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In-gel staining methods of G4 DNA and RNA structures. G4 DNA 和 RNA 结构的凝胶内染色法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2023-12-22 DOI: 10.1016/bs.mie.2023.12.002
Philipp Schult, Katrin Paeschke
{"title":"In-gel staining methods of G4 DNA and RNA structures.","authors":"Philipp Schult, Katrin Paeschke","doi":"10.1016/bs.mie.2023.12.002","DOIUrl":"10.1016/bs.mie.2023.12.002","url":null,"abstract":"<p><p>G-quadruplexes (G4) are functionally important nucleic acid structures, involved in many cellular pathways. They are often dynamically regulated in cells, which makes detecting them in vivo challenging and dependent on sophisticated technical equipment. Therefore, in vitro studies are commonly performed as a first step to confirm a candidate sequence folds into a G4. Several methods have been developed, each with its individual pros and cons. A highly accessible and quick approach, without the need for specialized equipment, is the detection of G4s in native gels using light-up probes. These molecules become fluorescent after specifically binding to G4s. Several different classes have been discovered, emitting light in various colors, and some possess specificity for certain G4 topologies, which makes them highly versatile tools for G4 visualization. Here, we will explore the general procedure using the light-up probe NMM on RNA G4s and discuss advantages and limitations of this method.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140194174","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
Single-molecule observation of G-quadruplex and R-loop formation induced by transcription. 单分子观测转录诱导的 G 型四联体和 R 型环的形成。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-16 DOI: 10.1016/bs.mie.2024.01.001
Jihee Hwang, Bradleigh Palmer, Sua Myong
{"title":"Single-molecule observation of G-quadruplex and R-loop formation induced by transcription.","authors":"Jihee Hwang, Bradleigh Palmer, Sua Myong","doi":"10.1016/bs.mie.2024.01.001","DOIUrl":"10.1016/bs.mie.2024.01.001","url":null,"abstract":"<p><p>Potential G-quadruplex forming sequences (PQS) are enriched in cancer-related genes and immunoglobulin class-switch recombination. They are prevalent in the 5'UTR of transcriptionally active genes, thereby contributing to the regulation of gene expression. We and others previously demonstrated that the PQS located in the non-template strand leads to an R-loop formation followed by a G-quadruplex (G4) formation during transcription. These structural changes increase mRNA production. Here, we present how single-molecule technique was used to observe cotranscriptional G4 and R-loop formation and to examine the impact on transcription, particularly for the initiation and elongation stages.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140194179","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
Construction and operation of high-resolution magnetic tape head tweezers for measuring single-protein dynamics under force. 用于测量单个蛋白质受力动态的高分辨率磁带头镊子的构造和操作。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-05 DOI: 10.1016/bs.mie.2023.12.010
Rafael Tapia-Rojo
{"title":"Construction and operation of high-resolution magnetic tape head tweezers for measuring single-protein dynamics under force.","authors":"Rafael Tapia-Rojo","doi":"10.1016/bs.mie.2023.12.010","DOIUrl":"10.1016/bs.mie.2023.12.010","url":null,"abstract":"<p><p>Mechanical forces are critical to protein function across many biological contexts-from bacterial adhesion to muscle mechanics and mechanotransduction processes. Hence, understanding how mechanical forces govern protein activity has developed into a central scientific question. In this context, single-molecule magnetic tweezers has recently emerged as a valuable experimental tool, offering the capability to measure single proteins over physiologically relevant forces and timescales. In this chapter, we present a detailed protocol for the assembly and operation of our magnetic tape head tweezers instrument, specifically tailored to investigate protein dynamics. Our instrument boasts a simplified microscope design and incorporates a magnetic tape head as the force-generating apparatus, facilitating precise force control and enhancing its temporal stability, enabling the study of single protein mechanics over extended timescales spanning several hours or even days. Moreover, its straightforward and cost-effective design ensures its accessibility to the wider scientific community. We anticipate that this technique will attract widespread interest within the growing field of mechanobiology and expect that this chapter will provide facilitated accessibility to this technology.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140140465","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
Force-fluorescence setup for observing protein-DNA interactions under load. 用于观察负载下蛋白质-DNA 相互作用的力荧光装置。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-16 DOI: 10.1016/bs.mie.2024.01.003
Jaehun Jung, Subin Kim, Sang-Hyun Rah, Jayil Lee, Min Ju Shon
