Frontiers in Bioengineering and Biotechnology
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2020
·
IF 3.644
Sun Jun,Lu Li-Bing,Liang Tian-Xin,Yang Li-Rong,Wu Jian-Ping
Pseudomonas putida (P. putida) KT2440 is a paradigmatic environmental-bacterium that possesses significant potential in synthetic biology, metabolic engineering and biodegradation applications. However, most genome editing methods of P. putida KT2440 depend on heterologous repair proteins and the provision of donor DNA templates, which is laborious and inefficient. In this report, an efficient cytosine base editing system was established by using cytidine deaminase (APOBEC1), enhanced specificity Cas9 nickase (eSpCas9ppD10A) and the uracil DNA glycosylase inhibitor (UGI). This constructed base editor converts C-G into T-A in the absence of DNA strands breaks and donor DNA templates. By introducing a premature stop codon in target spacers, we successfully applied this system for gene inactivation with an efficiency of 25–100% in various Pseudomonas species, including P. putida KT2440, P. aeruginosa PAO1, P. fluorescens Pf-5 and P. entomophila L48. We engineered an eSpCas9ppD10A-NG variant with a NG protospacer adjacent motif to expand base editing candidate sites. By modifying the APOBEC1 domain, we successfully narrowed the editable window to increase gene inactivation efficiency in cytidine-rich spacers. Additionally, multiplex base editing in double and triple loci was achieved with mutation efficiencies of 90–100% and 25–35%, respectively. Taken together, the establishment of a fast, convenient and universal base editing system will accelerate the pace of future research undertaken with P. putida KT2440 and other Pseudomonas species.
研究领域:
合成生物学
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代谢工程
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微生物遗传学
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生物技术
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DOI
PubMed
高影响因子
STAR Protocols
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2022
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IF 0
Meixia Wang,Fang Yan,Huanbin Zhou
Summary Base editing is a precision genome-editing approach that is widely utilized to generate single-nucleotide variants (SNVs) in genomes. Here, we present a protocol to perform targeted adenine (A)-to-guanine (G) substitution in rice using adenine base editor (ABE). We detail the design of sgRNA, CRISPR plasmid construction, rapid genetic transformation of rice, and genotyping of editing events. This protocol can be applied to cytosine base editing in rice as well. For complete details on the use and execution of this protocol, please refer to Yan et al. (2021). 1
研究领域:
植物生物学
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基因组编辑
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分子遗传学
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DOI
PubMed
JOURNAL OF CHROMATOGRAPHY A
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2024
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IF 3.8
Xiao-Dong Cheng,Zheng Zhang,Xue-xin Dai,Yun-Ping Li
A 3 μm undecylenic acid-functionalized stationary phase (UAS) was prepared for the separation of nucleosides and nucleobases using per aqueous liquid chromatography (PALC) and hydrophilic interaction liquid chromatography (HILIC). The retention behaviors of nucleosides and nucleobases in PALC and HILIC modes were explored by adjusting parameters such as water content, buffer concentration, pH of the mobile phase and column temperature. The experimental data and separation chromatogram demonstrated that PALC could provide retention comparable to that of HILIC for nucleosides and nucleobases. Comparative studies using diluted adenosine solutions evaluated theoretical plates and peak shape for the same retention factors (between 0.25 and 5.0) in PALC and HILIC. There was no buffer component in the mobile phases used to operate the comparisons. HILIC mode is more efficient for adenosine than PALC mode at low retention factors. It's the exact opposite phenomenon for high retention factors. It is proposed that the mass transfer of adenosine between the UAS, the water-rich layer and the ACN-rich mobile phase in HILIC is relatively slow. Given the significant use of toxic ACN in HILIC, PALC emerges as a safer and more effective alternative for separating nucleosides and nucleobases.
