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Selecting Fully-Modified XNA Aptamers Using Synthetic Genetics 利用合成遗传学选择全修饰的XNA适配体
Current protocols in chemical biology Pub Date : 2018-05-18 DOI: 10.1002/cpch.44
Alexander I. Taylor, Philipp Holliger
{"title":"Selecting Fully-Modified XNA Aptamers Using Synthetic Genetics","authors":"Alexander I. Taylor,&nbsp;Philipp Holliger","doi":"10.1002/cpch.44","DOIUrl":"10.1002/cpch.44","url":null,"abstract":"<p>This unit describes the application of “synthetic genetics,” i.e., the replication of xeno nucleic acids (XNAs), artificial analogs of DNA and RNA bearing alternative backbone or sugar congeners, to the directed evolution of synthetic oligonucleotide ligands (XNA aptamers) specific for target proteins or nucleic acid motifs, using a cross-chemistry selective exponential enrichment (X-SELEX) approach. Protocols are described for synthesis of diverse-sequence XNA repertoires (typically 10<sup>14</sup> molecules) using DNA templates, isolation and panning for functional XNA sequences using targets immobilized on solid phase or gel shift induced by target binding in solution, and XNA reverse transcription to allow cDNA amplification or sequencing. The method may be generally applied to select fully-modified XNA aptamers specific for a wide range of target molecules. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"10 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.44","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36243855","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 14
Design, Synthesis, and Application of the Trimethoprim-Based Chemical Tag for Live-Cell Imaging 基于甲氧苄啶的活细胞成像化学标签的设计、合成与应用
Current protocols in chemical biology Pub Date : 2018-04-26 DOI: 10.1002/9780470559277.ch130019
Chaoran Jing, Virginia W. Cornish
{"title":"Design, Synthesis, and Application of the Trimethoprim-Based Chemical Tag for Live-Cell Imaging","authors":"Chaoran Jing,&nbsp;Virginia W. Cornish","doi":"10.1002/9780470559277.ch130019","DOIUrl":"10.1002/9780470559277.ch130019","url":null,"abstract":"<p>Over the past decade, chemical tags have been developed to complement the use of fluorescent proteins in live-cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between <i>E. coli</i> dihydrofolate reductase and the antibiotic trimethoprim and was subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live-cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live-cell imaging. Alternate protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. <i>Curr. Protoc. Chem. Biol</i>. 5:131-155 © 2013 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31567609","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 5
Determining Lectin Specificity from Glycan Array Data Using Motif Segregation and GlycoSearch Software 利用Motif Segregation and GlycoSearch软件从Glycan Array数据中测定凝集素特异性
Current protocols in chemical biology Pub Date : 2018-04-26 DOI: 10.1002/9780470559277.ch130028
Doron Kletter, Zheng Cao, Marshall Bern, Brian Haab
{"title":"Determining Lectin Specificity from Glycan Array Data Using Motif Segregation and GlycoSearch Software","authors":"Doron Kletter,&nbsp;Zheng Cao,&nbsp;Marshall Bern,&nbsp;Brian Haab","doi":"10.1002/9780470559277.ch130028","DOIUrl":"10.1002/9780470559277.ch130028","url":null,"abstract":"<p>The glycan array is a powerful tool for investigating the specificities of glycan-binding proteins. By incubating a glycan-binding protein on a glycan array, the relative binding to hundreds of different oligosaccharides can be quantified in parallel. Based on these data, much information can be obtained about the preference of a glycan-binding protein for specific subcomponents of oligosaccharides, or motifs. In many cases, the analysis and interpretation of glycan array data can be time consuming and imprecise if done manually. Recently, GlycoSearch software was developed to facilitate the analysis and interpretation of glycan array data based on two previously developed methods, Motif Segregation and Outlier Motif Analysis. Here, the principles behind this method and the use of this new tool for mining glycan array data are described. The automated, objective, and precise analysis of glycan array data should enhance the value of these data for a broad range of research applications. <i>Curr. Protoc. Chem. Biol</i>. 5:157-169 © 2013 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31567610","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 17
