Methods in enzymology最新文献_第4页

Design, construction and characterization of laccase-xylanase chimeras by insertional fusion. 插入融合漆酶-木聚糖酶嵌合体的设计、构建与表征。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-18 DOI: 10.1016/bs.mie.2025.01.044

Lucas F Ribeiro, Gilvan P Furtado, Marcos R Lourenzoni, Richard J Ward

引用次数: 0

Preface. 前言。

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Methods in enzymology Pub Date : 2025-01-01 DOI: 10.1016/S0076-6879(25)00251-4

Timothy Bugg, Juan Carro

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Targeted genome mining for natural product discovery. 针对天然产物发现的基因组挖掘。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-04-15 DOI: 10.1016/bs.mie.2025.03.005

José D D Cediel-Becerra, Marc G Chevrette

{"title":"Targeted genome mining for natural product discovery.","authors":"José D D Cediel-Becerra, Marc G Chevrette","doi":"10.1016/bs.mie.2025.03.005","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.03.005","url":null,"abstract":"Natural products are a rich source of bioactive compounds, which are encoded by the biosynthetic gene clusters (BGCs). Genome mining is an essential strategy for identifying and characterizing BGCs. Targeted genome mining excels in the identification of similar BGCs based on key biosynthetic enzymes across multiple genomes. This chapter details the use of both manual and automated targeted genome mining to identify members of the FK-family BGCs (rapamycin, FK520/506). We describe the process of selecting query proteins, evaluating genomic context, and determining the presence of putative BGCs. Additionally, to streamline the manual process, we used GATOR-GC, a computational tool that identifies similar BGCs using required and optional proteins, performs comparative genomic analysis, deduplicates redundant BGCs, and generates visualizations of gene conservation and BGC diversity. Applying this approach, we showed how to identify FK-family members, both by looking into the cluster conservation diagrams, and the clustered heatmap summarizing all-vs-all BGC comparisons. The methods outlined here can be adapted for mining other natural product families, offering a scalable framework for uncovering novel biosynthetic pathways. Beyond natural product discovery, GATOR-GC provides broader applications for analyzing gene cluster conservation, organization, and evolutionary patterns.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"717 ","pages":"267-298"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144619077","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Editing specificity of ADAR isoforms. ADAR同工型的编辑特异性。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.021

Cornelia Vesely, Michael F Jantsch

{"title":"Editing specificity of ADAR isoforms.","authors":"Cornelia Vesely, Michael F Jantsch","doi":"10.1016/bs.mie.2024.11.021","DOIUrl":"10.1016/bs.mie.2024.11.021","url":null,"abstract":"Adenosine to inosine deaminases acting on RNA (ADARs) enzymes are found in all metazoa. Their sequence and protein organization is conserved but also shows distinct differences. Moreover, the number of ADAR genes differs between organisms, ranging from one in flies to three in mammals. The distinct isoforms of ADARs and their specific roles determine the complexity of A-to-I RNA editing, its regulation and the versatility of these enzymes. Understanding the different isoform-specific functions and targets will provide a deeper understanding of the diverse biological processes influenced by ADARs, either through ADAR editing of dsRNAs or the interaction with RNAs and proteins. The detailed identification and assigning of isoform-specific targets is a crucial step towards our understanding of functional differences amongst ADAR isoforms and will help us to understand their individual implications for health and disease. This chapter delves into unique characteristics and functional implications of ADAR isoforms. We describe the ectopic overexpression in editing free cells and the use of RNA immunoprecipitation coupled with sequencing to determine isoform-specific interactions with RNAs and their editing sites. Additionally, we discuss new challenges in editing detection by different ADARs in the context of other modifications and provide ideas for potentially better methods to determine the \"true editome\".","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"77-98"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143053006","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

TaqMan RT-qPCR for tRNA half quantification. TaqMan RT-qPCR用于tRNA半定量。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.010

Megumi Shigematsu, Takuya Kawamura, Yohei Kirino

{"title":"TaqMan RT-qPCR for tRNA half quantification.","authors":"Megumi Shigematsu, Takuya Kawamura, Yohei Kirino","doi":"10.1016/bs.mie.2024.11.010","DOIUrl":"10.1016/bs.mie.2024.11.010","url":null,"abstract":"When quantifying tRNA-derived short non-coding RNAs (sncRNAs), two key considerations must be addressed. First, sequencing analyses have revealed significant heterogeneity in the lengths and terminal sequences of tRNA-derived sncRNAs. Second, within the total RNA fraction, these sncRNAs coexist with more abundant mature tRNAs and their precursors (pre-tRNAs), which share identical sequences with the sncRNAs. While accurate quantification of individual tRNA-derived sncRNAs is crucial for research on these molecules, these two factors make it challenging to achieve with standard RT-qPCR, stem-loop RT-qPCR, and northern blot. We have developed a TaqMan RT-qPCR method that specifically quantifies tRNA half molecules. Here we describe a detailed and recently updated protocol in which an adaptor is ligated to the target tRNA half, and the TaqMan probe targets the boundaries of the tRNA half and adaptor, ensuring specific quantification without cross-reacting with corresponding mature tRNA or pre-tRNA. Our method utilizes only commercially available reagents and is broadly applicable for quantifying tRNA halves and other sncRNAs in diverse samples, including clinical specimens such as human plasma.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"155-170"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11947946/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Nanopore sequencing to detect A-to-I editing sites. 纳米孔测序检测A-to-I编辑位点。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.028

Jia Wei Joel Heng, Meng How Tan

引用次数: 0

Using CRISPR for viral nucleic acid detection. 利用CRISPR进行病毒核酸检测。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-01 DOI: 10.1016/bs.mie.2025.01.031

