Methods in enzymology最新文献_第5页

Methylated polyamines derivatives and antizyme-related effects. 甲基化多胺衍生物和抗酶相关作用。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-21 DOI: 10.1016/bs.mie.2025.01.072

Maxim A Khomutov, Arthur I Salikhov, Olga A Smirnova, Vladimir A Mitkevich, Alex R Khomutov

引用次数: 0

Targeting polyamine metabolism in an ex vivo prostatectomy model. 在体外前列腺切除术模型中靶向多胺代谢。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-11 DOI: 10.1016/bs.mie.2025.01.070

Hayley C Affronti, Aryn M Rowsam, Spencer R Rosario, Dominic J Smiraglia

{"title":"Targeting polyamine metabolism in an ex vivo prostatectomy model.","authors":"Hayley C Affronti, Aryn M Rowsam, Spencer R Rosario, Dominic J Smiraglia","doi":"10.1016/bs.mie.2025.01.070","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.070","url":null,"abstract":"Ex vivo models allow for testing drug efficacy and patient response, yet it remains a challenge to develop representative 3D cultures for prostate cancer. Tissue explant models offer a more clinically relevant alternative to organoids due to their ability to provide adequate tissue quantities, maintain tumor-stromal interactions and metabolic activity, and their relatively inexpensive culturing conditions. In this chapter we outline a protocol for culturing patient prostatectomy tumors for up to 7 days on dental sponges soaked in either control or drug containing media for evaluating drug efficacy. Further, we describe the preparation of tissue samples for downstream immunohistochemistry and metabolic analysis. We have tested the efficacy of a combination therapy targeting polyamine metabolism, which is dysregulated in prostate cancer, using this patient tumor explant model. We found that activating polyamine catabolism in combination with inhibition of methionine salvage was effective at inducing target protein expression, reducing intratumoral polyamines, and inducing apoptosis in a majority of the patient samples tested. Additionally, we were able to confirm drug induced effects were specific to the malignant prostate epithelial cells. This ex vivo prostatectomy model lends itself to both targeted metabolite analyses as well as more comprehensive metabolomic analyses. This method can be applied to strategies aiming to target metabolic pathways in solid tumor diseases.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"231-239"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094332","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

Whole-cell biocatalysis with Myxococcus xanthus. 黄粘球菌的全细胞生物催化。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2025.01.005

Lea Winand, Markus Nett

{"title":"Whole-cell biocatalysis with Myxococcus xanthus.","authors":"Lea Winand, Markus Nett","doi":"10.1016/bs.mie.2025.01.005","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.005","url":null,"abstract":"Biocatalysis has become an attractive complement to conventional chemical catalysis in the field of organic synthesis. This trend is due to the excellent chemo-, regio- and stereoselectivity of biocatalysis, which typically avoids the formation of unwanted by-products as well as cross-reactivity between reactants. Since the use of isolated enzymes and their cofactors can be associated with high costs, there is a continued interest in whole-cell biocatalysts. Applications range from the production of chiral building blocks to the derivatization of natural products and the generation of new drug candidates for the pharmaceutical and fine chemical industries. The bacterium Myxococcus xanthus is an emerging cell factory, especially for the production of antibiotics and other bioactive natural products. Not only possesses M. xanthus a considerable tolerance toward xenobiotics, but it is also a metabolically versatile host providing important building blocks for natural product biosynthesis. In this chapter, we describe procedures for the whole-cell biocatalysis with M. xanthus. This also involves methods for the construction of expression plasmids and their transfer into M. xanthus.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"714 ","pages":"219-237"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144009658","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

Genome mining of RiPPs driven by highly efficient pathway reconstruction methods. 高效途径重建方法驱动的RiPPs基因组挖掘。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-14 DOI: 10.1016/bs.mie.2025.01.059

Hengqian Ren

{"title":"Genome mining of RiPPs driven by highly efficient pathway reconstruction methods.","authors":"Hengqian Ren","doi":"10.1016/bs.mie.2025.01.059","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.059","url":null,"abstract":"Ribosomally synthesized and post-translationally modified peptides (RiPPs) constitute an emerging family of natural products, with arising interest in their biosynthetic diversity and therapeutic potentials. Advances in genome sequencing and bioinformatics have significantly accelerated the identification of RiPP biosynthetic gene clusters (BGCs) from genome sequences, however, deciphering the products of these BGCs remains challenging, primarily due to their highly diverse biological origins and elusive genetic regulation machineries. This chapter describes the use of pathway reconstruction approaches for exploring the biosynthetic potential of cryptic RiPP BGCs. Specifically, a plug-and-play pathway refactoring workflow is described, which can effectively rewire the underlying regulatory systems in target BGCs, ensuring their expression in genetically tractable organisms. In addition, the Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE), a method capable of cloning large DNA fragments into selected expression vectors, is provided as an alternative way for investigating BGCs with intricate gene arrangements. Due to their high efficiency and robustness, these methods would be of interest to those working on the genome mining of RiPPs, as well as other families of natural products.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"717 ","pages":"175-197"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144619070","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

Genome mining with hypothetical proteins. 用假设的蛋白质进行基因组挖掘。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-06-18 DOI: 10.1016/bs.mie.2025.04.004

Grace E Kenney

引用次数: 0

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