Chemoenzymatic Site-Specific Reversible Immobilization and Labeling of Proteins from Crude Cellular Extract Without Prior Purification Using Oxime and Hydrazine Ligation 使用肟和联氨连接未经事先纯化的粗细胞提取物的化学酶位点特异性可逆固定和蛋白质标记
Current protocols in chemical biology Pub Date : 2018-04-26 DOI: 10.1002/9780470559277.ch120247
Mohammad M. Mahmoodi, Mohammad Rashidian, Jonathan K. Dozier, Mark D. Distefano
{"title":"Chemoenzymatic Site-Specific Reversible Immobilization and Labeling of Proteins from Crude Cellular Extract Without Prior Purification Using Oxime and Hydrazine Ligation","authors":"Mohammad M. Mahmoodi,&nbsp;Mohammad Rashidian,&nbsp;Jonathan K. Dozier,&nbsp;Mark D. Distefano","doi":"10.1002/9780470559277.ch120247","DOIUrl":"10.1002/9780470559277.ch120247","url":null,"abstract":"<p>In a facile and potentially general method for protein modification at the C-terminus, aldehyde-modified proteins, obtained from enzymatic protein prenylation, react rapidly with hydrazide and aminooxy surfaces and fluorophores at neutral pH and in micromolar concentration ranges of reagents. This strategy was used for fluorescent labeling of eGFP-CVIA, as a model protein, with aminooxy and hydrazide fluorophores or PEGs, and immobilization onto and subsequent release of the protein from hydrazide-functionalized agarose beads using hydrazone-oxime exchange. This method is described in detail here and provides site-specifically PEGylated or fluorescently labeled proteins starting from crude cellular extract in three steps: prenylation, capture, and release. <i>Curr. Protoc. Chem. Biol</i>. 5:89-109 © 2013 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/9780470559277.ch120247","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31568258","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 11
Spatiotemporal Control of Gene Expression in Mammalian Cells and in Mice Using the LightOn System 利用LightOn系统对哺乳动物细胞和小鼠基因表达的时空调控
Current protocols in chemical biology Pub Date : 2018-04-26 DOI: 10.1002/9780470559277.ch120267
Xianjun Chen, Xue Wang, Zengmin Du, Zhengcai Ma, Yi Yang
{"title":"Spatiotemporal Control of Gene Expression in Mammalian Cells and in Mice Using the LightOn System","authors":"Xianjun Chen,&nbsp;Xue Wang,&nbsp;Zengmin Du,&nbsp;Zhengcai Ma,&nbsp;Yi Yang","doi":"10.1002/9780470559277.ch120267","DOIUrl":"10.1002/9780470559277.ch120267","url":null,"abstract":"<p>A light-switchable transgene system could be a powerful optogenetic tool for the precise manipulation of spatiotemporal gene expression in multicellular organisms. We have developed the LightOn system, which consists of a single chimeric protein (GAVPO) that can homodimerize and bind to promoters upon exposure to blue light, activating transcription of a target gene. This article describes protocols for precise control of gene expression in mammalian cells and mice using the LightOn system. These protocols can be carried out in an ordinary laboratory, as both liposome-mediated transfection and hydrodynamic tail vein injection are routine methods that can easily transfer the LightOn system to mammalian cells and mouse liver, respectively. The illumination equipment can also be easily obtained. The LightOn system can provide a robust, convenient means to control the expression of a gene of interest, with unprecedented temporal and spatial accuracy in manipulating an extremely broad range of biological processes. <i>Curr. Protoc. Chem. Biol</i>. 5:111-129 © 2013 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/9780470559277.ch120267","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31567608","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 28
ALARM NMR for HTS Triage and Chemical Probe Validation 警报核磁共振为HTS分类和化学探针验证
