Methods in enzymology最新文献_第9页

An in vitro cytidine deaminase assay to monitor APOBEC activity on DNA. 体外胞苷脱氨酶测定监测APOBEC对DNA的活性。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-07 DOI: 10.1016/bs.mie.2024.11.037

Ambrocio Sanchez, Rémi Buisson

{"title":"An in vitro cytidine deaminase assay to monitor APOBEC activity on DNA.","authors":"Ambrocio Sanchez, Rémi Buisson","doi":"10.1016/bs.mie.2024.11.037","DOIUrl":"10.1016/bs.mie.2024.11.037","url":null,"abstract":"APOBEC enzymes promote the deamination of cytosine (C) to uracil (U) in DNA to defend cells against viruses but also serve as a predominant source of mutations in cancer genomes. This protocol describes an assay to monitor APOBEC deaminase activity in vitro on a synthetic DNA oligonucleotide. The method described here focuses specifically on APOBEC3B to illustrate the different steps of the assay. However, the protocol can be applied to monitor the DNA deaminase activity of any other member of the APOBEC family, such as APOBEC3A. This assay involves preparing APOBEC3B-expressing cell extract or purifying APOBEC3B by immunoprecipitation, followed by incubation with a single-stranded DNA containing a TpC motif. The deaminated cytosine is then removed by recombinant Uracil DNA Glycosylase present in the reaction to form an abasic site. The abasic site creates a weakness in the DNA's backbone, causing the DNA to be cleaved under high temperatures and alkaline conditions. Denaturing gel electrophoresis is used to separate cleaved DNA from full-length DNA, enabling the quantification of the percentage of deamination induced by APOBEC3B. This protocol can be used to determine the presence of APOBEC and the regulation of APOBEC activity in specific cell lines, to study substrate preference targeted by different members of the APOBEC family and different APOBEC mutants, or to determine the efficiency and specificity of inhibitor compounds against APOBEC enzymes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"713 ","pages":"201-219"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12083365/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144064144","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

Quantification of spermine oxidase (SMOX) activity in tissues by HPLC. 高效液相色谱法测定组织中精胺氧化酶（SMOX）活性。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-17 DOI: 10.1016/bs.mie.2025.01.075

Jackson R Foley, Cassandra E Holbert

{"title":"Quantification of spermine oxidase (SMOX) activity in tissues by HPLC.","authors":"Jackson R Foley, Cassandra E Holbert","doi":"10.1016/bs.mie.2025.01.075","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.075","url":null,"abstract":"The polyamine oxidases, SMOX and PAOX, are enzymes involved in the normal metabolism of polyamines. Both enzymes are implicated in numerous human diseases including cancer, reperfusion injury, and neurodegenerative diseases. The ability to directly measure the activity of these enzymes is imperative in understanding their role in human health and disease. Most assays currently used to measure both SMOX and PAOX activity use a coupled reaction with horse radish peroxidase (HRP). These assays cannot be used when evaluating certain compounds for potential polyamine oxidase inhibition if these compouds also affect HRP activity. Additionally, since most assays use H2O2 production as an indicator of oxidase activity they cannot be used to evaluate polyamine oxidase activity in the presence of iron or other divalent metals. This prevents the use of these assays to evaluate polyamine oxidase activity in tissue samples. Here we describe the protocols for determining polyamine oxidase activity in an HRP-independent manner via an HPLC-based assay allowing for evaluation of both compounds that may interfere with HRP activity and polyamine oxidase activity in tissue samples.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"183-199"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094327","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

Cell-based determination of HDAC10-mediated polyamine deacetylase activity. 基于细胞的hdac10介导的多胺去乙酰酶活性测定。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-22 DOI: 10.1016/bs.mie.2025.01.047

Ishika Gupta, Ashley Nwafor, Robert A Casero, Tracy Murray Stewart

引用次数: 0

Delivery of genome editors with engineered virus-like particles. 基因编辑器与工程病毒样颗粒的传递。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-09 DOI: 10.1016/bs.mie.2025.01.007

Christopher Lu, Yuanhang Li, Jacob Ryan Cummings, Samagya Banskota

引用次数: 0

Expression and purification of bacterial dye-decolorizing peroxidases using Escherichia coli. 利用大肠杆菌表达和纯化细菌染料脱色过氧化物酶。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-04-16 DOI: 10.1016/bs.mie.2025.03.004

Hegne Pupart, Priit Eek

引用次数: 0

Optimized protocols to measure stability of ligninolytic enzymes. 优化方案测量木质素分解酶的稳定性。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-04-15 DOI: 10.1016/bs.mie.2025.01.043

Vânia Brissos, Paulo Durão, Carolina F Rodrigues, Eduardo P Melo, Lígia O Martins

