Chemical & Biomedical Imaging最新文献_第10页

Duplex Unwinding Mechanism of Coronavirus MERS-CoV nsp13 Helicase 冠状病毒MERS-CoV nsp13解旋酶的双解旋机制

Chemical & Biomedical Imaging Pub Date : 2024-12-19 DOI: 10.1021/cbmi.4c0007710.1021/cbmi.4c00077

Wei Hao, Xiao Hu, Qixin Chen, Bo Qin, Zhiqi Tian, Ziheng Li, Pengjiao Hou, Rong Zhao, Hamza Balci*, Sheng Cui* and Jiajie Diao*,

{"title":"Duplex Unwinding Mechanism of Coronavirus MERS-CoV nsp13 Helicase","authors":"Wei Hao, Xiao Hu, Qixin Chen, Bo Qin, Zhiqi Tian, Ziheng Li, Pengjiao Hou, Rong Zhao, Hamza Balci*, Sheng Cui* and Jiajie Diao*, ","doi":"10.1021/cbmi.4c0007710.1021/cbmi.4c00077","DOIUrl":"https://doi.org/10.1021/cbmi.4c00077https://doi.org/10.1021/cbmi.4c00077","url":null,"abstract":"The COVID-19 pandemic has underscored the importance of in-depth research into the proteins encoded by coronaviruses (CoV), particularly the highly conserved nonstructural CoV proteins (nsp). Among these, the nsp13 helicase of severe pathogenic MERS-CoV, SARS-CoV-2, and SARS-CoV is one of the most preserved CoV nsp. Utilizing single-molecule FRET, we discovered that MERS-CoV nsp13 unwinds DNA in distinct steps of about 9 bp when ATP is employed. If a different nucleotide is introduced, these steps diminish to 3–4 bp. Dwell-time analysis revealed 3–4 concealed steps within each unwinding process, which suggests the hydrolysis of 3–4 dTTP. Combining our observations with previous studies, we propose an unwinding model of CoV nsp13 helicase. This model suggests that the elongated and adaptable 1B-stalk of nsp13 may enable the 1B remnants to engage with the unwound single-stranded DNA, even as the helicase core domain has advanced over 3–4 bp, thereby inducing accumulated strain on the nsp13-DNA complex. Our findings provide a foundational framework for determining the unwinding mechanism of this unique helicase family.","PeriodicalId":53181,"journal":{"name":"Chemical & Biomedical Imaging","volume":"3 2","pages":"111–122 111–122"},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/cbmi.4c00077","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143473686","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Duplex Unwinding Mechanism of Coronavirus MERS-CoV nsp13 Helicase. 冠状病毒MERS-CoV nsp13解旋酶的双解旋机制

Chemical & Biomedical Imaging Pub Date : 2024-12-19 eCollection Date: 2025-02-24 DOI: 10.1021/cbmi.4c00077

Wei Hao, Xiao Hu, Qixin Chen, Bo Qin, Zhiqi Tian, Ziheng Li, Pengjiao Hou, Rong Zhao, Hamza Balci, Sheng Cui, Jiajie Diao

{"title":"Duplex Unwinding Mechanism of Coronavirus MERS-CoV nsp13 Helicase.","authors":"Wei Hao, Xiao Hu, Qixin Chen, Bo Qin, Zhiqi Tian, Ziheng Li, Pengjiao Hou, Rong Zhao, Hamza Balci, Sheng Cui, Jiajie Diao","doi":"10.1021/cbmi.4c00077","DOIUrl":"10.1021/cbmi.4c00077","url":null,"abstract":"The COVID-19 pandemic has underscored the importance of in-depth research into the proteins encoded by coronaviruses (CoV), particularly the highly conserved nonstructural CoV proteins (nsp). Among these, the nsp13 helicase of severe pathogenic MERS-CoV, SARS-CoV-2, and SARS-CoV is one of the most preserved CoV nsp. Utilizing single-molecule FRET, we discovered that MERS-CoV nsp13 unwinds DNA in distinct steps of about 9 bp when ATP is employed. If a different nucleotide is introduced, these steps diminish to 3-4 bp. Dwell-time analysis revealed 3-4 concealed steps within each unwinding process, which suggests the hydrolysis of 3-4 dTTP. Combining our observations with previous studies, we propose an unwinding model of CoV nsp13 helicase. This model suggests that the elongated and adaptable 1B-stalk of nsp13 may enable the 1B remnants to engage with the unwound single-stranded DNA, even as the helicase core domain has advanced over 3-4 bp, thereby inducing accumulated strain on the nsp13-DNA complex. Our findings provide a foundational framework for determining the unwinding mechanism of this unique helicase family.","PeriodicalId":53181,"journal":{"name":"Chemical & Biomedical Imaging","volume":"3 2","pages":"111-122"},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11863148/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143525159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Mini crRNA-Mediated CRISPR/Cas12a System Enhanced Imaging of Multiple MicroRNAs in Cells Mini crrna介导的CRISPR/Cas12a系统增强了细胞中多种microrna的成像

