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Current Protocols in Nucleic Acid Chemistry最新文献

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A Photocrosslinking-Based RNA Chemical Proteomics Approach to Profile m6 A-Regulated Protein-RNA Interactions. 基于光交联的RNA化学蛋白质组学方法研究m6 A调控的蛋白质-RNA相互作用。
Current Protocols in Nucleic Acid Chemistry Pub Date : 2018-12-01 Epub Date: 2018-11-08 DOI: 10.1002/cpnc.69
A Emilia Arguello, Tharan Srikumar, Ralph E Kleiner
{"title":"A Photocrosslinking-Based RNA Chemical Proteomics Approach to Profile m<sup>6</sup> A-Regulated Protein-RNA Interactions.","authors":"A Emilia Arguello,&nbsp;Tharan Srikumar,&nbsp;Ralph E Kleiner","doi":"10.1002/cpnc.69","DOIUrl":"https://doi.org/10.1002/cpnc.69","url":null,"abstract":"<p><p>Post-transcriptional modifications play an important role in RNA biology. In particular, the addition of small chemical groups to the nucleobases of mRNA can affect how modified transcripts are processed in the cell, thereby impacting gene expression programs. In order to study the molecular mechanisms underlying these modifications, it is necessary to characterize their 'readers', that is, proteins that directly bind to these modifications to mediate their functional consequences; this is a major challenge because we lack approaches to precisely manipulate RNA chemistry in the cell and because protein-modified RNA interactions can be low affinity. In this unit, we describe in detail a photocrosslinking-based RNA chemical proteomics approach to profile the protein-modified RNA interactome modulated by N<sup>6</sup> -methyladenosine (m<sup>6</sup> A), the most abundant internal modification in eukaryotic mRNA. First, we present protocols for the synthesis and characterization of short, diazirine-containing synthetic RNA probes, followed by a description of their use in mass spectrometry-based proteomics with HeLa cell lysate and a short commentary on data analysis and result interpretation. © 2018 by John Wiley & Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.69","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36659522","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
Synthesis of Cytokinins via Enzymatic Arsenolysis of Purine Nucleosides. 嘌呤核苷酶解法合成细胞分裂素。
Current Protocols in Nucleic Acid Chemistry Pub Date : 2018-12-01 Epub Date: 2018-10-09 DOI: 10.1002/cpnc.61
Vladimir E Oslovsky, Mikhail S Drenichev, Cyril S Alexeev, Pavel N Solyev, Roman S Esipov, Sergey N Mikhailov
{"title":"Synthesis of Cytokinins via Enzymatic Arsenolysis of Purine Nucleosides.","authors":"Vladimir E Oslovsky,&nbsp;Mikhail S Drenichev,&nbsp;Cyril S Alexeev,&nbsp;Pavel N Solyev,&nbsp;Roman S Esipov,&nbsp;Sergey N Mikhailov","doi":"10.1002/cpnc.61","DOIUrl":"https://doi.org/10.1002/cpnc.61","url":null,"abstract":"<p><p>This unit describes an effective method for the preparation of natural cytokinins and their synthetic derivatives based on enzymatic cleavage of the N-glycosidic bond of N<sup>6</sup> -substituted adenosine or O<sup>6</sup> -substituted inosine derivatives in the presence of purine nucleoside phosphorylase (PNP) and Na<sub>2</sub> HAsO<sub>4</sub> . The arsenolysis reaction is irreversible due to the hydrolysis of the resulting α-D-ribose-1-arsenate. As a result, the desired products are formed in near-quantitative yields, as indicated by high-performance liquid chromatography (HPLC) analysis, and can easily be isolated. In the strategy used here, the ribose residue acts as a protective group. © 2018 by John Wiley & Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.61","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36609827","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: 3
In-Cell NMR Spectroscopy of Nucleic Acids in Human Cells 人类细胞中核酸的细胞内核磁共振光谱
