Methods in enzymology最新文献_第9页

Chemoenzymatic labeling of RNA using terminal uridylyl transferase and bioorthogonal click chemistry. 利用末端尿苷基转移酶和生物正交点击化学对RNA进行化学酶标记。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-01 DOI: 10.1016/bs.mie.2026.05.007

Swagata Dutta, Arindam Pal, Seergazhi G Srivatsan

{"title":"Chemoenzymatic labeling of RNA using terminal uridylyl transferase and bioorthogonal click chemistry.","authors":"Swagata Dutta, Arindam Pal, Seergazhi G Srivatsan","doi":"10.1016/bs.mie.2026.05.007","DOIUrl":"10.1016/bs.mie.2026.05.007","url":null,"abstract":"The ability of native and engineered nucleic acid-processing enzymes to incorporate clickable nucleotide substrates has greatly advanced bioorthogonal labeling of nucleic acids, overcoming the limitations of conventional solid-phase oligonucleotide (ON) synthesis. In this chemoenzymatic approach, template-dependent polymerases routinely enable the incorporation of nucleotides bearing small reactive handles. The resulting nucleic acids undergo chemoselective reactions, such as azide-alkyne cycloaddition, inverse-electron-demand Diels-Alder, or Staudinger ligation, to install desired functionalities. Alternatively, the promiscuity of template-independent transferases, such as terminal uridylyl transferase (TUTase), provides access to site-specific labeling of RNA ONs at the 3'-end. In this methods chapter, we detail protocols for incorporating azide-modified UTP analogs into short RNA ONs and highly structured CRISPR guide RNAs (sgRNAs) using the terminal uridylyl transferase SpCID1. We describe methods to control the enzyme's incessant incorporation behavior and enable subsequent click functionalization of the RNAs. Finally, we demonstrate remodeling of the CRISPR system via synthesis of azide-modified sgRNAs, which when complexed with dCas9, recruit azide groups to specific gene targets for post-hybridization functionalization.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"731 ","pages":"223-252"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148330973","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 incorporation of a BCN-bearing amino acid for cell-surface protein labeling via tetrazine ligation. 携带bcn的氨基酸的遗传整合，通过四氮连接用于细胞表面蛋白标记。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-21 DOI: 10.1016/bs.mie.2026.05.020

Yawei Liu, Sicong Yao, Lei Zhang, Yu-Hsuan Tsai

引用次数: 0

Practical guidelines for the AMI-Isonitrile ligation. ami -异腈结扎的实用指南。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-01 DOI: 10.1016/bs.mie.2026.05.014

Ian Warm, Athanasios Markos, Helma Wennemers

引用次数: 0

Studying bacterial glycans with azidosugars and bioorthogonal chemistry. 叠氮糖与细菌聚糖的研究及生物正交化学。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-25 DOI: 10.1016/bs.mie.2026.05.015

Aniqa Chowdhury, Divya Bhargava, Karen D Moulton, Danielle H Dube

引用次数: 0

SMART deep learning tools to accelerate the characterization of natural product structures from their NMR data. SMART深度学习工具，加速天然产物结构的表征，从他们的核磁共振数据。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-09-27 DOI: 10.1016/bs.mie.2025.08.022

Byeol Ryu, Myeong Ji Kim, Wangdong Xu, Chen Zhang, Mingxun Wang, Hyunwoo Kim, Garrison W Cottrell, William H Gerwick

引用次数: 0

Predicting lasso peptide structure with LassoPred. 用lasopred预测套索肽结构。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-02-21 DOI: 10.1016/bs.mie.2026.01.037

Xingyu Ouyang, Xinchun Ran, Dantong Zhu, Zhongyue John Yang

{"title":"Predicting lasso peptide structure with LassoPred.","authors":"Xingyu Ouyang, Xinchun Ran, Dantong Zhu, Zhongyue John Yang","doi":"10.1016/bs.mie.2026.01.037","DOIUrl":"10.1016/bs.mie.2026.01.037","url":null,"abstract":"Lasso peptides (LaPs) are a structurally unique class of ribosomally synthesized and post-translationally modified peptides (RiPPs), characterized by a threaded rotaxane topology that confers exceptional stability and a broad spectrum of bioactivities. Despite increasing interest in LaPs as antimicrobial and therapeutic agents, accurate structure prediction remains a major challenge due to the scarcity of homologous templates and the failure of mainstream prediction tools to capture their knotted topology. In this chapter, we present LassoPred, a modular, machine learning-guided computational pipeline for high-throughput 3D structure prediction of lasso peptides from sequence alone. LassoPred integrates support vector machine classifiers trained on ESM2 embeddings to annotate critical topological features-namely, the isopeptide ring and plug residues-and a topology-aware structure constructor that assembles and refines atomic models via homology modeling, residue mutation, and energy minimization. The tool is implemented in Python, compatible with AMBER and PyMOL, and accessible via a public web interface, enabling both expert and non-specialist users to submit sequences and retrieve optimized structural models. LassoPred demonstrates near-experimental accuracy while reducing prediction time to minutes, expanding known lasso peptide structural coverage from fewer than 50 experimentally determined structures to over 4000 genome-mined models. The pipeline is further extensible to engineered LaP variants and potentially other RiPP families. By bridging the gap between sequence discovery and structural insight, LassoPred facilitates downstream applications in enzymology, structural biology, synthetic biology, and therapeutic design.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"730 ","pages":"91-106"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148006061","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

