Current Protocols in Nucleic Acid Chemistry最新文献

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. © 2018 by John Wiley & Sons, Inc.

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. XSI uses gold nanocrystal labels site-specifically attached to a macromolecule and measures the scattering interference from pairs of heavy metal labels. The recorded signal can directly be transformed into a distance distribution between the two probes. We describe the underlying concepts, present a detailed protocol for preparing samples and recording XSI data, and provide a custom-written graphical user interface to facilitate XSI data analysis. © 2018 by John Wiley & Sons, Inc.

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. © 2018 by John Wiley & Sons, Inc.

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. The protocol is based on the Illumina platform and allows easy multiplexing of samples and the removal of sequencing reads that are PCR duplicates. © 2018 by John Wiley & Sons, Inc.

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. For these DNA and RNA systems, the oligonucleotides are post-synthetically labeled, specifically after solid-phase synthesis by chemical means, with a fluorescent dye using copper(I)-catalyzed cycloaddition at the 2′ position of single uridines. In order to functionalize oligonucleotides with several different labels, an on-resin method is required to ensure the necessary selectivity. This unit describes two different CuAAC (“click”) approaches—in solution (post-synthetic) and on solid phase (during synthesis)—for the attachment of fluorophores to the 2′ position of DNA. © 2018 by John Wiley & Sons, Inc.

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. Then, theoretical VCD spectra of stable conformers are calculated and averaged on the basis of their Boltzmann population. Finally, the obtained experimental and theoretical VCD spectra are compared qualitatively or quantitatively. The agreement between these spectra leads to determination of the stereochemistry of the studied molecule. This protocol may also be useful for analyzing the conformation of nucleosides. © 2018 by John Wiley & Sons, Inc.

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. © 2018 by John Wiley & Sons, Inc.

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.

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 ¹H, ¹³C, and ³¹P 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. Each method described here is extensively documented and annotated with tips and observations to facilitate their application. Sections are devoted to sample preparation, NMR experiments and setup, resonance assignment, structure generation protocols, evaluation, tips that may be useful, and software sources. © 2018 by John Wiley & Sons, Inc.

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. 2′-OMeUtaPS is also potent at delivering an uncharged antisense polyA-tailed PMO sequence in muscle cells of the mdx mouse model of muscular dystrophy; targeting the polyA-tailed PMO sequence against a splice site of the pre-mRNA encoding mutated dystrophin triggers an alternate splicing event that results in excision of the mutated exon (exon 23) from the pre-mRNA and production of functional dystrophin, as demonstrated by agarose gel electrophoresis. © 2018 by John Wiley & Sons, Inc.