Current Protocols in Nucleic Acid Chemistry Pub Date : 2018-11-29 DOI: 10.1002/cpnc.71
Pavlina Viskova, Daniel Krafcik, Lukas Trantirek, Silvie Foldynova-Trantirkova
{"title":"In-Cell NMR Spectroscopy of Nucleic Acids in Human Cells","authors":"Pavlina Viskova,&nbsp;Daniel Krafcik,&nbsp;Lukas Trantirek,&nbsp;Silvie Foldynova-Trantirkova","doi":"10.1002/cpnc.71","DOIUrl":"10.1002/cpnc.71","url":null,"abstract":"<p>In-cell NMR spectroscopy is a unique tool that enables the study of the structure and dynamics of biomolecules as well as their interactions in the complex environment of living cells at near-to-atomic resolution. In this article, detailed instructions are described for setting up an in-cell NMR experiment for monitoring structures of DNA oligonucleotides introduced into nuclei of living human cells via tailored electroporation. Detailed step-by-step protocols for both the preparation of an in-cell NMR sample as well as protocols for conducting essential control experiments including flow cytometry and confocal microscopy are described. The strengths and limitations of in-cell NMR experiments are discussed. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.71","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36733160","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
Label-Free Electrophoretic Mobility Shift Assay (EMSA) for Measuring Dissociation Constants of Protein-RNA Complexes 用于测量蛋白质- rna复合物解离常数的无标记电泳迁移率转移测定(EMSA)
Current Protocols in Nucleic Acid Chemistry Pub Date : 2018-11-21 DOI: 10.1002/cpnc.70
Minguk Seo, Li Lei, Martin Egli
{"title":"Label-Free Electrophoretic Mobility Shift Assay (EMSA) for Measuring Dissociation Constants of Protein-RNA Complexes","authors":"Minguk Seo,&nbsp;Li Lei,&nbsp;Martin Egli","doi":"10.1002/cpnc.70","DOIUrl":"10.1002/cpnc.70","url":null,"abstract":"<p>The electrophoretic mobility shift assay (EMSA) is a well-established method to detect formation of complexes between proteins and nucleic acids and to determine, among other parameters, equilibrium constants for the interaction. Mixtures of protein and nucleic acid solutions of various ratios are analyzed via polyacrylamide gel electrophoresis (PAGE) under native conditions. In general, protein–nucleic acid complexes will migrate more slowly than the free nucleic acid. From the distributions of the nucleic acid components in the observed bands in individual gel lanes, quantitative parameters such as the dissociation constant (<i>K<sub>d</sub></i>) of the interaction can be measured. This article describes a simple and rapid EMSA that relies either on precast commercial or handcast polyacrylamide gels and uses unlabeled protein and nucleic acid. Nucleic acids are instead detected with SYBR Gold stain and band intensities established with a standard gel imaging system. We used this protocol specifically to determine <i>K<sub>d</sub></i> values for complexes between the PAZ domain of Argonaute 2 (Ago2) enzyme and native and chemically modified RNA oligonucleotides. EMSA-based equilibrium constants are compared to those determined with isothermal titration calorimetry (ITC). Advantages and limitations of this simple EMSA are discussed by comparing it to other techniques used for determination of equilibrium constants of protein-RNA interactions, and a troubleshooting guide is provided. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.70","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36704651","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: 18
Issue Information TOC 发布信息TOC
Current Protocols in Nucleic Acid Chemistry Pub Date : 2018-11-15 DOI: 10.1002/cpnc.64
