Methods in enzymology最新文献_第6页

Methods for purification and characterization of nicked tRNAs. nick trna的纯化和表征方法。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2024.11.004

Bruno Costa, Valentina Blanco, Alfonso Cayota, Juan Pablo Tosar

引用次数: 0

Use of xylose reductase as a cofactor enhancing system for in vivo biocatalysis. 利用木糖还原酶作为辅助因子增强系统进行体内生物催化。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-05 DOI: 10.1016/bs.mie.2025.01.022

Chalermroj Sutthaphirom, Pimchai Chaiyen

引用次数: 0

Enzyme expression in Cupriavidus necator H16 for whole-cell biocatalysis. Cupriavidus necator H16全细胞生物催化酶的表达。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-25 DOI: 10.1016/bs.mie.2025.01.079

Matteo Vajente, Mattia Ghirardi, Sandy Schmidt

{"title":"Enzyme expression in Cupriavidus necator H16 for whole-cell biocatalysis.","authors":"Matteo Vajente, Mattia Ghirardi, Sandy Schmidt","doi":"10.1016/bs.mie.2025.01.079","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.079","url":null,"abstract":"Climate change is an urgent and collective challenge, and new processes to synthesize complex molecules in a more sustainable way are highly desirable. Biocatalysis can be a strong player in this field, due to the specificity of enzymes and their ability to catalyze complex reactions at mild conditions. However, these reactions often require the regeneration of expensive cofactors in order to obtain relevant amounts of product. In vivo biocatalysis offers a solution to this problem by plugging the reaction in the microbial metabolism, which supplies the necessary energy. In particular, Cupriavidus necator H16 (C. necator H16) is an attractive microbial chassis due to its versatility and its lithoautotrophic metabolism. Its O2-tolerant soluble hydrogenase (SH) can be used to regenerate nicotinamide cofactors in an atom-efficient manner, without the creation of undesired side products. This hydrogenase has already been used as a cofactor regeneration system in vitro, but examples of in vivo biocatalysis are scarce due to the time-consuming genetic engineering process of C. necator H16. In this book chapter, we present a strategy for the engineering of C. necator from plasmid cloning (using a recently developed expression plasmid) to protein expression of a model oxidoreductase. This pipeline allows for rapid and streamlined strain engineering, which can aid the discovery and development of future in vivo biocatalytic processes using C. necator H16.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"714 ","pages":"195-218"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144033334","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

Purification and in vivo, cell-free, and in vitro characterization of CRISPR-Cas12a2. CRISPR-Cas12a2的纯化及体内、无细胞和体外鉴定。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-07 DOI: 10.1016/bs.mie.2025.01.032

Friso T Schut, Thomson Hallmark, Oleg Dmytrenko, Ryan N Jackson, Chase L Beisel

{"title":"Purification and in vivo, cell-free, and in vitro characterization of CRISPR-Cas12a2.","authors":"Friso T Schut, Thomson Hallmark, Oleg Dmytrenko, Ryan N Jackson, Chase L Beisel","doi":"10.1016/bs.mie.2025.01.032","DOIUrl":"10.1016/bs.mie.2025.01.032","url":null,"abstract":"The CRISPR-associated (Cas) nuclease Cas12a2 from Sulfuricurvum sp. PC08-66 (SuCas12a2) binds RNA targets with a complementary guide (g)RNA. Target RNA binding causes a major conformational rearrangement in Cas12a2 that activates a RuvC nuclease domain to collaterally cleave RNA, ssDNA and dsDNA, arresting growth and providing population-level immunity. Here, we report in vivo, cell-free, and in vitro methods to characterize the collateral cleavage activity of SuCas12a2 as well as a procedure for gRNA design. As part of the in vivo methods, we describe how to capture growth arrest through plasmid interference and induction of an SOS DNA damage response in the bacterium Escherichia coli. We further apply cell-free transcription-translation to affirm collateral cleavage activity triggered by an expressed RNA target. Finally, as part of the in vitro methods, we describe how to purify active nuclease and subsequently conduct biochemical cleavage assays. In total, the outlined methods should accelerate the exploration of SuCas12a2 and other related Cas nucleases, revealing new features of CRISPR biology and helping develop new CRISPR technologies for molecular diagnostics and other applications.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"143-181"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692750","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

