Methods in enzymology最新文献_第3页

How to engineer giant enzymes: A methodology for mutagenesis of polyketide synthases in native hosts. 如何设计巨型酶：原生宿主中聚酮合酶的诱变方法。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-07 DOI: 10.1016/bs.mie.2025.02.007

Susanna Kushnir, Uschi Hübner, Frank Schulz

引用次数: 0

cP-RNA-seq for tRNA half sequencing. cP-RNA-seq用于tRNA半测序。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.002

Megumi Shigematsu, Justin Gumas, Yohei Kirino

{"title":"cP-RNA-seq for tRNA half sequencing.","authors":"Megumi Shigematsu, Justin Gumas, Yohei Kirino","doi":"10.1016/bs.mie.2024.11.002","DOIUrl":"10.1016/bs.mie.2024.11.002","url":null,"abstract":"Although RNA-seq data are becoming more widely available for biomedical research, most datasets for short non-coding RNAs (sncRNAs) primarily focus on microRNA analysis using standard RNA-seq, which captures only sncRNAs with 5'-phosphate (5'-P) and 3'-hydroxyl (3'-OH) ends. Standard RNA-seq fails to sequence sncRNAs with different terminal phosphate states, including tRNA halves, the most abundant class of tRNA-derived sncRNAs that play diverse roles in various biological processes. tRNA halves are produced through the endoribonucleolytic cleavage of mature tRNA anticodon loops. The responsible endoribonucleases, such as Angiogenin, commonly leave a 2',3'-cyclic phosphate (cP) at the 3'-end of 5'-tRNA halves and forms a 5'-OH end of 3'-tRNA halves, making them incompatible with standard RNA-seq. We developed a method named \"cP-RNA-seq\" that selectively amplifies and sequences tRNA halves and other cP-containing sncRNAs. Here we describe a detailed and recently updated cP-RNA-seq protocol. In this method, the 3'-end of all sncRNAs, except those containing a cP, are cleaved through periodate treatment after phosphatase treatment. Consequently, adaptor ligation and cDNA amplification steps are exclusively applied to cP-containing sncRNAs. Our cP-RNA-seq only requires commercially available reagents and is broadly applicable for the global identification of tRNA halves and other cP-containing sncRNA repertoires in various transcriptomes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"135-153"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11938272/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425655","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

Assay for ribosome stimulation of angiogenin nuclease activity. 核糖体刺激血管生成素核酸酶活性的测定。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.007

Emily Sholi, Anna B Loveland, Andrei A Korostelev

{"title":"Assay for ribosome stimulation of angiogenin nuclease activity.","authors":"Emily Sholi, Anna B Loveland, Andrei A Korostelev","doi":"10.1016/bs.mie.2024.11.007","DOIUrl":"10.1016/bs.mie.2024.11.007","url":null,"abstract":"Angiogenin (RNase 5) is an unusual member of the RNase A family with very weak RNase activity and a preference for tRNA. The tRNAs cleaved by angiogenin are thought to have a variety of roles in cellular processes including translation reprogramming, apoptosis, angiogenesis, and neuroprotection. We recently demonstrated that angiogenin is potently activated by the cytoplasmic 80S ribosome. Angiogenin's binding to the ribosome rearranges the C-terminus of the protein, opening the active site for the cleavage of tRNA delivered to the ribosomal A site which angiogenin occupies. Here, we describe the biochemical procedure to test angiogenin's activation by the ribosome using the assay termed the Ribosome Stimulated Angiogenin Nuclease Assay (RiSANA). RiSANA can be used to test the activity of wild-type or mutant angiogenin, or other RNases, against different tRNAs and with different ribosome complexes. For example, given that angiogenin has been implicated in anti-microbial activity, we tested the ability of bacterial 70S ribosomes to stimulate angiogenin activity and found that the E. coli ribosome does not stimulate angiogenin. We also assayed whether angiogenin's closest homolog, RNase 4, could be stimulated by the ribosome, but unlike angiogenin this enzyme was not further activated by the ribosome. The RiSANA assay promises to reveal new aspects of angiogenin mechanism and may aid in the development of new diagnostic tools and therapeutics.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"381-404"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11839171/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425704","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

Analysis of metal-dependent DNA nicking activities by Cas endonucleases. 利用Cas内切酶分析金属依赖性DNA刻蚀活性。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-07 DOI: 10.1016/bs.mie.2025.01.034

Giang T Nguyen, Akshara Raju, Dipali G Sashital

引用次数: 0

Bacterial directed evolution of CRISPR base editors. CRISPR碱基编辑器的细菌定向进化。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-08 DOI: 10.1016/bs.mie.2025.01.003

Reilly Q Mach, Shannon M Miller

引用次数: 0

Obstacles in quantifying A-to-I RNA editing by Sanger sequencing. Sanger测序定量A-to-I RNA编辑的障碍。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-02 DOI: 10.1016/bs.mie.2024.11.032

