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Current Protocols in Nucleic Acid Chemistry

Arivazhagan Rajendran, Narumi Shigi, Jun Sumaoka, Makoto Komiyama
The human genome is highly susceptible to various modifications, lesions, and damage. To analyze lesions and proteins bound to a defined region of the human genome, the genome should be fragmented at desired sites and the region of interest should be isolated. The few available methods for isolating a desired region of the human genome have serious drawbacks and can only be applied to specific sequences or require tedious experimental procedures. We have recently developed a novel method to isolate a desired fragment of the genome released by site-specific scission of DNA using a pair of pseudo-complementary peptide nucleic acids (pcPNAs) and S1 nuclease...
February 12, 2019: Current Protocols in Nucleic Acid Chemistry
Kousuke Sato, Akira Matsuda
Straightforward and efficient methods for the synthesis of 2-amino-4-fluoropyridine-C-nucleoside (dF P) and the solid-phase synthesis of oligodeoxynucleotides containing dF P using a phosphoramidite are described. The synthesis of dF P is achieved by cross-coupling between a nucleobase (2-amino-4-fluoro-3,5-diiodopyridine) and sugar moieties. Its 3'-O-phosphoramidite is obtained by deiodination, 5'-O-protection, and 3'-O-phosphitilation in three steps. The phosphoramidite unit is compatible for the synthesis of oligonucleotides on solid-phase according to conventional phosphoramidite chemistry...
February 12, 2019: Current Protocols in Nucleic Acid Chemistry
Sung Ju Cho, Arash Ghorbani-Choghamarani, Yoshio Saito, Robert H E Hudson
The detailed synthetic protocols for the preparation of phosphoramidite reagents compatible with standard, automated oligonucleotide synthesis for the 2'-deoxy- and ribo-6-phenylpyrrolocyitidine are reported. Each protocol starts with the parent nucleoside and prepares the 5'-O-dimethoxytrityl-N4 -benzoyl-5-iodocytosine derivative for the nucleobase modification chemistry. The key step is the direct formation of 6-phenylpyrrolocytosine aglycon via a sequential, one-pot Pd-catalyzed Sonogashira-type cross- coupling followed by a 5-endo-dig cyclization...
February 6, 2019: Current Protocols in Nucleic Acid Chemistry
Arivazhagan Rajendran, Narumi Shigi, Jun Sumaoka, Makoto Komiyama
By combining a pair of pseudo-complementary peptide nucleic acids (pcPNAs) with S1 nuclease, a novel tool to cut DNA at a predetermined site can be obtained. Complementary pcPNAs invade the DNA duplex and base pair to each strand of a target site, creating single-stranded regions that are cleaved by S1 nuclease. The scission site can be freely modulated by the design of pcPNAs. This method can be used to cleave a single site in the human genome. This protocol presents experimental details for site-selective scission using this versatile new tool...
February 5, 2019: Current Protocols in Nucleic Acid Chemistry
Hari K Akula, Mahesh K Lakshman
Two approaches for C4 modifications of silyl-protected thymidine, 2'-deoxyuridine, and 3'-azido-2',3'-dideoxythymidine (AZT) are described. In both, nucleoside amide activation with 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and DBU yields O4 -(benzotriazol-1-yl) derivatives. These in situ-formed intermediates are reacted with various nucleophiles, resulting in C4 modifications. In the two-step, one-pot approach, the O4 -(benzotriazol-1-yl) nucleoside intermediates are initially produced by reactions of the nucleosides with BOP and DBU in THF...
January 28, 2019: Current Protocols in Nucleic Acid Chemistry
Pratibha P Ghodke, Matthew E Albertolle, Kevin M Johnson, F Peter Guengerich
O6 -Alkylguanine DNA-alkyltransferase (AGT), a DNA repair protein, can form crosslinks with DNA. The AGT-DNA crosslinks are known to be mutagenic when AGT is heterologously expressed in Escherichia coli, as well as in mammalian cells. To understand the biological consequences, reliable access to AGT-oligonucleotide crosslinks is needed. This article describes the synthesis and characterization of site-specific AGT-oligonucleotide crosslinks at the N2-position of deoxyguanosine and N6-position of deoxyadenosine...
January 18, 2019: Current Protocols in Nucleic Acid Chemistry
Pavlina Viskova, Daniel Krafcik, Lukas Trantirek, Silvie Foldynova-Trantirkova
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...
November 29, 2018: Current Protocols in Nucleic Acid Chemistry
Minguk Seo, Li Lei, Martin Egli
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 (Kd ) of the interaction can be measured...
