Current Protocols in Nucleic Acid Chemistry最新文献

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 N⁶ -methyladenosine (m⁶ 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.

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 N⁶ -substituted adenosine or O⁶ -substituted inosine derivatives in the presence of purine nucleoside phosphorylase (PNP) and Na₂ HAsO₄ . 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.

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 & Sons, Inc.

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 (K_d) 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 K_d 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 & Sons, Inc.

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′-O-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 & Sons, Inc.

This article describes a straight-forward chemical method for the synthesis of nucleoside-5′-O-tetraphosphates, such as cytosine-, guanosine-, adenosine-, and uridine-5′-O-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 N-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′-O-tetraphosphates were obtained in yields of 84% to 86%. © 2018 by John Wiley & Sons, Inc.

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-D-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-N-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 & Sons, Inc.

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 & Sons, Inc.

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-O-acetyl-2,3,5-tri-O-benzoyl-β-D-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 & Sons, Inc.