{"title":"Force-fluorescence setup for observing protein-DNA interactions under load.","authors":"Jaehun Jung, Subin Kim, Sang-Hyun Rah, Jayil Lee, Min Ju Shon","doi":"10.1016/bs.mie.2024.01.003","DOIUrl":"10.1016/bs.mie.2024.01.003","url":null,"abstract":"<p><p>This chapter explores advanced single-molecule techniques for studying protein-DNA interactions, particularly focusing on Replication Protein A (RPA) using a force-fluorescence setup. It combines magnetic tweezers (MT) with total internal reflection fluorescence (TIRF) microscopy, enabling detailed observation of DNA behavior under mechanical stress. The chapter details the use of DNA hairpins and bare DNA to examine RPA's binding dynamics and its influence on DNA's mechanical properties. This approach provides deeper insights into RPA's role in DNA replication, repair, and recombination, highlighting its significance in maintaining genomic stability.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140140467","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
Magnetic nano-tweezer for interrogating mechanosensitive signaling proteins in space and time. 用于在空间和时间上检测机械敏感信号蛋白的磁性纳米镊子。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-20 DOI: 10.1016/bs.mie.2024.01.009
Minsuk Kwak
{"title":"Magnetic nano-tweezer for interrogating mechanosensitive signaling proteins in space and time.","authors":"Minsuk Kwak","doi":"10.1016/bs.mie.2024.01.009","DOIUrl":"10.1016/bs.mie.2024.01.009","url":null,"abstract":"<p><p>Spatiotemporal interrogation of signal transduction at the single-cell level is necessary to understand how extracellular cues are converted into biochemical signals and differentially regulate cellular responses. Using single-cell perturbation tools such as optogenetics, specific biochemical cues can be delivered to selective molecules or cells at any desired location and time. By measuring cellular responses to provided perturbations, investigators have decoded and deconstructed the working mechanisms of a variety of neuroelectric and biochemical signaling processes. However, analogous methods for deciphering the working mechanisms of mechanosensitive signaling by regulating mechanical inputs to cell receptors have remained elusive. To address this unmet need, we have recently developed a nanotechnology-based single-cell and single-molecule perturbation tool, termed mechanogenetics, that enables precise spatial and mechanical control over genetically encoded cell-surface receptors in live cells. This tool combines a magnetofluorescent nanoparticle (MFN) actuator, which provides precise spatial and mechanical signals to receptors via target-specific one-to-one interaction, with a micromagnetic tweezers that remotely controls the force exerted on a single nanoparticle. This chapter provides comprehensive experimental protocols of mechanogenetics consisting of four stages: (i) chemical synthesis of MFNs, (ii) bio-conjugation and purification of monovalent MFNs, (iii) establishment of cells with genetically encoded mechanosensitive proteins, and (iv) modular targeting and control of cell-surface receptors in live cells. The entire procedure takes up to 1 week. This mechanogenetic tool can be generalized to study many outstanding questions related to the dynamics of cell signaling and transcriptional control, including the mechanism of mechanically activated receptor.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140140471","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
Magnetic tweezers in cell mechanics. 细胞力学中的磁镊。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-05 DOI: 10.1016/bs.mie.2023.12.007
Claudia Tanja Mierke
{"title":"Magnetic tweezers in cell mechanics.","authors":"Claudia Tanja Mierke","doi":"10.1016/bs.mie.2023.12.007","DOIUrl":"10.1016/bs.mie.2023.12.007","url":null,"abstract":"<p><p>The chapter provides an overview of the applications of magnetic tweezers in living cells. It discusses the advantages and disadvantages of magnetic tweezers technology with a focus on individual magnetic tweezers configurations, such as electromagnetic tweezers. Solutions to the disadvantages identified are also outlined. The specific role of magnetic tweezers in the field of mechanobiology, such as mechanosensitivity, mechano-allostery and mechanotransduction are also emphasized. The specific usage of magnetic tweezers in mechanically probing cells via specific cell surface receptors, such as mechanosensitive channels is discussed and why mechanical probing has revealed the opening and closing of the channels. Finally, the future direction of magnetic tweezers is presented.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140140473","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
Vanadium haloperoxidases as noncanonical terpene synthases. 作为非典型萜烯合成酶的钒卤过氧化物酶
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-17 DOI: 10.1016/bs.mie.2024.03.024
Jackson T Baumgartner, Lia I Lozano Salazar, Lukas A Varga, Gabriel H Lefebre, Shaun M K McKinnie
{"title":"Vanadium haloperoxidases as noncanonical terpene synthases.","authors":"Jackson T Baumgartner, Lia I Lozano Salazar, Lukas A Varga, Gabriel H Lefebre, Shaun M K McKinnie","doi":"10.1016/bs.mie.2024.03.024","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.024","url":null,"abstract":"<p><p>Vanadium-dependent haloperoxidases (VHPOs) are a unique family of enzymes that utilize vanadate, an aqueous halide ion, and hydrogen peroxide to produce an electrophilic halogen species that can be incorporated into electron rich organic substrates. This halogen species can react with terpene substrates and trigger halonium-induced cyclization in a manner reminiscent of class II terpene synthases. While not all VHPOs act in this capacity, several notable examples from algal and actinobacterial species have been characterized to catalyze regio- and enantioselective reactions on terpene and meroterpenoid substrates, resulting in complex halogenated cyclic terpenes through the action of single enzyme. In this article, we describe the expression, purification, and chemical assays of NapH4, a difficult to express characterized VHPO that catalyzes the chloronium-induced cyclization of its meroterpenoid substrate.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469500","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