{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpnc.64","DOIUrl":"10.1002/cpnc.64","url":null,"abstract":"","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.64","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50902115","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Synthesis and Application of Interstrand Cross-Linked Duplexes by Covalently Linking a Pair of Abasic Sites 共价连接一对碱基的链间交联双链化合物的合成及应用
Current Protocols in Nucleic Acid Chemistry Pub Date : 2018-10-13 DOI: 10.1002/cpnc.63
Yu Hirano, Naoshi Kojima, Yasuo Komatsu
{"title":"Synthesis and Application of Interstrand Cross-Linked Duplexes by Covalently Linking a Pair of Abasic Sites","authors":"Yu Hirano,&nbsp;Naoshi Kojima,&nbsp;Yasuo Komatsu","doi":"10.1002/cpnc.63","DOIUrl":"10.1002/cpnc.63","url":null,"abstract":"<p>Interstrand cross-linking of DNA or RNA inhibits the double strands from dissociating into single strands. This article contains detailed procedures for the synthesis of a novel interstrand cross-linker that comprises a bis-aminooxy naphthalene derivative and a description of its use in the preparation of sequence-specific interstrand cross-linked oligonucleotide duplexes. The interstrand cross-linker covalently connects a pair of apurinic/apyrimidinic sites in DNA/RNA duplexes with bis(aminooxy) groups. The resulting oxime linkages are stable under physiological conditions and greatly improve the thermal stability of the duplex. In addition, we construct a novel anti-miRNA oligonucleotide (AMO) flanked by interstrand cross-linked 2′-<i>O</i>-methylated RNA duplexes (CLs). AMO flanked by CLs at the 5′- and 3′-termini exhibited high inhibition activity toward miRNA function in cells. The novel interstrand cross-linker indicates potent activity and is applicable in biophysical studies, oligonucleotide therapeutics, and materials science. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.63","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36625166","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: 1
Synthesis of Nucleoside-5′-O-Tetraphosphates from Activated Trimetaphosphate and Nucleoside-5′-O-Monophosphates 活化三甲基磷酸和核苷-5′- o -单磷酸合成核苷-5′- o -四磷酸
Current Protocols in Nucleic Acid Chemistry Pub Date : 2018-10-11 DOI: 10.1002/cpnc.62
Samy Mohamady, Scott D. Taylor
{"title":"Synthesis of Nucleoside-5′-O-Tetraphosphates from Activated Trimetaphosphate and Nucleoside-5′-O-Monophosphates","authors":"Samy Mohamady,&nbsp;Scott D. Taylor","doi":"10.1002/cpnc.62","DOIUrl":"10.1002/cpnc.62","url":null,"abstract":"<p>This article describes a straight-forward chemical method for the synthesis of nucleoside-5′-<i>O</i>-tetraphosphates, such as cytosine-, guanosine-, adenosine-, and uridine-5′-<i>O</i>-tetraphosphates, starting from the corresponding nucleoside monophosphates and trimetaphosphate, a readily available and inexpensive starting material. The procedure involves reacting the tri(tetrabutylammonium) salt of trimetaphosphate with mesitylenesulfonyl chloride and <i>N</i>-methylimidazole. The resulting activated cyclic trimetaphosphate is reacted with the tetrabutylammonium salts of nucleoside monophosphates. After quenching the reaction with buffer and high-performance liquid chromatography purification, the desired nucleoside-5′-<i>O</i>-tetraphosphates were obtained in yields of 84% to 86%. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.62","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36615492","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
Pd/PTABS: An Efficient Water-Soluble Catalytic System for the Amination of 6-Chloropurine Ribonucleoside and Synthesis of Alogliptin Pd/PTABS: 6-氯嘌呤核糖核苷胺化及阿格列汀合成的高效水溶性催化体系
Current Protocols in Nucleic Acid Chemistry Pub Date : 2018-08-21 DOI: 10.1002/cpnc.58
Shatrughn Bhilare, Siva Sankar Murthy Bandaru, Anant R. Kapdi, Yogesh S. Sanghvi, Carola Schulzke
