerase with the hydrogels. During PCR amplification, temperature cycling can destroy the noncovalent bonds, thereby releasing Taq DNA polymerase from the hydrogels to synthesize DNA. The release rate, regulated through the variation of BSA concentration, allows for velocity-controlled amplification of DNA. As the amount of DNA products exceeds that of the Taq DNA polymerase released from hydrogels, a linear amplification replaces the exponential PCR amplification. When the amplification velocity begins to decrease, the time needed for reaching a plateau increase. This provides a broader detection window for the analysis of the end products, which correspond to the difference compared with the initial DNA concentration. The number of primers is continuously reduced with increasing cycles, which further causes a decline in the DNA amplification efficiency. The difference in the initial DNA template may cause a different consumption of primers, which can cause errors in the Ct values in the case of qPCR. Based on the slopes of the linear amplification curve, the Ct values can be calibrated to reduce this error, and a more accurate detection of the initial content of the target DNA can be implemented.
研究领域:
分子生物学
·
生物技术
·
生物化学
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DOI
高影响因子
NATURE BIOTECHNOLOGY
·
2021
·Vol. 39
·期 12
·1548-1555
·
IF 54.908
Fan Chuyao,Deng Qiang,Zhu Ting F.
Natural DNA is exquisitely evolved to store genetic information. The chirally inverted l -DNA, possessing the same informational capacity but resistant to biodegradation, may serve as a robust, bioorthogonal information repository. Here we chemically synthesize a 90-kDa high-fidelity mirror-image Pfu DNA polymerase that enables accurate assembly of a kilobase-sized mirror-image gene. We use the polymerase to encode in l -DNA an 1860 paragraph by Louis Pasteur that first proposed a mirror-image world of biology. We realize chiral steganography by embedding a chimeric d -DNA/ l -DNA key molecule in a d -DNA storage library, which conveys a false or secret message depending on the chirality of reading. Furthermore, we show that a trace amount of an l -DNA barcode preserved in water from a local pond remains amplifiable and sequenceable for 1 year, whereas a d -DNA barcode under the same conditions could not be amplified after 1 day. These next-generation mirror-image molecular tools may transform the development of advanced mirror-image biology systems and pave the way for the realization of the mirror-image central dogma and exploration of their applications.
研究领域:
分子生物学
·
合成生物学
·
生物技术
·
信息存储
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产品关联
DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2019
·Vol. 186
·期 9
·1-9
·
IF 5.479
Chen Dongli,Zhang Meng,Zhou Fenyue,Hai Hong,Li Jianping
An electrochemiluminescent (ECL) biosensor is described for the determination of the breast cancer biomarker microRNA. The method is based on the amplification via target cyclic regeneration through a system of hairpin DNA probes, primers, and Klenow fragment of DNA polymerases combined with CdTe quantum dots (QDs) and gold nanoparticles. The assay is performed by exploiting the luminescence properties of CdTe-QDs and K 2 S 2 O 8 as a co-reactive agent to increase the ECL signal. It was successfully applied to ECL-based detection of a 20-mer microRNA. The sensor has a linear response in the 0.1 fM to 0.2 pM microRNA concentration range and a detection limit as low as 33 aM. The assay has been applied to the determination of microRNA spiked in serum samples, and recoveries ranged from 94.4 to 100.5%.
A novel electroluminescence biosensor based on the amplification of target cyclic regeneration is described. It is achieved by using a system of hairpin DNA probes, primers, and Klenow fragment of DNA polymerases combined with CdTe QDs and Au NPs, and was successfully applied to microRNA detection.
研究领域:
生物传感器
·
癌症研究
·
纳米技术
·
分子诊断
·
电化学
✓ 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）