{"title":"Optimized protocols to measure stability of ligninolytic enzymes.","authors":"Vânia Brissos, Paulo Durão, Carolina F Rodrigues, Eduardo P Melo, Lígia O Martins","doi":"10.1016/bs.mie.2025.01.043","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.043","url":null,"abstract":"Biocatalysis is considered a critical component for developing a sustainable bioeconomy, and stability is a crucial enzyme property for biotechnological and industrial applications. Enzymes with higher thermostability are more durable and desirable in industrial settings due to their resilience across various operational conditions, which helps reduce overall enzyme costs. Understanding an enzyme's thermal stability ensures its long-term efficacy and performance. Thermodynamic stability reflects the equilibrium between the native, functional protein, and unfolded state, and the kinetic or long-term stability is associated with the irreversible inactivation of the enzyme. Therefore, the thermostability of biocatalysts can be characterized by their melting temperature (Tm) when 50 % of the enzyme is unfolded and the half-life time (t1/2), reporting the time gap to the loss of 50 % of the activity at a specific temperature. This parameter is crucial for assessing the feasibility of an enzymatic-based (bio)process, as it indicates the enzyme's temperature-dependent deactivation and operational stability over time. The optimum temperature of an enzyme (Topt) usually reflects its (thermo)stability, particularly the stability of the native state. Here, we describe protocols for accessing the thermodynamic and kinetic stability of different ligninolytic enzymes, including laccases and DyP-type peroxidases. We provide practical examples and emphasize the challenges encountered during experimental procedures and data analysis. While these protocols are tailored to these specific enzymes, they can be broadly applied to other proteins and enzymes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"716 ","pages":"157-197"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144294090","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

Biochemical reconstitution of a type I-B CRISPR-associated transposon. I-B型crispr相关转座子的生化重组。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-26 DOI: 10.1016/bs.mie.2025.01.042

Shukun Wang, Leifu Chang

引用次数: 0

Preparation of high-purity RNPs of CRISPR-based DNA base editors. 基于crispr的DNA碱基编辑器高纯度RNPs的制备。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-27 DOI: 10.1016/bs.mie.2025.01.019

Mitchell J McAndrew, Madeleine B King, Audrone Lapinaite

{"title":"Preparation of high-purity RNPs of CRISPR-based DNA base editors.","authors":"Mitchell J McAndrew, Madeleine B King, Audrone Lapinaite","doi":"10.1016/bs.mie.2025.01.019","DOIUrl":"10.1016/bs.mie.2025.01.019","url":null,"abstract":"Since their introduction, CRISPR-based DNA base editors (BEs) have become essential in the field of precision genome editing, revolutionizing the correction of pathogenic SNPs for both basic research and therapeutic applications. As this technology advances, more laboratories are implementing these tools into their workflow. The delivery of BEs as BE-guide RNA complexes (RNPs), rather than as mRNA or plasmids, has been shown to exhibit lower off-target effects, establishing it as the preferred method of delivery. However, there are no protocols describing in detail how to obtain high-purity and highly active BE RNPs. Here, we offer a comprehensive guide for the expression, purification, RNP reconstitution, and in vitro activity assessment of TadA-based BEs. The protocol includes guidance on performing activity assays using commercial denaturing gels, which is convenient and uses standard molecular biology equipment. This allows for rapid quality control testing of reconstituted BE RNPs prior to more expensive and time-consuming in vivo genome editing experiments. Overall, this protocol aims to empower more laboratories to generate tailored BE RNPs for diverse in vitro and in vivo applications.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"277-315"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692825","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

Mouse models for understanding physiological functions of ADARs. 用于了解ADARs生理功能的小鼠模型。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-09 DOI: 10.1016/bs.mie.2024.11.024

Qinyi Zhang, Carl R Walkley

引用次数: 0

tRNA-derived RNAs that form tetramolecular assemblies. 形成四分子组合的trna衍生的rna。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-11-22 DOI: 10.1016/bs.mie.2024.11.014

Prakash Kharel

{"title":"tRNA-derived RNAs that form tetramolecular assemblies.","authors":"Prakash Kharel","doi":"10.1016/bs.mie.2024.11.014","DOIUrl":"10.1016/bs.mie.2024.11.014","url":null,"abstract":"Transfer RNA (tRNA)-derived small RNAs (tDRs) are emerging as a novel class of regulatory molecules with significant implications in gene expression and cellular processes. These tDRs are generated through precise cleavage of precursor or mature tRNAs and can function in a sequence dependent manner or structure dependent manner. Recent studies have uncovered a unique subset of tDRs that can form tetramolecular assemblies, adding a new layer of complexity to their functional repertoire. Tetramolecular tDRs exhibit remarkable stability and functional diversity, influencing processes such as translation regulation, stress response, and cellular signaling. The assembly of these tDRs into tetramers is facilitated by guanine-rich sequence motifs which promote intermolecular interactions essential for their structure and biological activity. Understanding the formation, structural dynamics, and functional roles of tetramolecular tDRs offers new insights into tDR-mediated gene regulation and the potential development of RNA-based therapeutic strategies. This article aims to discuss a set of biochemical, biophysical, and reporter assay-based techniques that can be used to characterize G-quadruplex structures formed by tDRs.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"47-63"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425835","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