研究领域:
分析化学
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生物化学
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产品关联
DOI
PubMed
高影响因子
MOLECULES
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2017
·Vol. 22
·期 6
·918
·
IF 2.861
Shaoqing Zhu,Sheng Guo,Jin-ao Duan,Dawei Qian,Hui Yan,Xiuxiu Sha,Zhenhua Zhu
To explore the nutrients in roots ofAngelica sinensis(Angelicae Sinensis Radix, ASR), a medicinal and edible plant, and evaluate its nutritional value, a rapid and reliable UHPLC-TQ-MS method was established and used to determine the potential nutritional compounds, including nucleosides, nucleobases and amino acids, in 50 batches of ASR samples obtained using two drying methods. The results showed that ASR is a healthy food rich in nucleosides, nucleobases and amino acids, especially arginine. The total average content of nucleosides and nucleobases in all ASR samples was 3.94 mg/g, while that of amino acids reached as high as 61.79 mg/g. Principle component analysis showed that chemical profile differences exist between the two groups of ASR samples prepared using different drying methods, and the contents of nutritional compounds in samples dried with the tempering-intermittent drying processing method (TIDM) were generally higher than those dried using the traditional solar processing method. The above results suggest that ASR should be considered an ideal healthy food and TIDM could be a suitable drying method for ASR when taking nucleosides, nucleobases and amino acids as the major consideration for their known human health benefits.Keywords:Angelica sinensis;nucleosides;nucleobases;amino acids;UHPLC-TQ-MS
研究领域:
植物化学
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营养学
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中药学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS
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2013
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IF 2.947
Fengtao Li,Jin-ao Duan,Dawei Qian,Sheng Guo,Yuhua Ding,Xunhong Liu,Yefei Qian,Yunru Peng,Yijun Ren,Yu Chen
A rapid and sensitive hydrophilic interaction liquid chromatography method was developed for the identification and quantitative determination of 17 nucleosides and nucleobases by using ultra-high performance liquid chromatography separation system coupled to triple quadrupole mass spectrometer. Sufficient separation of the target compounds was achieved on an Acquity BEH Amide column (2.1 mm × 100 mm, 1.7 μm) in a single run of 9 min. The LOD and LOQ were in the range of 0.017–50.00 ng ml −1 and 0.056–146.00 ng ml −1 , respectively. Then, the method was applied for the analysis of nucleosides and nucleobases in Elaphuri Davidiani Cornu and Cervi Cornu . Multivariate statistical analysis was also used to investigate the differences in the nucleosides and nucleobases profiles among the samples. The present study also provided helpful information for quality control of Elaphuri Davidiani Cornu and Cervi Cornu .
研究领域:
分析化学
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药学
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DOI
PubMed
高影响因子
ACS Sensors
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2021
·
IF 7.711
Wenjun Zhong,Qiufang Yang,Kerui Fang,Dan Xiao,Cuisong Zhou
The simultaneous discrimination of let-7 microRNAs (miRNAs) would greatly facilitate the early diagnosis and prognosis monitoring of diseases. In this work, a molecular beacon DNA probe was designed to be able to flip out its mismatched cytosine base within the α-hemolysin (α-HL) latch and generate completely separated blocking currents to identify the single-base difference. As a result, the characteristic blocking current of fully matched MB/let-7a and single-base mismatched MB/let-7f was 84.30 ± 0.92 and 87.05 ± 0.86% (confidence level P 95%), respectively. Let-7 miRNA family let-7a and let-7f were completely simultaneously discriminated, which could be attributed to the following strengths. (1) The statistic distribution of blocking current is extremely concentrated with a small relative standard deviation (RSD) of less than 1% and a narrow distribution range. (2) Complete separation is achieved with a high separation resolution of 1.54. (3) The cytosine base flipping out within the α-HL latch provides a universal labeling-free strategy to simultaneously discriminate the single-base mismatch. Overall, the target let-7f sequences were detected with a linear range from 0.001 to 10 pM in human serum samples containing 200 nM let-7a. Great potential has been demonstrated for precise detection, early diagnosis, and prognosis monitoring of diseases related to single-base difference.
研究领域:
纳米技术
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生物物理学
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分子生物学
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医学诊断
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产品关联
DOI
PubMed
高影响因子
BIOMATERIALS
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2023
·
IF 14
Quan Zou,Yi Lu,Bo Qing,Na Li,Ting Zhou,Jinbin Pan,Xuejun Zhang,Xuening Zhang,Yupeng Chen,Shao-Kai Sun
CRISPR-based base editors (BEs) are powerful tools for precise nucleotide substitution in a wide range of organisms, but spatiotemporal control of base editing remains a daunting challenge. Herein, we develop a photoactivatable base editor (Mag-ABE) for spatiotemporally controlled genome editing in vivo for the first time. The base editing activity of Mag-ABE can be activated by blue light for spatiotemporal regulation of both EGFP reporter gene and various endogenous genes editing. Meanwhile, the Mag-ABE prefers to edit A4 and A5 positions rather than to edit A6 position, showing the potential to decrease bystander editing of traditional adenine base editors. After integration with upconversion nanoparticles as a light transducer, the Mag-ABE is further applied for near-infrared (NIR) light-activated base editing of liver in transgenic reporter mice successfully. This study opens a promising way to improve the operability, safety, and precision of base editing.
