ANALYTICAL CHEMISTRY
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2021
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IF 6.986
Jinhua Shang,Shanshan Yu,Yingying Chen,Yuhui Gao,Chen Hong,Fengzhe Li,Fuan Wang
Polynucleotide kinase (PNK) shows an in-depth correlationship with DNA repair and metabolism processes. The in situ visualization of intracellular PNK revealed an extremely biological significance in supplementing reliable and quantitative information on its spatiotemporal distribution in live cells. Herein, we developed a versatile cascaded DNA amplification circuit through the integration of catalytic DNA assembly and hybridization chain reaction circuits and realized the accurate evaluation of intracellular PNK activity via the Förster resonance energy transfer (FRET) principle. Initially, without PNK, trigger T was firmly caged in the PNK-recognizing hairpin HT, resulting in no disturbance of the concatenated circuit. However, with the introduction of PNK, the 5′-OH terminal of PNK-addressing HT was phosphorylated, then the phosphorylated HT could be subsequently digested by λ exonuclease (λ Exo) to produce trigger T of the cascaded DNA circuit. As a result, the integrated circuit was stimulated to produce an amplified FRET signal for quantitatively monitoring the activity of PNK. Due to the λ Exo-specific digestion of 5′-phosphate DNA and the high signal gain of the cascade circuit, our proposed strategy enables the sensitive analysis of PNK activity in vitro and in complex biological samples. Furthermore, our PNK-sensing platform was extensively explored in HeLa cells for realizing reliable intracellular PNK imaging and thus showed high potential in the future diagnosis and treatment of kinase-related diseases.
研究领域:
分子生物学
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生物化学
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医学诊断
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DOI
PubMed
高影响因子
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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2021
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IF 3.575
Ligang Xing,Yanmei Zhao,Mingzhu Gong,Xia Liu,Yuhui Zhang,Dan Li,Zefeng He,Ping Yan,Jidong Yang
In this study, Graphene Oxide (GO) was used to screen the binding with the aptamers of L-carnitine chiral enantiomers . The ssDNA library was prepared by the method of Lambda exonuclease . In addition, a simple casing device was designed to improve the purification and recovery efficiency of the small ssDNA fragments in the process of screening. Finally, more than 160,000 aptamer sequences were obtained by high-throughput sequencing. We determined the strongest affinity aptamer sequence, CA04, by the Resonance Rayleigh scattering (RRS) technology. We also analyzed the key binding sites (in the 16th position case) of the truncated aptamer sequence CAD10. Interestingly, we found that aptamer CA10 and CA06 were both C-rich bases through sequence alignment and analysis, and the aptamer CA10 was confirmed that the CA10 and CA06 were formed under acidic conditions (pH 4.5) by CD spectrum and ESI-MS analysis. The interaction between gold nanoparticles (AuNPs) and functionalized aptamer CA10 was analyzed. We used Site-directed mutagenesis design and QGRS Mapper to optimize aptamer CA10, where an optimal aptamer CA10-03 were obtained after affinity analysis. It is also proved to be an effective method to obtain stronger affinity aptamer. Meanwhile, Native-PAGE and UV spectrum analysis were performed on the mutation sequences, and the interaction with ThT was analyzed. Finally, it is hoped that my study can provide help for later identification and detection of L-carnitine .
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 Analysis and Testing
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2024
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IF 5.5
Xiao Qi,Liu Shuai,Wang Yali,Li Huihao,Yang Mingli,Fang Yi,Chen Sijing,Huang Shan
Human T-cell lymphophilic virus type 1 (HTLV-1), the known retrovirus causing cancer in humans, is closely associated with adult T-cell leukemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis. Due to its ability to evade the host's defense mechanisms, early tracking of HTLV-1 becomes crucial. In this study, we integrate λ-Exonuclease (λ-Exo)-assisted target recycling with a terminal deoxynucleotidyl transferase (TdT)-mediated template-free DNA extension process to develop an electrochemical analysis platform for the specific and sensitive detection of HTLV-1 DNA. During the λ-Exo-assisted target recycling, HTLV-1 DNA is recognized by hairpin DNA (Hp-DNA), forming double-stranded DNA (dsDNA) through DNA hybridization. The dsDNA, featuring blunt 5' terminal phosphorylation, is cleaved by λ-Exo, generating abundant short output sequence (sDNA). HTLV-1 DNA is released, initiating a cyclic hybridization-cleavage process. Subsequently, thiol-labelled capture DNA (CP-DNA) assembled on gold electrode surface captures a substantial amount of the generated sDNA, forming CP-DNA-sDNA nanostructures. When TdT and dNTPs are present on the electrode surface, the 3'-OH terminal of sDNA extends to generate long single-stranded DNA (ssDNA) structure. Methylene blue (MB) is selected as the electrochemical signal molecule. MB not only binds with ssDNA but also interacts specifically with dsDNA, resulting in a significantly enhanced electrochemical signal on modified electrode surface. The detection limit of HTLV-1 DNA is as low as 19 amol/L ( S / N = 3) when the two signal amplification strategies are combined. The analysis platform exhibits excellent analytical performance and holds promise as a novel tool for the early tracing and diagnosis of HTLV-1 DNA.