Characterizations of the β-amyloid (Aβ)-membrane interaction intermediates. β-淀粉样蛋白(Aβ)-膜相互作用中间体的表征。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-28 DOI: 10.1016/bs.mie.2026.01.028

Maurine K Kengwerere, Tingyao Wang, Wei Qiang

引用次数: 0

Methods for characterizing the individual filament structures of amyloid peptide assemblies using atomic force microscopy. 用原子力显微镜表征淀粉样肽组合的单个纤维结构的方法。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-02-03 DOI: 10.1016/bs.mie.2026.01.026

Afsheen Shahbaz, Samantha L Weetman, Claudia Chitty, Wei-Feng Xue

{"title":"Methods for characterizing the individual filament structures of amyloid peptide assemblies using atomic force microscopy.","authors":"Afsheen Shahbaz, Samantha L Weetman, Claudia Chitty, Wei-Feng Xue","doi":"10.1016/bs.mie.2026.01.026","DOIUrl":"10.1016/bs.mie.2026.01.026","url":null,"abstract":"A wide range of distinctive helical filamentous amyloid structures self-assembled from monomeric peptide or protein building blocks are found both in nature and in human disease states. Amyloid nano-fibrils are also being developed and synthetically made as novel peptide-based nanomaterials. In humans, accumulation of a range of amyloid structures, for example those formed from the amyloid-beta peptides in Alzheimer's disease, play a crucial role in the pathology of neurodegenerative and metabolic diseases. The diverse range of amyloid structures found is a manifestation of the amyloid structural polymorphism phenomenon. This is where different filament structures are assembled even from the same peptide or protein precursors. Due to the structural diversity of amyloid fibrils that can be found even in the same sample or the same disease state, an experimental method that allows structural analysis of individual amyloid filaments is required to understand the relationships between the polymorphic structures and the biological and physicochemical properties they elicit. Here, a method with a detailed protocol to analyze the structures of individual amyloid filament assemblies by topological Atomic Force Microscopy (AFM) imaging and Contact-Point Reconstruction AFM (CPR-AFM) image analysis is described. This approach to resolve the 3D shapes of amyloid polymorphs, one individual fibril at a time, allows mapping of the polymorphic landscapes of amyloid assemblies. It serves as an inexpensive, fast and effective experimental tool for individual filament level structural analysis, and offers new, exciting opportunities in elucidating population distributions of heterogeneous amyloid samples, rare amyloid structures within the populations, and structural variations between or within individual filaments. These are all key parts to experimental developments in therapeutic discovery and novel bio-nanomaterials applications.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"729 ","pages":"281-308"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776315","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

Tagging lipoproteins in mycobacteria via metabolic incorporation of modified fatty acids. 通过修饰脂肪酸的代谢结合标记分枝杆菌中的脂蛋白。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-02-11 DOI: 10.1016/bs.mie.2026.01.023

Kindra L Becker, Julian P Maceren, Lia A Parkin, Jessica C Seeliger

{"title":"Tagging lipoproteins in mycobacteria via metabolic incorporation of modified fatty acids.","authors":"Kindra L Becker, Julian P Maceren, Lia A Parkin, Jessica C Seeliger","doi":"10.1016/bs.mie.2026.01.023","DOIUrl":"10.1016/bs.mie.2026.01.023","url":null,"abstract":"Lipoproteins are lipid-modified proteins and critical components of the mycobacterial cell envelope with diverse functions related to virulence and survival. In Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, lipoproteins account for ∼3 % of the Mtb proteome, yet their precise functions remain poorly characterized. Prior experimental efforts have addressed the validation and preliminary functional annotation of specific lipoproteins. Whereas these studies elucidated aspects of lipoprotein localization and involvement in host-pathogen interactions, the methodologies employed do not allow further analysis such as targeted enrichment or on a proteome-wide level. To overcome these limitations, we developed a method in which modified fatty acids are metabolically incorporated into lipoproteins. These fatty acids are functionalized with click-compatible and photo-activatable chemical handles, thereby enabling downstream detection and identification of lipoproteins. The method described here provides a facile and versatile approach for further investigating mycobacterial lipoprotein synthesis, identity, and function.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"727 ","pages":"161-177"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147326739","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

In vitro methods for studying protein-lipid droplet interactions. 体外研究蛋白-脂滴相互作用的方法。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-02-16 DOI: 10.1016/bs.mie.2026.01.021

Julie Eichler, Catherine Tomasetto, Guillaume Drin, Fabien Alpy

引用次数: 0