Chemical & Biomedical Imaging Pub Date : 2024-12-19 DOI: 10.1021/cbmi.4c0008910.1021/cbmi.4c00089

Xiaolong Chen, Qianyi You, Yawen Guo, Jing Zhang, Qiao Hu, Chaowang Huang, Zhili Wu, Junjiang Deng, Jing Xu, Peng Zhao* and Mingdong Hu*,

{"title":"Mini crRNA-Mediated CRISPR/Cas12a System Enhanced Imaging of Multiple MicroRNAs in Cells","authors":"Xiaolong Chen, Qianyi You, Yawen Guo, Jing Zhang, Qiao Hu, Chaowang Huang, Zhili Wu, Junjiang Deng, Jing Xu, Peng Zhao* and Mingdong Hu*, ","doi":"10.1021/cbmi.4c0008910.1021/cbmi.4c00089","DOIUrl":"https://doi.org/10.1021/cbmi.4c00089https://doi.org/10.1021/cbmi.4c00089","url":null,"abstract":"RNA imaging in live cells can provide comprehensive information on the expression, localization, degradation, storage, and regulation of RNA in cells, which is crucial for basic biology and clinical research. Our previous research finds that slicing the facilitated crRNA in the typical CRISPR/Cas12a system at a fitted site did not affect its trans-cleavage activity, which was previously reported to be triggered by targeted ssDNA or dsDNA, and a mini crRNA-mediated CRISPR/Cas12a (MCM-CRISPR/Cas12a) system was proposed. Here, we further apply it to enhanced imaging of MicroRNAs in cells by designing the activator in the system as a molecular beacon (MB), which can form a hybrid double-stranded structure of DNA/RNA with the targeted MicroRNA. When targeted MicroRNA is present, the hairpin structure of the MB is opened and the system emits fluorescence. Simultaneously, the DNA-RNA formed by the targeted MicroRNA and MB activates the trans-cleavage activity of LbCas12a, partially cleaving the single-stranded DNA extended from the MB and further enhancing the fluorescence intensity of the system. We designed the MCM-CRISPR/Cas12a system for miRNA-21, miRNA-155, and miRNA-10b and successfully applied it for sensitive and specific imaging of these MicroRNAs both inside and outside cells. This study provides a new idea for the sensitive and specific imaging of multiple MicroRNAs within cells, which is important for studying the distribution and dynamic changes of MicroRNAs in cells.","PeriodicalId":53181,"journal":{"name":"Chemical & Biomedical Imaging","volume":"3 6","pages":"369–378 369–378"},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/cbmi.4c00089","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144337995","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Long Fluorescence Lifetime Probe for Labeling of Gram-Negative Bacteria. 一种用于标记革兰氏阴性菌的长荧光寿命探针。

Chemical & Biomedical Imaging Pub Date : 2024-12-05 eCollection Date: 2025-01-27 DOI: 10.1021/cbmi.4c00066

Assel Baibek, Zuzanna Konieczna, Muhammed Üçüncü, Zainab S Alghamdi, Richa Sharma, Mathew H Horrocks, Mark Bradley