{"title":"Pd/PTABS: An Efficient Water-Soluble Catalytic System for the Amination of 6-Chloropurine Ribonucleoside and Synthesis of Alogliptin","authors":"Shatrughn Bhilare,&nbsp;Siva Sankar Murthy Bandaru,&nbsp;Anant R. Kapdi,&nbsp;Yogesh S. Sanghvi,&nbsp;Carola Schulzke","doi":"10.1002/cpnc.58","DOIUrl":"10.1002/cpnc.58","url":null,"abstract":"<p>The synthesis and catalytic applications of the highly water-soluble ligand 7-phospha-1,3,5-triaza-admantane butane sultonate (PTABS) has been described. The synthesized PTABS ligand along with palladium acetate exhibits excellent reactivity towards the amination reaction of 6-chloro-9-(β-<span>D</span>-ribofuranosyl)-9H-purine at ambient temperature. This protocol offers an advantage over the previously published procedures for the amination of 6-chloropurine nucleoside furnishing 6-<i>N</i>-substituted adenosine analogues. The validation of the present strategy has been demonstrated via synthesis of a uracil-based, anti-diabetic drug alogliptin. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.58","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36413839","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: 9
Preparation of Pyrimidine Alkenyl Acyclic Nucleoside Phosphonoamidates 嘧啶烯基无环核苷膦酸酯的制备
Current Protocols in Nucleic Acid Chemistry Pub Date : 2018-08-13 DOI: 10.1002/cpnc.56
Elisa Pileggi, Michaela Serpi, Fabrizio Pertusati
{"title":"Preparation of Pyrimidine Alkenyl Acyclic Nucleoside Phosphonoamidates","authors":"Elisa Pileggi,&nbsp;Michaela Serpi,&nbsp;Fabrizio Pertusati","doi":"10.1002/cpnc.56","DOIUrl":"10.1002/cpnc.56","url":null,"abstract":"<p>This synthetic protocol describes two strategies for the preparation of pyrimidine alkenyl acyclic nucleoside phosphonoamidates (ANPs), including linear and trisubstituted alkenyl derivatives. For the first procedure, a bis-trimethylsilyl ester of the parent alkenyl ANPs is the key intermediate that reacts with the desired amino acid ester and aryl alcohol. For the second procedure, an allyl phosphonoamidate bearing the ProTide promoieties is the key synthon employed as olefin partner for a cross-metathesis reaction with an alkylated nucleobase. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.56","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36392414","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
6-Chloropurine Ribonucleosides from Chloropyrimidines: One-Pot Synthesis 从氯嘧啶中提取的6-氯嘌呤核糖核苷:一锅合成
Current Protocols in Nucleic Acid Chemistry Pub Date : 2018-08-13 DOI: 10.1002/cpnc.57
Renaud Zelli, Waël Zeinyeh, Jean-Luc Décout
{"title":"6-Chloropurine Ribonucleosides from Chloropyrimidines: One-Pot Synthesis","authors":"Renaud Zelli,&nbsp;Waël Zeinyeh,&nbsp;Jean-Luc Décout","doi":"10.1002/cpnc.57","DOIUrl":"10.1002/cpnc.57","url":null,"abstract":"<p>A one-pot glycosylation and cyclization procedure is described for the synthesis of 6-chloropurine ribonucleosides from chloropyrimidines. From such a procedure and modification of the obtained chloropurine ribonucleosides, many drug candidates or molecular tools for biological study designed from their similarity to naturally occurring nucleosides could be obtained. The synthesis begins by preparation of several amidinoaminochloropyrimidines as precursors for the one-pot procedure. Then, by adding trimethylsilyl trifluoromethanesulfonate (TMSOTf) to a mixture of a pyrimidine and 1-<i>O</i>-acetyl-2,3,5-tri-<i>O</i>-benzoyl-β-<span>D</span>-ribose, different 6-chloropurine ribonucleosides are obtained. This methodology allows the straightforward introduction of an alkyl substituent at position 8 of purine ribonucleosides, which then can be functionalized at positions 2 and 6. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.57","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36392417","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
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