Methods in enzymology最新文献

CellREADR: An ADAR-based RNA sensor-actuator device. CellREADR：一种基于adar的RNA传感器致动器设备。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-10 DOI: 10.1016/bs.mie.2024.11.027

Xiaolu Yang, Kehali Woldemichael, Xiao Guo, Shengli Zhao, Yongjun Qian, Z Josh Huang

{"title":"CellREADR: An ADAR-based RNA sensor-actuator device.","authors":"Xiaolu Yang, Kehali Woldemichael, Xiao Guo, Shengli Zhao, Yongjun Qian, Z Josh Huang","doi":"10.1016/bs.mie.2024.11.027","DOIUrl":"10.1016/bs.mie.2024.11.027","url":null,"abstract":"RNAs are central mediators of genetic information flow and gene regulation that underlie diverse cell types and cell states across species. Thus, methods that can sense and respond to RNA profiles in living cells will have broad applications in biology and medicine. CellREADR - Cell access through RNA sensing by Endogenous ADAR (adenosine deaminase acting on RNA), is a programmable RNA sensor-actuator technology that couples the detection of a cell-defining RNA to the translation of an effector protein to monitor and manipulate the cell. The CellREADR RNA device consists of a 5' sensor region complementary to a cellular RNA and a 3' protein payload coding region. Payload translation is gated by the removal of a STOP codon in the sensor region upon base pairing with the cognate cellular RNA through an ADAR-mediated A-to-I editing mechanism ubiquitous to metazoan cells. CellREADR thus represents a new generation of programmable RNA device for monitoring and manipulating animal cells in ways that are simple, versatile, and generalizable across tissues and species. Here, we describe a detailed procedure for implementing CellREADR experiments in cell culture systems and in animals. The procedure includes sensor and payload design, cloning, validation and characterization in mammalian cell cultures. The in vivo protocol focuses on AAV-based delivery of CellREADR through expression vectors using brain tissue as an example. We describe current best practices and various experimental controls.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"207-227"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143053003","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

Bioinformatics of simultaneous, quantitative measurements of full-length tRNA and tRNA fragments by MSR sequencing. 生物信息学的同时，定量测量全长度tRNA和tRNA片段的MSR测序。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-11-22 DOI: 10.1016/bs.mie.2024.11.009

Luke R Frietze, Tao Pan

引用次数: 0

Low-error RNA sequencing techniques for detecting RNA editing by APOBECs: Circular RNAseq assay and safe-sequencing system (SSS). 用于检测APOBECs RNA编辑的低误差RNA测序技术：环状RNA测序法和安全测序系统（SSS）。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-10 DOI: 10.1016/bs.mie.2024.12.004

Shanshan Wang, Benjamin Fixman, Xiaojiang S Chen

{"title":"Low-error RNA sequencing techniques for detecting RNA editing by APOBECs: Circular RNAseq assay and safe-sequencing system (SSS).","authors":"Shanshan Wang, Benjamin Fixman, Xiaojiang S Chen","doi":"10.1016/bs.mie.2024.12.004","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.12.004","url":null,"abstract":"Cytidine-to-Uridine (C-to-U) RNA editing is a post-transcriptional modification essential for various biological processes. APOBEC deaminases mediate C-to-U editing which play critical role in cellular function and regulation. Advances in next-generation sequencing (NGS) technologies and analytical tools have provided powerful means to assess RNA editing activities and their physiological implications. However, inherent errors in NGS workflows-including reverse transcription, PCR amplification, and sequencing-complicate the detection of actual editing events. With error rates ranging from 10-2 to 10-3 per nucleotide, these technical artifacts can obscure APOBEC-mediated editing events occurring at similar frequencies. To address these challenges, in this chapter, we describe two established and optimized RNA sequencing strategies explicitly designed to detect low-frequency RNA editing events accurately while distinguishing them from NGS-associated errors. These methods are termed \"circular RNA Sequencing Assay\" and \"Safe-Sequencing System (SSS)\" and enable the reliable identification of RNA editing events (and also somatic mutations) at or below typical error thresholds.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"713 ","pages":"15-30"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144064146","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

Genetic manipulation of the betaproteobacterial genera Thauera and Aromatoleum. betaproteobacter属Thauera和Aromatoleum的遗传操作。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-28 DOI: 10.1016/bs.mie.2025.01.009

Dominik Hege, Yvonne Gemmecker, Lina Clermont, Ivana Aleksic, Gabriela Oleksy, Maciej Szaleniec, Johann Heider

引用次数: 0

Discovery, production and characterization of bacterial aryl-alcohol oxidases. 细菌芳基醇氧化酶的发现、生产和表征。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-18 DOI: 10.1016/bs.mie.2025.01.025

Paula Cinca-Fernando, Christian Ascaso-Alegre, Patricia Ferreira, Juan Mangas-Sánchez

引用次数: 0

Ultrahigh-throughput screening assay for PET-degrading enzymes. pet降解酶的超高通量筛选试验。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-31 DOI: 10.1016/bs.mie.2025.01.021

Álvaro Lorente-Arévalo, María Gimeno-Pérez, Carmen Ortega, James Finnigan, Simon Charnock, Aurelio Hidalgo