{"title":"A Long Fluorescence Lifetime Probe for Labeling of Gram-Negative Bacteria.","authors":"Assel Baibek, Zuzanna Konieczna, Muhammed Üçüncü, Zainab S Alghamdi, Richa Sharma, Mathew H Horrocks, Mark Bradley","doi":"10.1021/cbmi.4c00066","DOIUrl":"10.1021/cbmi.4c00066","url":null,"abstract":"Bacterial resistance, primarily stemming from misdiagnosis, misuse, and overuse of antibacterial medications in humans and animals, is a pressing issue. To address this, we focused on developing a fluorescent probe for the detection of bacteria, with a unique feature-an exceptionally long fluorescence lifetime, to overcome autofluorescence limitations in biological samples. The polymyxin-based probe (ADOTA-PMX) selectively targets Gram-negative bacteria and used the red-emitting fluorophore azadioxatriangulenium (with a reported fluorescence lifetime of 19.5 ns). Evaluation of ADOTA-PMX's bacterial labeling efficacy revealed strong specificity for Gram-negative bacteria, and full spectral fluorescence lifetime imaging microscopy demonstrated the potential of ADOTA-PMX for bacterial imaging applications. The probe exhibited a lifetime of 4.5 ns when bound to bacteria, possibly indicating interactions with the bacterial outer membrane. Furthermore, the fluorescence lifetime measurements of ADOTA-PMX labeled bacteria could be performed using a benchtop fluorimeter without the need of sophisticated microscopes. This study represents the first targeted probe for fluorescence lifetime imaging, offering sensitivity for detecting Gram-negative bacteria and enabling multiplexing via fluorescence lifetime imaging.","PeriodicalId":53181,"journal":{"name":"Chemical & Biomedical Imaging","volume":"3 1","pages":"45-50"},"PeriodicalIF":0.0,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11775855/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143069088","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Long Fluorescence Lifetime Probe for Labeling of Gram-Negative Bacteria 一种用于标记革兰氏阴性菌的长荧光寿命探针

Chemical & Biomedical Imaging Pub Date : 2024-12-05 DOI: 10.1021/cbmi.4c0006610.1021/cbmi.4c00066

Assel Baibek, Zuzanna Konieczna, Muhammed Üçüncü, Zainab S. Alghamdi, Richa Sharma, Mathew H. Horrocks and Mark Bradley*,

{"title":"A Long Fluorescence Lifetime Probe for Labeling of Gram-Negative Bacteria","authors":"Assel Baibek, Zuzanna Konieczna, Muhammed Üçüncü, Zainab S. Alghamdi, Richa Sharma, Mathew H. Horrocks and Mark Bradley*, ","doi":"10.1021/cbmi.4c0006610.1021/cbmi.4c00066","DOIUrl":"https://doi.org/10.1021/cbmi.4c00066https://doi.org/10.1021/cbmi.4c00066","url":null,"abstract":"Bacterial resistance, primarily stemming from misdiagnosis, misuse, and overuse of antibacterial medications in humans and animals, is a pressing issue. To address this, we focused on developing a fluorescent probe for the detection of bacteria, with a unique feature─an exceptionally long fluorescence lifetime, to overcome autofluorescence limitations in biological samples. The polymyxin-based probe (ADOTA-PMX) selectively targets Gram-negative bacteria and used the red-emitting fluorophore azadioxatriangulenium (with a reported fluorescence lifetime of 19.5 ns). Evaluation of ADOTA-PMX’s bacterial labeling efficacy revealed strong specificity for Gram-negative bacteria, and full spectral fluorescence lifetime imaging microscopy demonstrated the potential of ADOTA-PMX for bacterial imaging applications. The probe exhibited a lifetime of 4.5 ns when bound to bacteria, possibly indicating interactions with the bacterial outer membrane. Furthermore, the fluorescence lifetime measurements of ADOTA-PMX labeled bacteria could be performed using a benchtop fluorimeter without the need of sophisticated microscopes. This study represents the first targeted probe for fluorescence lifetime imaging, offering sensitivity for detecting Gram-negative bacteria and enabling multiplexing via fluorescence lifetime imaging.","PeriodicalId":53181,"journal":{"name":"Chemical & Biomedical Imaging","volume":"3 1","pages":"45–50 45–50"},"PeriodicalIF":0.0,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/cbmi.4c00066","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143087372","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Chemical & Biomedical Imaging Pub Date : 2024-11-25

Xiaotong Chen, Yajing Jiang, Jiaxin Liu, Yu Tian, Yifan Deng, Xiaoqiong Li*, Wenbo Wu*, Ruoyu Zhang* and Yulin Deng*,

引用次数: 0

Chemical & Biomedical Imaging Pub Date : 2024-11-25

Giulia Tedeschi, Mariana X. Navarro, Lorenzo Scipioni, Tanvi K. Sondhi, Jennifer A. Prescher* and Michelle A. Digman*,

引用次数: 0

Chemical & Biomedical Imaging Pub Date : 2024-11-25

Richard Ortega, Mónica Fernández-Monreal, Noémie Pied, Stéphane Roudeau, Peter Cloetens and Asuncion Carmona*,

引用次数: 0

Chemical & Biomedical Imaging Pub Date : 2024-11-25

William J. Tipping*, Karen Faulds and Duncan Graham*,