Maaike S Grimm, Cameron Myhrvold

{"title":"Using CRISPR for viral nucleic acid detection.","authors":"Maaike S Grimm, Cameron Myhrvold","doi":"10.1016/bs.mie.2025.01.031","DOIUrl":"10.1016/bs.mie.2025.01.031","url":null,"abstract":"Pathogenic microorganisms, such as viruses, have threatened human health and will continue to contribute to future epidemics and pandemics, highlighting the importance of developing effective diagnostics. To contain viral outbreaks within populations, fast and early diagnosis of infected individuals is essential. Although current standard methods are highly sensitive and specific, like RT-qPCR, some can have slow turnaround times, which can hinder the prevention of viral transmission. The discovery of CRISPR-Cas systems in bacteria and archaea initially revolutionized the world of genome editing. Intriguingly, CRISPR-Cas enzymes also have the ability to detect nucleic acids with high sensitivity and specificity, which sparked the interest of researchers to also explore their potential in diagnosis of viral pathogens. In particular, the CRISPR-Cas13 system has been used as a tool for detecting viral nucleic acids. Cas13's capability to detect both target RNA and non-specific RNAs has led to the development of detection methods that leverage these characteristics through designing specific detection read-outs. Optimization of viral sample collection, amplification steps and the detection process within the Cas13 detection workflow has resulted in assays with high sensitivity, rapid turnaround times and the capacity for large-scale implementation. This review focuses on the significant innovations of various CRISPR-Cas13-based viral nucleic acid detection methods, comparing their strengths and weaknesses while highlighting Cas13's great potential as a tool for viral diagnostics.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"245-275"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692834","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A quick guide to evaluating prime editing efficiency in mammalian cells. 评估哺乳动物细胞初始编辑效率的快速指南。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-31 DOI: 10.1016/bs.mie.2025.01.016

Chengfang Liu, Sifan Cheng, Junjie Zhu, Lina Zhou, Jia Chen

{"title":"A quick guide to evaluating prime editing efficiency in mammalian cells.","authors":"Chengfang Liu, Sifan Cheng, Junjie Zhu, Lina Zhou, Jia Chen","doi":"10.1016/bs.mie.2025.01.016","DOIUrl":"10.1016/bs.mie.2025.01.016","url":null,"abstract":"According to the Clinvar database, modeling the diseases associated with pathogenic mutations requires the installation of base substitutions, small insertions or deletions. Prime editor (PE) was recently developed to precisely install any base substitutions and/or small insertions/deletions (indels) in mammalian cells and animals without requiring DSBs or donor DNA templates. PE also offers greater editing and targeting flexibility compared to other precision CRISPR editing methods because the versatile editing information is encoded in the reverse-transcription template of its prime editing guide RNA. However, optimal PE system selection and experimental design can be complex, and there are various factors that can affect PE efficiency. This chapter serves as a rapid entry-level guideline for the application of PE, providing an experimental framework for using PE at a specific genomic locus. RUNX1 was selected as a representative target site to illustrate the detailed methodology for constructing PE plasmids and the process of transfecting these plasmids into 293FT cells. We further examined the efficiency of PE-mediated genome editing in mammalian cells by using next-generation sequencing.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"419-436"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692734","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Restoration of G to A mutated transcripts using the MS2-ADAR1 system. 利用MS2-ADAR1系统恢复G到A突变转录本。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-05 DOI: 10.1016/bs.mie.2024.11.031

Sonali Bhakta, Toshifumi Tsukahara

{"title":"Restoration of G to A mutated transcripts using the MS2-ADAR1 system.","authors":"Sonali Bhakta, Toshifumi Tsukahara","doi":"10.1016/bs.mie.2024.11.031","DOIUrl":"10.1016/bs.mie.2024.11.031","url":null,"abstract":"Site-directed RNA editing (SDRE) holds significant promise for treating genetic disorders resulting from point mutations. Gene therapy, for common genetic illnesses is becoming more popular and, although viable treatments for genetic disorders are scarce, stop codon mutation-related conditions may benefit from gene editing. Effective SDRE generally depends on introducing many guideRNA molecules relative to the target gene; however, large ratios cannot be achieved in the context of gene therapy applications. Gene-encoded information can be altered, and functionally diverse proteins produced from a single gene by restoration of point-mutated RNA molecules using SDRE. Adenosine deaminase acting on RNA (ADAR) is an RNA-editing enzyme, that can specifically convert adenosine (A) residues to inosines (I), which are translated as guanosine (G). MS2 system along with ADAR1 deaminase domain can target a particular A and repair G to A mutations. In this study, we used the RNA binding MS2 coat protein fused with the ADAR1 deaminase domain controlled by the CMV promoter, and a 19 bp guide RNA (complementary to the target mRNA sequence) engineered with 6 × MS2 stem-loops downstream or 1 × MS2 stem-loop (double MS2) on either side, controlled by the U6 promoter. When the EGFP TGG codon (tryptophan) was altered to an amber (TAG), opal (TGA), or ochre (TAA) stop codon, the modified ADAR1 deaminase domain could convert A-to-I (G) at the edited sites. It is anticipated that successful establishment of this technique will result in a new era in gene therapy, allowing remarkably efficient gene repair, even in vivo.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"229-240"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Drosophila melanogaster imaginal disc assays to study the polyamine transport system. 黑腹果蝇影像盘试验研究多胺转运系统。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-06 DOI: 10.1016/bs.mie.2025.01.038

Shannon L Nowotarski, Justin R DiAngelo

引用次数: 0