研究领域:
分子生物学
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医学诊断
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生物化学
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高影响因子
Analytical Methods
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2016
·Vol. 8
·期 20
·4119-4126
·
IF 1.915
Lin Lin,Dongmin Shi,Quanfa Li,Guangfeng Wang,Xiaojun Zhang
Determination of nucleotide kinase activity is valuable due to its importance in regulating nucleic acid metabolism. In this work, a novel and simple colorimetric method for discriminative detection of T4 polynucleotide kinase (PNK) through a MnO2 nanosheet-3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric system is developed. In aqueous solution at pH 7.4, the MnO2 nanosheet, a mimic enzyme, rapidly converts colourless TMB to a blue oxidized form. In the presence of T4PNK, a hairpin DNA could be phosphorylated and further cleaved by λ exonuclease (λ exo) and the ssDNA fragments would adsorb on the surface of the MnO2 nanosheet, resulting in the inhibition of the catalytic effect of the MnO2 nanosheet to the oxidation of TMB and weakening of the blue color of oxidized TMB with the absorption peak centred at 652 nm. The presence of T4PNK can in this way easily be detected with the naked eye, based on the obvious colour change. The experimental results revealed that the advanced strategy was sensitive for detecting T4PNK in the concentration range from 0.005 to 10 U mL−1 with a detection limit of 0.005 U mL−1. In addition, the relative standard deviation was 3.92% in 3 repetitive assays of 10 U mL−1 T4PNK, indicating that the reproducibility of this strategy was acceptable. By comparison with other relevant fluorogenic assays, the sensitivity of the developed chromogenic assays was satisfactory. The present approach opens the possibility for the design of simple and sensitive colorimetric assays for polynucleotide kinase using inexpensive and available TMB as a universal chromogenic compound. Therefore, besides desirable sensitivity and selectivity, due to the colorimetric system, the developed method for T4PNK detection also showed cost-effective, reliable and simplified operations and strategy, which has considerable potential for biological process research.
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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高影响因子
JOURNAL OF ELECTROANALYTICAL CHEMISTRY
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2020
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IF 3.807
Jingxiu Mao,Xin Chen,Huahuang Xu,Xueqin Xu
T4 polynucleotide kinase (T4 PNK), a bifunctional repair enzyme, catalyzes the transfer of γ-phosphate residues from adenosine triphosphate (ATP) to 5′- hydroxyl termini in nucleic acids，which plays an essential role in regulating DNA phosphorylation modes during the repair of damaged DNA. Herein, a sensitive DNAzyme-driven DNA walker biosensor was developed for amplified electrochemical detection of T4 PNK activity and inhibition based on T4 PNK-triggered phosphorylation reaction and λ exonuclease (λ exo) cleavage. A novel functional hairpin-shaped swing arm DNA (SA) containing a DNA sequence of the Pb 2+ -dependent DNAzyme is designed to specifically bind to and hydrolyze the substrate strand (rSS) sequence that can drive DNA walker. Upon the reaction of SA， T4 PNK and ATP, λ exo removes the mononucleotides from the stem of the phosphorylated SA, unfolding the hairpin structure and initiating DNAzyme-driven DNA walker. Subsequently, the DNA walker can repeatedly bind and cleave rSS with the aid of Pb 2+ , releasing the methylene blue (MB) - labeled fragment of rSS. The T4 PNK activity can be facilely monitored by recording the electrochemical signal change of MB. The proposed biosensor exhibited a broad linear range from 0.01 U mL −1 to 15 U mL −1 with a low detection limit of 0.001 U mL −1 . The proposed biosensor has also been utilized to screen the T4 PNK inhibitors and exhibited good performance in real sample analysis, making such a strategy hold potential promise in biological fields and clinical diagnosis.