Bioinformatic analysis of microbial type terpene synthase genes in plants. 植物中微生物型萜烯合成酶基因的生物信息学分析。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-12 DOI: 10.1016/bs.mie.2024.02.014
Xinlu Chen, Jin Han, Feng Chen
{"title":"Bioinformatic analysis of microbial type terpene synthase genes in plants.","authors":"Xinlu Chen, Jin Han, Feng Chen","doi":"10.1016/bs.mie.2024.02.014","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.02.014","url":null,"abstract":"<p><p>Plants are prolific producers of terpenoids. Terpenoid biosynthesis is initiated by terpene synthases (TPS). In plants, two types of terpenes synthase genes are recognized: typical plant TPS genes and microbial-terpene synthase like-genes (MTPSL). While TPS genes are ubiquitous in land plants, MTPSL genes appear to be restricted to non-seed land plants. Evolutionarily, TPS genes are specific to land plants, whereas MTPSL genes have related counterparts in other organisms, especially fungi and bacteria. The presence of microbial type TPS in plants, fungi and bacteria, with the latter two often being associated with plants, poses a challenge in accurately identifying bona fide MTPSL genes in plants. In this chapter, we present bioinformatic procedures designed to identify MTPSL genes in sequenced plant genomes and/or transcriptomes. Additionally, we outline validation methods for confirming the identified microbial-type TPS genes as genuine plant genes. The method described in this chapter can also be adopted to analyze microbial type TPS in organisms other than plants.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469484","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
Structural biology of terpene synthases. 萜烯合成酶的结构生物学。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-31 DOI: 10.1016/bs.mie.2024.03.012
Baiying Xing, Zhenyu Lei, Zhaoye Bai, Guowei Zang, Yuxian Wang, Chenyu Zhang, Minren Chen, Yucheng Zhou, Jiahao Ding, Donghui Yang, Ming Ma
{"title":"Structural biology of terpene synthases.","authors":"Baiying Xing, Zhenyu Lei, Zhaoye Bai, Guowei Zang, Yuxian Wang, Chenyu Zhang, Minren Chen, Yucheng Zhou, Jiahao Ding, Donghui Yang, Ming Ma","doi":"10.1016/bs.mie.2024.03.012","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.012","url":null,"abstract":"<p><p>Structural biology research of terpene synthases (TSs) has provided a useful basis to understand their catalytic mechanisms in producing diverse terpene products with polycyclic ring systems and multiple chiral centers. However, compared to the large numbers of>95,000 terpenoids discovered to date, few structures of TSs have been solved and the understanding of their catalytic mechanisms is lagging. We here (i) introduce the basic catalytic logic, the structural architectures, and the metal-binding conserved motifs of TSs; (ii) provide detailed experimental procedures, in gene cloning and plasmid construction, protein purification, crystallization, X-ray diffraction data collection and structural elucidation, for structural biology research of TSs; and (iii) discuss the prospects of structure-based engineering and de novo design of TSs in generating valuable terpene molecules, which cannot be easily achieved by chemical synthesis.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469497","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
Structural studies of catalytic peptides using molecular dynamics simulations. 利用分子动力学模拟对催化肽进行结构研究。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-06 DOI: 10.1016/bs.mie.2024.01.019
Parth Rathee, Sreerag N Moorkkannur, Rajeev Prabhakar
{"title":"Structural studies of catalytic peptides using molecular dynamics simulations.","authors":"Parth Rathee, Sreerag N Moorkkannur, Rajeev Prabhakar","doi":"10.1016/bs.mie.2024.01.019","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.01.019","url":null,"abstract":"<p><p>Many self-assembling peptides can form amyloid like structures with different sizes and morphologies. Driven by non-covalent interactions, their aggregation can occur through distinct pathways. Additionally, they can bind metal ions to create enzyme like active sites that allow them to catalyze diverse reactions. Due to the non-crystalline nature of amyloids, it is quite challenging to elucidate their structures using experimental spectroscopic techniques. In this aspect, molecular dynamics (MD) simulations provide a useful tool to derive structures of these macromolecules in solution. They can be further validated by comparing with experimentally measured structural parameters. However, these simulations require a multi-step process starting from the selection of the initial structure to the analysis of MD trajectories. There are multiple force fields, parametrization protocols, equilibration processes, software and analysis tools available for this process. Therefore, it is complicated for non-experts to select the most relevant tools and perform these simulations effectively. In this chapter, a systematic methodology that covers all major aspects of modeling of catalytic peptides is provided in a user-friendly manner. It will be helpful for researchers in this critical area of research.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141180261","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
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