Current protocols in chemical biology Pub Date : 2018-04-09 DOI: 10.1002/cpch.35
Jayme L. Dahlin, Matthew Cuellar, Gurpreet Singh, Kathryn M. Nelson, Jessica M. Strasser, Todd Rappe, Youlin Xia, Gianluigi Veglia, Michael A. Walters
{"title":"ALARM NMR for HTS Triage and Chemical Probe Validation","authors":"Jayme L. Dahlin,&nbsp;Matthew Cuellar,&nbsp;Gurpreet Singh,&nbsp;Kathryn M. Nelson,&nbsp;Jessica M. Strasser,&nbsp;Todd Rappe,&nbsp;Youlin Xia,&nbsp;Gianluigi Veglia,&nbsp;Michael A. Walters","doi":"10.1002/cpch.35","DOIUrl":"10.1002/cpch.35","url":null,"abstract":"<p>Nonspecific target engagement by test compounds and purported chemical probes is a significant source of assay interference and promiscuous bioactivity in high-throughput screening (HTS) and chemical biology. Most counter-screens for thiol-reactive compounds utilize mass spectrometry or fluorescence detection, as well as nonproteinaceous reporters like glutathione that may not always approximate the reactivity of protein side chains. By contrast, <span>a</span> <span>L</span>a <span>a</span>ssay to detect <span>r</span>eactive <span>m</span>olecules by <span>n</span>uclear <span>m</span>agnetic <span>r</span>esonance (ALARM NMR) is an industry-developed protein-based [<sup>1</sup>H-<sup>13</sup>C]-heteronuclear multiple quantum coherence NMR counter-screen to identify nonspecific protein interactions by test compounds by reporting their tendencies to modulate the human La antigen conformation. This article is a user's guide to the production of the <sup>13</sup>C-labeled La antigen reporter protein and reaction of test compounds with this reporter protein, as well as the collection and analysis of characteristic NMR spectra. Combined with other assay interference counter-screens, this assay will enhance chemical biology by helping researchers better prioritize chemical matter, which will increase the number of tractable HTS screening actives and aid in the development of better chemical probes. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.35","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36333859","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 20
PCR Amplification of Base-Modified DNA 碱基修饰DNA的PCR扩增
Current protocols in chemical biology Pub Date : 2018-04-09 DOI: 10.1002/cpch.33
Elena Eremeeva, Piet Herdewijn
{"title":"PCR Amplification of Base-Modified DNA","authors":"Elena Eremeeva,&nbsp;Piet Herdewijn","doi":"10.1002/cpch.33","DOIUrl":"10.1002/cpch.33","url":null,"abstract":"<p>An efficient PCR amplification of various templates (short 57-mer, random 67- and 82-mer, and long DNA) with base-modified nucleoside triphosphates is presented here. Using 5-substituted pyrimidine and 7-substituted-7-deaza- or 8-substituted purine nucleoside triphosphates as substrates for thermostable DNA polymerases [<i>Taq</i> and Vent (exo<sup>–</sup>)], successful PCR amplification of partially or entirely modified DNA libraries and long DNA constructs (up to 1.5 kb) is achieved. Visualization of double-stranded PCR product formation is improved through the use of primers with different fluorescent labels. This allows one to monitor the efficiency of modified substrate incorporation and the enzymatic recognition of the modified template during PCR. The redesigned fully base-modified DNA (denoted ‘DZA’) can be utilized for the straightforward production of diverse libraries for <i>in vitro</i> selection of aptamer and catalytic nucleic acids as well as for the synthesis of artificial genetic templates, replicons, or complex vectors. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.33","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36339626","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Design and Functional Characterization of Synthetic E3 Ubiquitin Ligases for Targeted Protein Depletion 靶向蛋白去除合成E3泛素连接酶的设计与功能表征
Current protocols in chemical biology Pub Date : 2018-04-09 DOI: 10.1002/cpch.37
Morgan R. Baltz, Erin A. Stephens, Matthew P. DeLisa
