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Current Protocols in Nucleic Acid Chemistry

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https://www.readbyqxmd.com/read/29927111/rna-seq-for-bacterial-gene-expression
#1
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
https://www.readbyqxmd.com/read/29927110/recording-and-analyzing-nucleic-acid-distance-distributions-with-x-ray-scattering-interferometry-xsi
#2
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
https://www.readbyqxmd.com/read/29927103/time-resolved-hydroxyl-radical-footprinting-of-rna-with-x-rays
#3
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
https://www.readbyqxmd.com/read/29927099/synthesis-of-chimeric-oligonucleotides-having-modified-internucleotide-linkages-via-an-automated-h-phosphonate-phosphoramidite-approach
#4
Danielle Vlaho, Masad J Damha
This article describes an automated solid-phase approach for the synthesis of chimeric oligonucleotides containing phosphoramidate-modified internucleotide linkages. An optimized H-phosphonate synthetic cycle is combined with the commonly used phosphoramidite approach to obtain oligonucleotides comprising blocks having various types of internucleotide linkages. This article is specific to the synthesis of oligonucleotides having phosphoramidate modifications, but is adaptable to permit the incorporation of other modified linkages accessible through H-phosphonate diester intermediates...
June 2018: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/29927126/synthesis-of-dye-modified-oligonucleotides-via-copper-i-catalyzed-alkyne-azide-cycloaddition-using-on-and-off-bead-approaches
#5
Christian Schwechheimer, Larissa Doll, Hans-Achim Wagenknecht
Fluorescence molecular imaging is widely used to visualize and observe different biomolecules, in particular DNA and RNA, in vivo and in real time. Typically, DNA strands are tagged with only one fluorophore, and, in the case of molecular beacons, an additional quencher is conjugated, which bears the risk of false-positive or false-negative results because only fluorescence intensities at one fluorescence wavelength (color) are compared. To address this drawback, the concept of "DNA/RNA traffic lights," which is characterized by a fluorescence color change due to energy transfer between two dyes, was developed by our working group...
March 2018: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/29927125/stereochemistry-and-conformation-of-nucleosides-and-their-synthetic-precursors-by-vibrational-circular-dichroism
#6
Tohru Taniguchi, Kie Nakano, Kenji Monde
The chemistry of artificial nucleosides is associated with the difficulties in the characterization of the stereochemistry and conformation of their furanose ring moiety. This unit describes how to use vibrational circular dichroism (VCD) spectroscopy to identify the three-dimensional structure of nucleosides. The experimental part of this protocol is dedicated to obtain a VCD spectrum of a sample with high S/N ratio. The computational part generally starts with a conformational search using molecular mechanics and the following structural optimization by density functional theory calculation...
March 2018: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/29927124/nmr-structure-determination-for-oligonucleotides
#7
Alexander M Spring-Connell, Marina Evich, Markus W Germann
NMR spectroscopy is a versatile tool for determining the structure and dynamics of nucleic acids under solution conditions. In this unit, we provide an overview and detail of the experiments and methods used in our laboratory to determine the structure of oligonucleotides at natural abundance, thus limiting our approach to 1 H, 13 C, and 31 P NMR techniques. Isotopic labeling is heavily used in RNA NMR studies, however, labeling of DNA is still less common and, if modified nucleotides are investigated, is exceptionally expensive or not feasible...
March 2018: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/29927123/synthesis-characterization-and-function-of-an-rna-based-transfection-reagent
#8
Harsh V Jain, Jessica F Boehler, Kanneboyina Nagaraju, Serge L Beaucage
A synthetic 8-mer, amphipathic, trans-acting poly-2'-O-methyluridylic thiophosphate triester RNA element (2'-OMeUtaPS) can be prepared using solid-phase synthesis protocols. 2'-OMeUtaPS efficiently mediates the delivery of uncharged polyA-tailed phosphorodiamidate morpholino (PMO) sequences in HeLa pLuc 705 cells, as evidenced by flow cytometry measurements. In this cell line, 2'-OMeUtaPS-mediated transfection of an antisense polyA-tailed PMO sequence induces alternative splicing of an aberrant luciferase pre-mRNA splice site, leading to restoration of functional luciferase, as quantitatively measured using a typical luciferase assay...