TGIRT-seq to profile tRNA-derived RNAs and associated RNA modifications. TGIRT-seq分析trna衍生RNA和相关RNA修饰。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-11-22 DOI: 10.1016/bs.mie.2024.11.001

Abigail Grace Johnston, Monima Anam, Anindya Dutta, Zhangli Su

{"title":"TGIRT-seq to profile tRNA-derived RNAs and associated RNA modifications.","authors":"Abigail Grace Johnston, Monima Anam, Anindya Dutta, Zhangli Su","doi":"10.1016/bs.mie.2024.11.001","DOIUrl":"10.1016/bs.mie.2024.11.001","url":null,"abstract":"RNA modifications are key regulators for RNA processes. tRNA-derived RNAs are small RNAs with size between 15 and 50 bases long that are processed from mature or precursor tRNAs. Despite their more recent discovery, tRNA-derived RNAs have been found to play regulatory roles in many cellular processes including gene silencing, protein synthesis, stress response, and transgenerational inheritance. Furthermore, tRNA-derived RNAs are highly abundant in bodily fluids, posing as potential biomarkers. A unique feature of tRNA-derived RNAs is that they are rich in RNA modifications. Many of the RNA modifications on tRNA-derived RNAs disrupt Watson-Crick base pairing and will thus stall reverse transcriptase, such as N1-methyladenosine (m1A), N1-methylguanosine (m1G) and N2, N2-dimethylguanosine (m22G). These RNA modifications add another layer of regulation onto tRNA-derived RNAs' functions and are of interests for future research. However, these RNA modifications could also lead to lower detection of modification-containing RNAs in genome-wide small RNA sequencing analysis due to reverse transcriptase stall. To circumvent this bias, TGIRT (Thermostable Group II Intron Reverse Transcriptase) has been used to readthrough RNA modifications inserting mismatches. These mismatch signatures can then be used to precisely map the modification sites at base resolution. Here we describe the step-by-step experimental protocol to start with purified RNAs from cells or tissues and use TGIRT to make small RNA sequencing library for Illumina sequencing to profile the abundance of tRNA-derived RNAs and the associated RNA modifications.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"223-240"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11890191/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425832","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

Determining the biochemical function of type IV CRISPR ribonucleoprotein complexes and accessory proteins. 测定IV型CRISPR核糖核蛋白复合物和辅助蛋白的生化功能。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-11 DOI: 10.1016/bs.mie.2025.01.039

Andrew A Williams, Olivine Redman, Hannah Domgaard, Matthew J Armbrust, Ryan N Jackson

引用次数: 0

CRISPRoff epigenome editing for programmable gene silencing in human cell lines and primary T cells. CRISPRoff表观基因组编辑用于人类细胞系和原代T细胞的可编程基因沉默。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-06 DOI: 10.1016/bs.mie.2025.01.010

Rithu K Pattali, Izaiah J Ornelas, Carolyn D Nguyen, Da Xu, Nikita S Divekar, NunezJames K Nuñez

{"title":"CRISPRoff epigenome editing for programmable gene silencing in human cell lines and primary T cells.","authors":"Rithu K Pattali, Izaiah J Ornelas, Carolyn D Nguyen, Da Xu, Nikita S Divekar, NunezJames K Nuñez","doi":"10.1016/bs.mie.2025.01.010","DOIUrl":"10.1016/bs.mie.2025.01.010","url":null,"abstract":"The advent of CRISPR-based technologies has enabled the rapid advancement of programmable gene manipulation in cells, tissues, and whole organisms. An emerging platform for targeted gene perturbation is epigenetic editing, the direct editing of chemical modifications on DNA and histones that ultimately results in repression or activation of the targeted gene. In contrast to CRISPR nucleases, epigenetic editors modulate gene expression without inducing DNA breaks or altering the genomic sequence of host cells. Recently, we developed the CRISPRoff epigenetic editing technology that simultaneously establishes DNA methylation and repressive histone modifications at targeted gene promoters. Transient expression of CRISPRoff and the accompanying single guide RNAs in mammalian cells results in transcriptional repression of targeted genes that is memorized heritably by cells through cell division and differentiation. Here, we describe our protocol for the delivery of CRISPRoff through plasmid DNA transfection, as well as the delivery of CRISPRoff mRNA, into transformed human cell lines and primary immune cells. We also provide guidance on evaluating target gene silencing and highlight key considerations when utilizing CRISPRoff for gene perturbations. Our protocols are broadly applicable to other CRISPR-based epigenetic editing technologies, as programmable genome manipulation tools continue to evolve rapidly.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"517-551"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12052198/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692760","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