{"title":"Design and Functional Characterization of Synthetic E3 Ubiquitin Ligases for Targeted Protein Depletion","authors":"Morgan R. Baltz,&nbsp;Erin A. Stephens,&nbsp;Matthew P. DeLisa","doi":"10.1002/cpch.37","DOIUrl":"10.1002/cpch.37","url":null,"abstract":"<p>A number of techniques now exist for decreasing the expression of cellular proteins without the need for genomic modification. One such technique involves engineered protein chimeras that combine the ubiquitination activity of E3 ubiquitin ligases with the binding affinity and substrate specificity of designer binding proteins (DBPs). These chimeras, called “ubiquibodies,” are capable of selectively and controllably steering virtually any protein to the ubiquitin proteasome pathway (UPP) for degradation, making ubiquibodies a powerful addition to the protein knockout toolbox. A distinguishing feature of ubiquibodies is their modularity—simply swapping DBPs can generate a new ubiquibody with specificity for a different substrate protein. Moreover, by employing DBPs that recognize particular protein states (e.g., active versus inactive conformation, mutant versus wild-type, post-translational modification), it becomes possible to deplete certain protein subpopulations while sparing others. This protocol outlines the steps necessary to design and functionally evaluate ubiquibodies for customizable silencing of cellular proteins. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.37","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36337444","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 7
Preparation and Application of Enzyme-Nucleotide Conjugates 酶-核苷酸缀合物的制备及应用
Current protocols in chemical biology Pub Date : 2018-04-09 DOI: 10.1002/cpch.36
Moritz Welter, Andreas Marx
{"title":"Preparation and Application of Enzyme-Nucleotide Conjugates","authors":"Moritz Welter,&nbsp;Andreas Marx","doi":"10.1002/cpch.36","DOIUrl":"10.1002/cpch.36","url":null,"abstract":"<p>In this unit the preparation and application of enzyme-nucleotide conjugates is depicted. First, a modified nucleoside triphosphate is synthesized bearing a long and flexible linker equipped with a thiol group. The nucleotide is then reacted with maleimide-activated horseradish peroxidase to yield an enzyme-nucleotide conjugate, which due to the long linker, can be used as a substrate by DNA polymerases in primer extension reactions. Finally, an assay based on these findings is described that provides a fast and easy nucleic acid detection and genotyping platform. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.36","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36338594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Compartmentalized Self-Replication for Evolution of a DNA Polymerase DNA聚合酶进化的区隔自我复制
Current protocols in chemical biology Pub Date : 2018-04-09 DOI: 10.1002/cpch.34
Zhanar Abil, Andrew D. Ellington
{"title":"Compartmentalized Self-Replication for Evolution of a DNA Polymerase","authors":"Zhanar Abil,&nbsp;Andrew D. Ellington","doi":"10.1002/cpch.34","DOIUrl":"10.1002/cpch.34","url":null,"abstract":"<p>Compartmentalized self-replication (CSR) is an emulsion PCR-based method for the selection of DNA polymerases. <i>E. coli</i> host cells expressing a library of DNA polymerases are emulsified so that no more than a single cell is present in a single emulsion droplet. In a subsequent emulsion PCR step, the DNA polymerase protein, as well as the plasmid encoding it are released into the emulsion droplet and the genes that created the most active or abundant polymerase variants are exponentially amplified and can be passed to the next round of CSR. CSR is a powerful method for engineering of polymerases since it allows selection under a variety of conditions, including the use of non-standard substrates. In this unit, we provide a step-by-step procedure for the selection of polymerases, using as an example the selection of reverse transcriptase activity starting from a library of <i>Thermococcus kodakaraensis</i> (KOD) DNA polymerase variants. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.34","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36339627","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 6
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