March 2018: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/29927122/synthesis-of-n-6-substituted-adenosines-as-cytokinin-nucleosides
#9
Mikhail S Drenichev, Vladimir E Oslovsky, Vitali I Tararov, Sergey N Mikhailov
This unit describes preparation of N6 -substituted adenosines (cytokinin nucleosides), a unique class of compounds with a wide spectrum of biological activities. Regioselective alkylation of N6 -acetyl-2',3',5'-tri-O-acetyladenosine with alkyl halides under basic conditions or alcohols under Mitsunobu conditions followed by deprotection are the methods of choice for the preparation of the cytokinin nucleosides. The attractive feature of this strategy is the possibility of using a broad library of commercially available alkyl halides and alcohols under mild reaction conditions...
March 2018: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/29927121/synthesis-of-ena-nucleotides-and-ena-oligonucleotides
#10
Koji Morita, Makoto Koizumi
2'-O,4'-C-Ethylene-bridged nucleic acid (ENA) is a sugar-modified oligonucleotide with an ethylene bridge between the 2'-oxygen and 4'-carbon of ribose. ENA not only has as high binding affinity to complementary RNA as conventional bridged/locked nucleic acid, but also has much higher nuclease resistance in plasma, which makes it a promising candidate for antisense therapeutics. This unit presents detailed protocols for the synthesis and characterization of ENA nucleosides and oligonucleotides. © 2018 by John Wiley & Sons, Inc...
March 2018: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/29275540/synthesis-of-%C3%AE-nicotinamide-riboside-using-an-efficient-two-step-methodology
#11
Ning Zhang, Anthony A Sauve
A two-step chemical method for the synthesis of β-nicotinamide riboside (NR) is described. NR has achieved wide use as an NAD+ precursor (vitamin B3) and can significantly increase central metabolite NAD+ concentrations in mammalian cells. β-NR can be prepared with an efficient two-step procedure. The synthesis is initiated via coupling of commercially available 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose with ethyl nicotinate in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf). 1 H NMR showed that the product was formed with complete stereoselectivity to produce only the β-isomer in high yield (>90% versus starting sugar)...
December 24, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/29275539/synthesis-of-bipartite-tetracysteine-pna-probes-for-dna-in-situ-fluorescent-labeling
#12
Ge-Min Fang, Oliver Seitz
"Label-free" fluorescent probes that avoid additional steps or building blocks for conjugation of fluorescent dyes with oligonucleotides can significantly reduce the time and cost of parallel bioanalysis of a large number of nucleic acid samples. A method for the synthesis of "label-free" bicysteine-modified PNA probes using solid-phase synthesis and procedures for sequence-specific DNA in situ fluorescent labeling is described here. The concept is based on the adjacent alignment of two bicysteine-modified peptide nucleic acids on a DNA target to form a structurally optimized bipartite tetracysteine motif, which induces a sequence-specific fluorogenic reaction with commercially available biarsenic dyes, even in complex media such as cell lysate...
December 24, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/29275538/practical-synthesis-of-4-thioribonucleosides-from-l-arabinose-via-novel-reductive-ring-contraction-reaction-and-pummerer-type-thioglycosylation
#13
Hideaki Wakamatsu, Kozue Nitta, Nozomi Shoji, Yoshihiro Natori, Yukako Saito, Yuichi Yoshimura
The detailed practical synthesis of 4'-thionucleosides starting from L-arabinose is described here. 1,4-Anhydro-2,3-O-isopropylidene-4-thioribitol, which is the key intermediate for the synthesis of 4'-thionucleosides, is obtained from L-arabinose in several steps, including a novel reductive ring-contraction reaction. After oxidation of the key intermediate, the sulfoxide is subjected to Pummerer-type thioglycosylation in the presence of persilylated nucleobases to obtain the 4'-thioribonucleosides in good yield and β-selectively...
December 24, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/29275537/3-o-caged-2-deoxynucleoside-triphosphates-for-light-mediated-enzyme-catalyzed-template-independent-dna-synthesis
#14
Anu Stella Mathews, Haikang Yang, Carlo Montemagno
Synthesis, purification, and characterization of 3'-O-caged 2'-deoxyribonucleoside triphosphates (dNTPs), namely 3'-O-(2-nitrobenzyl)-2'-deoxy ribonucleoside triphosphates (NB-dNTPs) and 3'-O-(4,5-dimethoxy-2-nitrobenzyl)-2'-deoxy ribonucleoside triphosphates (DMNB-dNTPs), are discussed in detail. A total of eight 3'-O-caged dNTPs are synthesized with specific protocols depending on the nitrogenous base on the first carbon, i.e., adenine, guanine, thymine, and cytosine, as well as the photo-cleavable group, i...