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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临床诊断
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高影响因子
Biosensors-Basel
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2023
·Vol. 13
·期 10
·918
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IF 5.4
Zhen Liu,Chen Liu,Liqiong He,Jinquan Liu,Le Li,Shengyuan Yang,Yan Tan,Xing Liu,Xilin Xiao
Herein, an ultrasensitive DNAzyme-based fluorescence biosensor for detecting Cu2+was designed using the cascade signal amplification strategy, coupling λ-exonuclease-assisted target recycling and mismatched catalytic hairpin assembly (MCHA). In the designed detection system, the target, Cu2+, can activate the Cu2+-dependent DNAzyme to cause a cleavage reaction, releasing ssDNA (tDNA). Then,tDNA binds to hairpin DNA (H0) with an overhanging 5′-phosphorylated terminus to form dsDNA with a blunt 5′-phosphorylated terminus, which activates the dsDNA to be digested by λ-Exo and releasestDNA along with another ssDNA (iDNA). Subsequently, theiDNA initiates MCHA, which can restore the fluorescence of carboxyfluorescein (FAM) previously quenched by tetramethylrhodamine (TAMRA), resulting in a strong fluorescent signal. Furthermore, MCHA efficiently improves the signal-to-noise ratio of the detection system. More importantly,tDNA recycling can be achieved with the λ-Exo digestion reaction to release moreiDNA, efficiently amplifying the fluorescent signal and further improving the sensitivity to Cu2+with a detection limit of 60fM. The practical application of the developed biosensor was also demonstrated by detecting Cu2+in real samples, proving it to be an excellent analytical strategy for the ultrasensitive quantification of heavy metal ions in environmental water sources.Keywords:Cu2+detection;λ-exonuclease-assisted target recycling;mismatched catalytic hairpin assembly;cascade signal amplification;environmental pollutant
研究领域:
生物化学
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环境科学
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分析化学
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PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2019
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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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高影响因子
ANALYTICAL CHEMISTRY
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2018
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IF 6.042
Lin Cui,Juan Hu,Meng Wang,Xing-kang Diao,Chen-chen Li,Chun-yang Zhang
We demonstrate for the first time the development of a mimic peroxidase- and bismuth sulfide (Bi2S3) nanorod-based photoelectrochemical (PEC) biosensor for signal-on detection of polynucleotide kinase (PNK) on the basis of manganese-based mimic enzyme (MnME) catalytic precipitation. We use the hybrid film which consists of Bi2S3 nanorods and Au nanoparticles (AuNPs) as the immobilization matrix of capture probe. The capture probe on the modified electrode can specifically hybridize with the MnME@AuNPs-labeled signal probe to form the double-stranded DNA (dsDNA), generating a PEC biosensor. In the absence of PNK, MnME may stimulate the mimic enzyme catalytic precipitation onto the electrode surface, blocking the interfacial electron transfer and eventually leading to a low PEC signal. While in the presence of PNK, the dsDNA is phosphorylated and subsequently cleaved by lambda exonuclease to release the MnME@AuNPs conjugates from the electrode, leading to the decrease of catalytic precipitation on the surface of electrode and consequently the production of a high PEC signal. Notably, the MnME can be easily synthesized and possesses higher catalytic activity than the manganese-based mimic enzyme. This signal-on PEC biosensor exhibits high sensitivity with a detection limit of 1.27 × 10–5 U mL–1 and an extrembly large dynamic range of 5 orders of magnitude. Moreover, it can be applied for the screening of PNK inhibitors and accurate quantification of PNK activity in cancer cell extracts.
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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癌症诊断
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PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2013
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IF 5.695
Wenjiao Zhou,Xue Gong,Yun Xiang,Ruo Yuan,Yaqin Chai
The employment of DNAzyme probes for visual biodetections has received increasing interest recently due to the simple nature of this type of assay. However, achieving high sensitivity and detecting targets beyond nucleic acids remain two major challenges in DNAzyme-based visual detections. In this work, based on a new quadratic amplification strategy, we developed a sensitive and visual detection method for cytokines by using hairpin aptamer DNAzyme probes. The target cytokine, interferon γ (IFN-γ), associates with the aptamer sequences and unfolds the hairpin structure of the probes, leading to simultaneous recycling of the target IFN-γ (assisted by Bst-polymerase) and the DNA sequences (aided by λ exonuclease) to achieve quadratic amplification. This quadratic amplification results in the generation of numerous peroxidase-mimicking DNAzymes, which cause significantly intensified color change of the probe solution for highly sensitive detection of IFN-γ by the naked eye down to 50 pM. The proposed visual sensing method shows also high selectivity toward the target IFN-γ and can be performed in homogeneous solutions with using completely unmodified, synthetic aptamer DNAzyme probes. These distinct advantages of our developed assay protocol make it a potential platform for detecting various types of biomolecules with careful probe designs.
研究领域:
生物化学
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分析化学
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分子生物学
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生物技术
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DOI
PubMed
高影响因子
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