November 21, 2018: Current Protocols in Nucleic Acid Chemistry
A Emilia Arguello, Tharan Srikumar, Ralph E Kleiner
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...
December 2018: Current Protocols in Nucleic Acid Chemistry
Ruth Suchsland, Bettina Appel, Sabine Müller
We have developed two methods, in solution and on solid phase, that give easy access to trinucleotide phosphoramidites capable of undergoing coupling reactions by the solid-phase phosphoramidite approach. The solution protocol is characterized by application of 5'-O-dimethoxytrityl (DMT) and 3'-O-tert-butyldimethylsilyl (TBDMS) as a pair of orthogonal protecting groups and 2-cyanoethyl (CE) for protection of the phosphate. Starting with suitably functionalized monomers, synthesis proceeds in the 3'- to 5'-direction, delivering the fully protected trinucleotide...
December 2018: Current Protocols in Nucleic Acid Chemistry
M Benjamin Turner, Brooke A Anderson, George N Samaan, Michael Coste, Dillon D Burns, Byron W Purse
DEA tC is a tricyclic 2'-deoxycytidine analog that can be incorporated into oligonucleotides by solid-phase synthesis and that exhibits a large fluorescence enhancement when correctly base-paired with a guanine base in a DNA-DNA duplex. The synthesis of DEA tC begins with 5-amino-2-methylbenzothiazole and provides the DEA tC nucleobase analog over five synthetic steps. This nucleobase analog is then silylated using N,O-bis(trimethylsilyl)acetamide and conjugated to Hoffer's chlorosugar to provide the protected DEA tC nucleoside in good yield...
December 2018: Current Protocols in Nucleic Acid Chemistry
Yu Hirano, Naoshi Kojima, Yasuo Komatsu
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...
October 13, 2018: Current Protocols in Nucleic Acid Chemistry
Samy Mohamady, Scott D Taylor
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...
October 11, 2018: Current Protocols in Nucleic Acid Chemistry
Vladimir E Oslovsky, Mikhail S Drenichev, Cyril S Alexeev, Pavel N Solyev, Roman S Esipov, Sergey N Mikhailov
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...
October 9, 2018: Current Protocols in Nucleic Acid Chemistry
Shatrughn Bhilare, Siva Sankar Murthy Bandaru, Anant R Kapdi, Yogesh S Sanghvi, Carola Schulzke
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...
September 2018: Current Protocols in Nucleic Acid Chemistry
Renaud Zelli, Waël Zeinyeh, Jean-Luc Décout
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...
September 2018: Current Protocols in Nucleic Acid Chemistry
Elisa Pileggi, Michaela Serpi, Fabrizio Pertusati
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...
September 2018: Current Protocols in Nucleic Acid Chemistry
Line Dahl Poulsen, Jeppe Vinther
RNA sequencing (RNA-seq) has become the preferred method for global quantification of bacterial gene expression. With the continued improvements in sequencing technology and data analysis tools, the most labor-intensive and expensive part of an RNA-seq experiment is the preparation of sequencing libraries, which is also essential for the quality of the data obtained. Here, we present a straightforward and inexpensive basic protocol for preparation of strand-specific RNA-seq libraries from bacterial RNA as well as a computational pipeline for the data analysis of sequencing reads...
June 2018: Current Protocols in Nucleic Acid Chemistry
Thomas Zettl, Rhiju Das, Pehr A B Harbury, Daniel Herschlag, Jan Lipfert, Rebecca S Mathew, Xuesong Shi
Most structural techniques provide averaged information or information about a single predominant conformational state. However, biological macromolecules typically function through series of conformations. Therefore, a complete understanding of macromolecular structures requires knowledge of the ensembles that represent probabilities on a conformational free energy landscape. Here we describe an emerging approach, X-ray scattering interferometry (XSI), a method that provides instantaneous distance distributions for molecules in solution...
June 2018: Current Protocols in Nucleic Acid Chemistry
Yumeng Hao, Jen Bohon, Ryan Hulscher, Mollie C Rappé, Sayan Gupta, Tadepalli Adilakshmi, Sarah A Woodson
RNA footprinting by hydroxyl radical cleavage provides 'snapshots' of RNA tertiary structure or protein interactions that bury the RNA backbone. Generation of hydroxyl radicals with a high-flux synchrotron X-ray beam provides analysis on a short timescale (5-100 msec), which enables the structures of folding intermediates or other transient conformational states to be determined in biochemical solutions or cells. This article provides protocols for using synchrotron beamlines for hydroxyl radical footprinting...
June 2018: Current Protocols in Nucleic Acid Chemistry
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