December 24, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/29275536/palladium-catalyzed-synthesis-of-e-5-3-aminoallyl-uridine-5-o-triphosphates
#15
Muthian Shanmugasundaram, Annamalai Senthilvelan, Anilkumar R Kore
This unit describes a simple, reliable, and efficient chemical method for the synthesis of 5-(3-aminoallyl)-2'-deoxyuridine-5'-O-triphosphate (AA-dUTP) and 5-(3-aminoallyl)-uridine-5'-O-triphosphate (AA-UTP), starting from the corresponding nucleoside triphosphate. The presented strategy involves regioselective iodination of nucleoside triphosphate using N-iodosuccinimide followed by the palladium-catalyzed Heck coupling with allylamine to provide the corresponding (E)-5-aminoallyl-uridine-5'-O-triphosphate in good yields...
December 24, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28921499/synthesis-of-the-phosphoramidite-units-for-benzene-glycol-nucleic-acid
#16
Yusuke Maeda, Nazuki Niwa, Yoshihito Ueno
Benzene-glycol nucleic acid-DNA chimeras form thermally and thermodynamically stable duplexes with complementary RNAs, and have base-discriminating abilities. This unit describes the synthesis of four nucleoside analogs, an adenine, cytosine, thymine, and guanine analogs with base-benzene-glycol structure. The synthesis starts with conversion of (S)-mandelic acid in arylboronic acid derivative, common intermediate. Nucleobase coupling of the intermediate and phosphitylation afford to phosphoroamidite units...
September 18, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28921498/synthesis-of-4-selenoribonucleosides-the-building-blocks-of-4-selenorna-using-a-hypervalent-iodine
#17
Noriko Saito-Tarashima, Masashi Ota, Noriaki Minakawa
Herein is described a detailed protocol for the synthesis of 4'-selenoribonucleoside derivatives that involves the use of a hypervalent iodine species. These derivatives are versatile units for the preparation of 4'-selenoRNA. Large-scale synthesis of a 4-selenosugar starting from D-ribose is achieved in eight steps, including a final chromatographic purification. The resulting 4-selenosugar is then subjected to the one-pot Pummerer-like reaction using hypervalent iodine in the presence of silylated nucleobases...
September 18, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28921497/quantitative-analysis-of-nucleic-acid-stability-with-ligands-under-high-pressure-to-design-novel-drugs-targeting-g-quadruplexes
#18
Shuntaro Takahashi, Naoki Sugimoto
Nucleic acids (DNA and RNA) can form various non-canonical structures. Because some serious diseases are caused by the conformational change of G-quadruplex DNA structures, the development of ligands that bind and stabilize G-quadruplex DNA is of interest to the field of nucleic acid chemistry. Volumetric changes (ΔV) in the biomolecular reaction include the structural change of biomolecules and hydration behaviors, which provide information about the tertiary interaction between G-quadruplex DNA and ligands...
September 18, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28921496/nucleoside-o-methyl-h-phosphinates-novel-monomers-for-the-synthesis-of-methylphosphonate-oligonucleotides-using-h-phosphonate-chemistry
#19
Ondřej Kostov, Ondřej Páv, Ivan Rosenberg
This unit comprises the straightforward synthesis of protected 2'-deoxyribonucleoside-O-methyl-(H)-phosphinates in both 3'- and 5'-series. These compounds represent a new class of monomers compatible with the solid-phase synthesis of oligonucleotides using H-phosphonate chemistry and are suitable for the preparation of both 3'- and 5'-O-methylphosphonate oligonucleotides. The synthesis of 4-toluenesulfonyloxymethyl-(H)-phosphinic acid as a new reagent for the preparation of O-methyl-(H)-phosphinic acid derivatives is described...
September 18, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28921495/probing-rna-folding-pathways-by-rna-fingerprinting
#20
Sarah A Woodson
This unit provides protocols for using native polyacrylamide gel electrophoresis to distinguish folding and unfolding conformers of RNA. It is useful for studying conformers that can exchange in a period of minutes or seconds, and that are thus difficult to study by solution-based methods. Conformers that have been separated and immobilized in the gel matrix can be used to study catalytic activity with or without being eluted from the gel. The method can be applied to a wide variety of catalytic RNAs and RNA-protein complexes...
September 18, 2017: Current Protocols in Nucleic Acid Chemistry
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