Alla Fishman, Ayelet T Lamm

{"title":"Obstacles in quantifying A-to-I RNA editing by Sanger sequencing.","authors":"Alla Fishman, Ayelet T Lamm","doi":"10.1016/bs.mie.2024.11.032","DOIUrl":"10.1016/bs.mie.2024.11.032","url":null,"abstract":"Adenosine-to-Inosine (A-to-I) RNA editing is the most prevalent type of RNA editing, in which adenosine within a completely or largely double-stranded RNA (dsRNA) is converted to inosine by deamination. RNA editing was shown to be involved in many neurological diseases and cancer; therefore, detection of A-to-I RNA editing and quantitation of editing levels are necessary for both basic and clinical biomedical research. While high-throughput sequencing (HTS) is widely used for global detection of editing events, Sanger sequencing is the method of choice for precise characterization of editing site clusters (hyper-editing) and for comparing levels of editing at a particular site under different environmental conditions, developmental stages, genetic backgrounds, or disease states. To detect A-to-I editing events and quantify them using Sanger sequencing, RNA samples are reverse transcribed, cDNA is amplified using gene-specific primers, and then sequenced. The chromatogram outputs are then compared to the genomic DNA sequence. As editing occurs in the context of dsRNA, the reverse transcription step is performed at a temperature as high as 65 °C, using thermostable reverse transcriptase to open double-stranded structures. However, this measure alone is insufficient for transcripts possessing long stems comprised of hundreds of nucleotide pairs. Consequently, the editing levels detected by Sanger sequencing are significantly lower than those obtained by HTS, and the amplification yield is low. We suggest that the reverse transcription is biased towards unedited transcripts, and the severity of the bias is dependent on the transcript's secondary structure. Here, we show how this bias can be significantly reduced to allow reliable detection of editing levels and sufficient product yield.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"285-302"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052991","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

Hydrolytic endonucleolytic ribozyme (HYER): Systematic identification, characterization and potential application in nucleic acid manipulation. 水解核内溶核酶（HYER）：系统鉴定、表征及其在核酸操作中的潜在应用。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-06 DOI: 10.1016/bs.mie.2025.01.033

Zi-Xian Liu, Jun-Jie Gogo Liu

引用次数: 0

Biocatalysis: Important considerations for testing and evaluation of biocatalysts. 生物催化：生物催化剂测试和评价的重要考虑因素。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-25 DOI: 10.1016/bs.mie.2025.01.080

Dirk Tischler, Giovanni Davide Barone, Jose Munoz-Munoz, John M Woodley

引用次数: 0

Evaluation of platinum drug toxicity resulting from polyamine catabolism. 多胺分解代谢引起铂类药物毒性的评价。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-06 DOI: 10.1016/bs.mie.2025.01.065

Kamyar Zahedi, Sharon Barone, Manoocher Soleimani

{"title":"Evaluation of platinum drug toxicity resulting from polyamine catabolism.","authors":"Kamyar Zahedi, Sharon Barone, Manoocher Soleimani","doi":"10.1016/bs.mie.2025.01.065","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.065","url":null,"abstract":"Polyamines, spermidine (Spd) and Spermine (Spm), are polycations that serve a number of important biological functions. The tissue contents of polyamines are tightly regulated through their cellular import and export, as well as their metabolism (anabolism and catabolism). Polyamine catabolism in mediated via the spermidine/spermine N1-acetyltransferase (SAT1)/acetylpolyamine oxidase (APOX) cascade and oxidation of Spm by spermine oxidase (SMOX). The expression of SAT1 and SMOX increases in injured organs in response to trauma, ischemia/reperfusion, sepsis, and exposure to toxic compounds. Cisplatin is a highly effective chemotherapeutic agent that is used for the treatment of a variety of solid tumors. Its anti-tumor activity is mediated via its ability to form stable DNA adducts that inhibit the growth of actively proliferating cells. However, cisplatin also can lead to severe off-target deleterious effects (e.g., nephrotoxicity and ototoxicity), and because of such adverse effects the use of cisplatin has to be discontinued in many patients. Understanding and decoupling the therapeutic and toxic effects of cisplatin will lead to more effective use of this and other platinum-derived compounds in the treatment of cancer patients. Acute and chronic exposure to cisplatin in mice leads to severe renal tubular injuries and an increase in the expression of SAT1 and SMOX while the ablation of their genes in mice reduces the severity of nephrotoxic injuries caused by cisplatin. Furthermore, neutralization of the toxic by-products of polyamine degradation reduce the severity if cisplatin nephrotoxicity. These observations suggest that interventions targeting the adverse effects of enhanced polyamine catabolism may provide effective therapies by reducing the toxic effects of cisplatin without affecting its anti-neoplastic activity.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"93-116"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094196","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

Preface. 前言。

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Methods in enzymology Pub Date : 2025-01-01 DOI: 10.1016/S0076-6879(25)00204-6

Robert A Casero, Tracy Murray Stewart

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