Methods in enzymology最新文献_第5页

Aptazyme-directed A-to-I RNA editing. 适配体酶导向的A-to-I RNA编辑。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.022

Xilei Ai, Zhuo Tang

{"title":"Aptazyme-directed A-to-I RNA editing.","authors":"Xilei Ai, Zhuo Tang","doi":"10.1016/bs.mie.2024.11.022","DOIUrl":"10.1016/bs.mie.2024.11.022","url":null,"abstract":"As a promising therapeutic approach, the RNA editing process can correct pathogenic mutations and is reversible and tunable, without permanently altering the genome. RNA editing mediated by human ADAR proteins offers unique advantages, including high specificity and low immunogenicity. Compared to CRISPR-based gene editing techniques, RNA editing events are temporary, which can reduce the risk of long-term unintended side effects, making off-target edits less concerning than DNA-targeting methods. Moreover, ADAR-based RNA editing tools are less likely to elicit immune reactions because ADAR proteins are of human origin, and their small size makes them relatively easy to incorporate into gene therapy vectors, such as adeno-associated virus vectors (AAVs), which have limited space. Despite the promise of RNA editing as a therapeutic approach, precise temporal and spatial control of RNA editing is still lacking. Therefore, we have developed a small molecule-inducible RNA editing strategy by incorporating aptazymes into the guide RNA of the BoxB-λN-ADAR system. This chapter provides detailed protocols for targeted RNA editing by ADAR deaminases using aptazyme-based guide RNAs controlled by exogenous small molecules, marking the earliest use of aptazymes to regulate RNA editing strategies. Once small molecules are added or removed, aptazymes trigger self-cleavage to release the guide RNA, thus achieving small molecule-controlled RNA editing. To satisfy different RNA editing applications, we have realized the conditional activation and deactivation of A-to-I RNA editing of target mRNA using switch aptazymes. We provide step-by-step protocols for constructing guide RNA plasmids for regulatory purposes and conducting small molecule-induced RNA regulatory editing experiments in cells.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"267-283"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052979","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

Using Prime Editing Guide Generator (PEGG) for high-throughput generation of prime editing sensor libraries. 利用质数编辑引导生成器（peg）实现高通量生成质数编辑传感器库。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-06 DOI: 10.1016/bs.mie.2025.01.006

Samuel I Gould, Francisco J Sánchez-Rivera

引用次数: 0

Restoration of cytidine to uridine genetic code using an MS2-APOBEC1 artificial enzymatic approach. 利用MS2-APOBEC1人工酶法恢复胞苷对尿苷遗传密码的影响。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-15 DOI: 10.1016/bs.mie.2024.11.034

Sonali Bhakta, Toshifumi Tsukahara

{"title":"Restoration of cytidine to uridine genetic code using an MS2-APOBEC1 artificial enzymatic approach.","authors":"Sonali Bhakta, Toshifumi Tsukahara","doi":"10.1016/bs.mie.2024.11.034","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.034","url":null,"abstract":"By employing site-directed RNA editing (SDRE) to restore point-mutated RNA molecules, it is possible to change gene-encoded information and synthesize proteins with different functionality from a single gene. Thymine (T) to cytosine (C) point mutations cause various genetic disorders, and when they occur in protein-coding regions, C-to-uridine (U) RNA changes can lead to non-synonymous alterations. By joining the deaminase domain of apolipoprotein B messenger RNA (mRNA) editing catalytic polypeptide 1 (APOBEC1) with a guide RNA (gRNA) complementary to a target mRNA, we created an artificial RNA editase. We used an mRNA encoding blue fluorescent protein (BFP), obtained from the green fluorescent protein (GFP) gene through the introduction of a T > C mutation, as our target RNA. In a proof of principle experiment, we reverted the T > C mutation at the RNA level using our APOBEC1 site-directed RNA editing system, recovering GFP signal. Sanger sequencing of cDNA from transfected cells and polymerase chain reaction-restriction length polymorphism analysis validated this result, indicating an editing of approximately 21 %. Our successful development of an artificial RNA editing system using the deaminase APOBEC1, in conjunction with the MS2 system, may lead to the development of treatments for genetic diseases based on the restoration of specific types of wild type sequences at the mRNA level.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"713 ","pages":"271-285"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144018386","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

Ribozyme-mediated expression of tRNA-derived small RNAs in bacteria. 核糖酶介导的trna衍生小rna在细菌中的表达。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.003

Carmela Esposito, Anamaria Buzoianu, Marina Cristodero, Norbert Polacek

{"title":"Ribozyme-mediated expression of tRNA-derived small RNAs in bacteria.","authors":"Carmela Esposito, Anamaria Buzoianu, Marina Cristodero, Norbert Polacek","doi":"10.1016/bs.mie.2024.11.003","DOIUrl":"10.1016/bs.mie.2024.11.003","url":null,"abstract":"Transfer RNA-derived RNAs (tDRs) have emerged as important regulatory molecules found across all three domains of life. Despite their discovery over four decades ago, their biological significance has only recently begun to be elucidated. However, studying bacterial tDRs poses challenges due to technical limitations in assessing their in vivo functionality. To address this, we established a novel approach utilizing a self-cleaving Twister ribozyme to express tDRs in Escherichia coli. Specifically, we employed the type P1 Sva1-1 Twister ribozyme, to generate tDRs with genuine 3' ends. Our method involves the inducible expression of tDRs by incorporating the desired tDR sequence into a plasmid construct downstream of two lac operators and upstream of the Twister ribozyme. Upon induction with IPTG and transcription of the construct, the Twister ribozyme undergoes self-cleavage, thus producing tDRs with defined 3' ends. As a proof of principle, we demonstrated the in vivo application of our novel method by expressing and analyzing two stress-induced tRNA halves in E. coli. Overall, our method offers a valuable tool for studying tDRs in bacteria to shed light on their regulatory roles in cellular processes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"65-83"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425825","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

En masse evaluation of RNA guides (EMERGe) for ADARs. RNA指南（EMERGe）对ADARs的整体评价。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-02 DOI: 10.1016/bs.mie.2024.11.030

Prince J Salvador, Natalie M Dugan, Randall Ouye, Peter A Beal

{"title":"En masse evaluation of RNA guides (EMERGe) for ADARs.","authors":"Prince J Salvador, Natalie M Dugan, Randall Ouye, Peter A Beal","doi":"10.1016/bs.mie.2024.11.030","DOIUrl":"10.1016/bs.mie.2024.11.030","url":null,"abstract":"Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in duplex RNA, and through the delivery of guide RNAs, can be directed to edit specific adenosine sites. As ADARs are endogenously expressed in humans, their editing capacities hold therapeutic potential and allow us to target disease-relevant sequences in RNA through the rationale design of guide RNAs. However, current design principles are not suitable for difficult-to-edit target sites, posing challenges to unlocking the full therapeutic potential of this approach. This chapter discusses how we circumvent this barrier through an in vitro screening method, En Masse Evaluation of RNA Guides (EMERGe), which enables comprehensive screening of ADAR substrate libraries and facilitates the identification of editing-enabling guide strands for specific adenosines. From library generation and screening to next generation sequencing (NGS) data analysis to verification experiments, we describe how a sequence of interest can be identified through this high-throughput screening method. Furthermore, we discuss downstream applications of selected guide sequences, challenges in maximizing library coverage, and potential to couple the screen with machine learning or deep learning models.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"131-152"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12014283/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143053011","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

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。

4区生物学

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