Methods in enzymology最新文献_第4页

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

4区生物学

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。

4区生物学

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。

4区生物学

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。

4区生物学

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

Methylated polyamines derivatives and antizyme-related effects. 甲基化多胺衍生物和抗酶相关作用。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-21 DOI: 10.1016/bs.mie.2025.01.072

Maxim A Khomutov, Arthur I Salikhov, Olga A Smirnova, Vladimir A Mitkevich, Alex R Khomutov

引用次数: 0

Targeting polyamine metabolism in an ex vivo prostatectomy model. 在体外前列腺切除术模型中靶向多胺代谢。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-11 DOI: 10.1016/bs.mie.2025.01.070

Hayley C Affronti, Aryn M Rowsam, Spencer R Rosario, Dominic J Smiraglia

{"title":"Targeting polyamine metabolism in an ex vivo prostatectomy model.","authors":"Hayley C Affronti, Aryn M Rowsam, Spencer R Rosario, Dominic J Smiraglia","doi":"10.1016/bs.mie.2025.01.070","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.070","url":null,"abstract":"Ex vivo models allow for testing drug efficacy and patient response, yet it remains a challenge to develop representative 3D cultures for prostate cancer. Tissue explant models offer a more clinically relevant alternative to organoids due to their ability to provide adequate tissue quantities, maintain tumor-stromal interactions and metabolic activity, and their relatively inexpensive culturing conditions. In this chapter we outline a protocol for culturing patient prostatectomy tumors for up to 7 days on dental sponges soaked in either control or drug containing media for evaluating drug efficacy. Further, we describe the preparation of tissue samples for downstream immunohistochemistry and metabolic analysis. We have tested the efficacy of a combination therapy targeting polyamine metabolism, which is dysregulated in prostate cancer, using this patient tumor explant model. We found that activating polyamine catabolism in combination with inhibition of methionine salvage was effective at inducing target protein expression, reducing intratumoral polyamines, and inducing apoptosis in a majority of the patient samples tested. Additionally, we were able to confirm drug induced effects were specific to the malignant prostate epithelial cells. This ex vivo prostatectomy model lends itself to both targeted metabolite analyses as well as more comprehensive metabolomic analyses. This method can be applied to strategies aiming to target metabolic pathways in solid tumor diseases.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"231-239"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094332","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

Whole-cell biocatalysis with Myxococcus xanthus. 黄粘球菌的全细胞生物催化。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2025.01.005

Lea Winand, Markus Nett

{"title":"Whole-cell biocatalysis with Myxococcus xanthus.","authors":"Lea Winand, Markus Nett","doi":"10.1016/bs.mie.2025.01.005","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.005","url":null,"abstract":"Biocatalysis has become an attractive complement to conventional chemical catalysis in the field of organic synthesis. This trend is due to the excellent chemo-, regio- and stereoselectivity of biocatalysis, which typically avoids the formation of unwanted by-products as well as cross-reactivity between reactants. Since the use of isolated enzymes and their cofactors can be associated with high costs, there is a continued interest in whole-cell biocatalysts. Applications range from the production of chiral building blocks to the derivatization of natural products and the generation of new drug candidates for the pharmaceutical and fine chemical industries. The bacterium Myxococcus xanthus is an emerging cell factory, especially for the production of antibiotics and other bioactive natural products. Not only possesses M. xanthus a considerable tolerance toward xenobiotics, but it is also a metabolically versatile host providing important building blocks for natural product biosynthesis. In this chapter, we describe procedures for the whole-cell biocatalysis with M. xanthus. This also involves methods for the construction of expression plasmids and their transfer into M. xanthus.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"714 ","pages":"219-237"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144009658","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