Fluorescent shift assay for APOBEC-mediated RNA editing. apobecc介导的RNA编辑的荧光位移试验。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-10 DOI: 10.1016/bs.mie.2024.12.002

Shanshan Wang, Benjamin Fixman, Xiaojiang S Chen

{"title":"Fluorescent shift assay for APOBEC-mediated RNA editing.","authors":"Shanshan Wang, Benjamin Fixman, Xiaojiang S Chen","doi":"10.1016/bs.mie.2024.12.002","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.12.002","url":null,"abstract":"Cytidine (C) to Uridine (U) RNA editing is a post-transcriptional modification that is involved in diverse biological processes. The APOBEC deaminase family acts in various cellular processes mostly through inducing C-to-U mutation in single-stranded RNA (or DNA). However, comparing the activity of different RNA editing enzymes to one another is difficult due to the limited number of systems that can provide direct and efficient readout. In this report, a system in which RNA editing directly prompts a change in the subcellular localization of a modified eGFP structure is described in detail. This approach allows us to compare relative fluorescence intensity based on the RNA editing level. When observed through a fluorescence detection system, like a scanning confocal microscope, the cellular nucleus can be readily identified using a DNA-binding stain, such as DAPI or Hoechst, so that the accurate calculation of the ratio of nuclear to cytosolic eGFP intensity can be applied for an individual cell. This method provides a useful and flexible tool to examine and quantify RNA editing activity within cells, and it is not only limited to APOBEC proteins, but can also be applied more generally to other RNA editing enzymatic assays.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"713 ","pages":"1-14"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144002689","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

Gene replacement therapy to restore polyamine metabolism in a Snyder-Robinson syndrome mouse model. 基因替代疗法恢复Snyder-Robinson综合征小鼠模型中的多胺代谢。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-16 DOI: 10.1016/bs.mie.2025.01.068

Oluwaseun Akinyele, Krystal B Tran, Marie A Johnson, Dwi U Kemaladewi

{"title":"Gene replacement therapy to restore polyamine metabolism in a Snyder-Robinson syndrome mouse model.","authors":"Oluwaseun Akinyele, Krystal B Tran, Marie A Johnson, Dwi U Kemaladewi","doi":"10.1016/bs.mie.2025.01.068","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.068","url":null,"abstract":"Polyamines, including putrescine, spermidine, and spermine, are organic cations essential for cell growth, proliferation, and tissue regeneration. Their levels are tightly regulated by a set of enzymes controlling their biosynthesis, catabolism, and interconversion. Dysregulation of polyamine metabolism is associated with a group of rare genetic neurodevelopmental disorders collectively known as \"polyaminopathies\", including Snyder-Robinson Syndrome (SRS). SRS is an X-linked recessive disorder caused by mutations in the SMS gene, which encodes the spermine synthase enzyme. The lack of spermine synthase leads to aberrant polyamine levels and neurological impairments, as observed in patients and animal models. Currently, there are no available treatment options for SRS. Due to its monogenic nature, SRS is an excellent candidate for gene replacement therapy. The recent success of Zolgensma in treating children with Spinal Muscular Atrophy and the establishment of Platform Vector Gene Therapy (Pave-GT) initiative at the National Institute of Health (NIH) offer a framework to adapt-and-apply the same gene delivery system for multiple rare disease gene therapies. This chapter outlines strategies for delivering a functional copy of the SMS gene using an adeno-associated viral (AAV) vector, as well as methods to evaluate the molecular efficacy of this approach in an SRS mouse model. Our ultimate goal is to establish a versatile platform for genetic interventions targeting SRS and other polyaminopathies.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"271-292"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094212","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

NMR spectroscopy: An invaluable tool to identify lignin structural modifications induced by oxidative enzymes. 核磁共振波谱：鉴定氧化酶诱导的木质素结构修饰的宝贵工具。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-26 DOI: 10.1016/bs.mie.2025.02.003

Nicolò Pajer, Umberto Danelon, Claudia Crestini

引用次数: 0