研究领域:
遗传学
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分子生物学
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生物技术
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产品关联
DOI
PubMed
高影响因子
Polymers
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2020
·Vol. 12
·期 2
·261
·
IF 3.426
Yu Ji,Qiang Yao,Weihong Cao,Yueying Zhao
The intumescent process of sodium (substituted) phenolates has been studied. The generation of hydrogen radical via a homolytic cleavage of the Ar–H bond and the subsequent hydroarylation of phenolates to cyclohexadienes along with cyclization and elimination reactions of cyclohexadienes are critical steps in the base promoted intumescence of phenols. The substituents show great influence on the intumescence of phenolates. Phenolates substituted with a weak electron donating group enable intumescence while those with an electron withdrawing group or strong electron donating group suppresses intumescence. This distinction can be justified by both electronic and steric effects of substituents on the generation of hydrogen radical and the degree of hydroarylation.Keywords:alkali;intumescence;substituent;hydroarylation;phenolate
研究领域:
化学
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材料科学
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DOI
高影响因子
JOURNAL OF ELECTROANALYTICAL CHEMISTRY
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2015
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IF 2.729
Shuang Ren,Huan Wang,Hongyi Zhang,Liqing Yu,Mingjie Li,Meng Li
Novel mesoporous carbon fibers (MCFs) were fabricated through a self-templating strategy and solution growth process. MCF modified glassy carbon electrode (MCF/GCE) was proposed for the direct electrocatalytic and simultaneous determination of four DNA bases (guanine (G), adenine (A), thymine (T) and cytosine (C)) using differential pulse voltammetry (DPV) measurement. The MCF/GCE exhibits satisfactory electrocatalytic activity by significantly enhancing the oxidation peak currents and lowering the oxidation potentials toward the four bases when compared with those on GCE. Especially, MCF greatly accelerates the electron transfer of pyrimidine base, which have high oxidation potentials and slow electron transfer kinetics. Moreover, MCF/GCE realizes the simultaneous determination of four DNA bases with low detection limits, high sensitivity and wide linear range. More important, the proposed electrode was successfully applied in the direct detection of bases in herring sperm DNA sample with satisfactory results. Besides, the MCF/GCE shows good long-term stability and reproducibility. These superior performances of MCF/GCE toward purine and pyrimidine DNA bases provide a good possibility for applying the MCF/GCE in our further DNA biosensor research.
研究领域:
生物化学
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分析化学
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DOI
高影响因子
SEPARATION SCIENCE AND TECHNOLOGY
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2015
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IF 1.171
Yuanchao Pei,Jianzhou Zhang,Xinxin Song,Ming Zhao,Jianji Wang
In this paper, a series of ionic liquids (ILs) contains cholinium as cations were prepared. These ILs were used as extractants to separate some model drug molecules. The partition coefficients of these drug molecules between ILs and aqueous solutions were determined. Furthermore, the influence of different extraction parameters was investigated: volume ratio, equilibrium time, pH values, temperature, and ILs structure. It is shown that these ILs are highly effective materials for the extraction of drug molecules. The results imply that these ILs may have potential in drug extraction or separation as a new type of media.
研究领域:
药学
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化学
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DOI
高影响因子
Analytical Methods
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2013
·Vol. 5
·期 22
·6395-6400
·
IF 1.855
Yilin Pan,Ping Xue,Xiang Li,Jianwei Chen,Jin Li
To establish an ultra-high performance liquid chromatography coupled with triple quadrupole mass (HILIC-UPLC-MS/MS) for determination of sixteen nucleosides and nucleobases in Isatidis Radix. Chromatographic separation was carried out on a Acquity UPLC BEH Amide column with gradient elution of acetonitrile (containing 0.1% acetic acid) and water (containing 0.8% acetic acid and 10 mmol L−1 ammonium acetate) at a flow rate of 0.3 mL min−1, the column temperature was set at 35 °C; Waters Xevo™ TQ worked in multiple reaction monitoring mode. Each component were separating in 11 min. All calibration curves were linear (r2 > 0.997) over the tested ranges. The limits of detection of these compounds were 0.02–42.54 ng mL−1. The limits of quantitation of these compounds were 0.05–98.18 ng mL−1. The average recoveries were in the range of 93.81–105.77% with RSD value less than 7.5%. The result showed that almost all of these Isatidis Radix were rich in nucleosides and nucleobases, but their contents were obviously various. The method was simple and fast with high precision, sensitivity and repeatability, which can be used for determination of this type class of nucleosides and nucleobases in other medicinal herbs.
研究领域:
药学
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分析化学
✓ 已验证
DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2013
·
IF 3.535
Xijing Huang,Yanlin Hao,Hanyin Wu,Qingquan Guo,Liangqia Guo,Jing Wang,Liangshuang Zhong,Tianran Lin,FengFu Fu,Guonan Chen
A simple and sensitive DNAzyme-based colorimetric sensor for detecting Hg 2+ based on T–Hg 2+ -T base pair and magnetic beads (MBs) is proposed. In the presence of Hg 2+ , capture probes (CPs) immobilized on MBs are hybridized with the signal probes (SPs) consisting of G-quadruplex sequences with the help of T–Hg 2+ -T base pairs. The SPs are separated by MBs and used to bind hemin for the formation of catalytically active hemin/G-quadruplex DNAzymes, which catalyzes the H 2 O 2 -mediated oxidation of ABTS to produce a blue-green-colored product. A linear relationship between the absorbance and Hg 2+ concentration is observed over a range of 10–200 nM with a limit of detection 5 nM. Taking advantage of MBs separation/collection process, the sensing system can be regenerated by using EDTA as the chelator.
研究领域:
生物化学
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环境科学
·
分析化学
✓ 已验证
产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）