{"title":"Ultrahigh-throughput screening assay for PET-degrading enzymes.","authors":"Álvaro Lorente-Arévalo, María Gimeno-Pérez, Carmen Ortega, James Finnigan, Simon Charnock, Aurelio Hidalgo","doi":"10.1016/bs.mie.2025.01.021","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.021","url":null,"abstract":"In recent years, several PET-degrading enzymes have been identified from both known microorganisms and metagenomic sources in response to the growing environmental issue of polyethylene terephthalate (PET) accumulation. Despite this progress, there is a limited number of (ultra)high-throughput screening methods for assessing PET-hydrolyzing activity without relying on surrogate substrates. This method utilizes the coupled activity of ketoreductases (KREDs) and diaphorase to produce a fluorescent compound (resorufin) in the presence of PET degradation products, offering a more direct and efficient screening approach. A metagenomic KRED was coupled with the diaphorase from Clostridium kluyveri to enable the detection of the hydrolysis of PET degradation products catalyzed by the Bacillus subtilis BS2 esterase. The coupled reaction was established in water-in-oil microdroplets, encapsulating a single E. coli cell per droplet, demonstrating its potential for use in the ultrahigh-throughput screening of metagenomic libraries or randomized libraries for directed evolution campaigns.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"714 ","pages":"489-503"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144028035","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 assays for AID/APOBECs and the identification of AID/APOBEC inhibitors. AID/APOBECs的生化检测及AID/APOBEC抑制剂的鉴定。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2024.12.001

Priyanka Govindarajan, Ying Zeng, Mani Larijani

{"title":"Biochemical assays for AID/APOBECs and the identification of AID/APOBEC inhibitors.","authors":"Priyanka Govindarajan, Ying Zeng, Mani Larijani","doi":"10.1016/bs.mie.2024.12.001","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.12.001","url":null,"abstract":"Activation-induced cytidine deaminase (AID) and apolipoprotein B-mRNA editing catalytic polypeptide 3 (APOBEC3 or A3) proteins belong to the AID/APOBEC family of cytidine deaminases. While AID mediates somatic hypermutation and class-switch recombination in adaptive immunity, A3s restrict viruses and retroelements by hypermutation. Mis-regulated expression and off-target activity of AID/A3 can cause genome-wide mutations promoting oncogenesis, immune evasion, and therapeutic resistance due to tumor and viral evolution. In these contexts, inhibition of AID/A3 represents a promising therapeutic approach. Competitive inhibition could be achieved with different strategies: one class would be small molecules that bind in the catalytic pocket (active site) and block access for the substrate cytidine. Another type of larger molecule inhibitor would bind the enzymes' surface more broadly and compete with the binding of the polynucleotide substrates prior to deamination catalysis. Several biochemical assays developed to assess AID/A3 activity can be employed to screen for potential inhibitors. These include in cellulo and in vitro activity-based as well as binding-based assays. In this chapter, we discuss the key considerations for designing robust enzyme assays and provide an overview of assays that we and others have established or modified for specific applications in AID/A3 enzymology, including measurement of inhibition. We provide detailed protocols for the two most widely used in vitro enzyme assays that directly measure the activities of purified AID/A3s on DNA and/or RNA substrates, namely, the gel-based alkaline cleavage assay and multiple variations of PCR/sequencing-based assays.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"713 ","pages":"163-200"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144001934","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

Methods to study polyamine metabolism during osteogenesis. 方法研究成骨过程中多胺代谢。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-20 DOI: 10.1016/bs.mie.2025.01.064

Amin Cressman, Fernando A Fierro

引用次数: 0

Expression, purification, and crystallization of "humanized" Danio rerio histone deacetylase 10 "HDAC10", the eukaryotic polyamine deacetylase. 真核生物多胺脱乙酰酶“人源化”丹尼奥河组蛋白脱乙酰酶10“HDAC10”的表达、纯化和结晶。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-11 DOI: 10.1016/bs.mie.2025.01.074

Juana Goulart Stollmaier, Corey J Herbst-Gervasoni, David W Christianson

引用次数: 0

Polyamine transport inhibitors: Methods and caveats associated with measuring polyamine uptake in mammalian cells. 多胺转运抑制剂：测量哺乳动物细胞中多胺摄取的方法和注意事项。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-02 DOI: 10.1016/bs.mie.2025.01.062

Alexandra Bunea, Otto Phanstiel

{"title":"Polyamine transport inhibitors: Methods and caveats associated with measuring polyamine uptake in mammalian cells.","authors":"Alexandra Bunea, Otto Phanstiel","doi":"10.1016/bs.mie.2025.01.062","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.062","url":null,"abstract":"Combination therapies which target both polyamine biosynthesis and polyamine transport have shown promise as anti-cancer strategies and as potentiators of the immune response. While polyamine biosynthesis inhibitors like difluoromethylornithine (DFMO) exist, cancers often escape via upregulated polyamine import. As a result, polyamine transport inhibitors (PTIs) are needed to inhibit polyamine uptake and create a 'full-court press' on polyamine metabolism. As new PTIs are developed, they need to be ranked for their ability to inhibit polyamine uptake. This paper describes three polyamine transport assays to evaluate polyamine transport inhibition. The first tests the ability of the PTI to inhibit the uptake of an anthracene-containing polyamine poison (Ant44). The second assay evaluates the ability of the PTI to inhibit the uptake of a rescuing dose of spermidine into DFMO-treated cells. The final assay is the gold standard for the field and involves determining the concentration of PTI needed to inhibit 50 % of the uptake of each of the radiolabeled native polyamines: 3H-putrescine, 3H-spermidine or 14C-spermine. These assays provide EC50 and IC50 values which allow a formal ranking of transport inhibition potency to aid in PTI selection.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"65-91"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094323","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