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A new family of hydrazone modified cytidine phosphoramidite building block was synthesized and incorporated into oligodeoxynucleotides to construct photoswitchable DNA strands. TheE‐Zisomerization triggered by the irradiation of blue light with a wavelength of 450 nm was investigated and confirmed by¹H NMR spectroscopy and HPLC in the contexts of both nucleoside and oligodeoxynucleotide. The light activatedZform isomer of this hydrazone‐cytidine with a six‐member intramolecular hydrogen bond was found to inhibit DNA synthesis in the primer extension model by usingBstDNA polymerase. In addition, the hydrazone modification caused the misincorporation of dATP together with dGTP into the growing DNA strand with similar selectivity, highlighting a potential G to A mutation. This work provides a novel functional DNA building block and an additional molecular tool that has potential chemical biology and biomedicinal applications to control DNA synthesis and DNA‐enzyme interactions using the cell friendly blue light irradiation.

 

This article contains detailed synthetic protocols for preparation of 5‐cyanomethyluridine (cnm⁵U) and 5‐cyanouridine (cn⁵U) phosphoramidites. The synthesis of the cnm⁵U phosphoramidite building block starts with commercially available 5‐methyluridine (m⁵C), followed by bromination of the 5‐methyl group to install the cyano moiety using TMSCN/TBAF. The cn⁵U phosphoramidite is obtained by regular Vorbrüggen glycosylation of the protected ribofuranose with silylated 5‐cyanouracil. These two modified phosphoramidites are suitable for synthesis of RNA oligonucleotides on solid phase using conventional amidite chemistry. Our protocol provides access to two novel building blocks for constructing RNA‐based therapeutics. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of cnm⁵U and cn⁵U phosphoramidites

Basic Protocol 2: Synthesis, purification, and characterization of cnm⁵U‐ and cn⁵U‐modified RNA oligonucleotides

 

This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid‐phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed‐phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC‐MS and LC‐MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC‐MS is employed first to identify RNA phosphorothioate modifications. An optimized LC‐MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides

Basic Protocol 2: Digestion of RNA samples extracted from cells

Basic Protocol 3: Detection and quantification of RNA phosphorothioate modifications by mass spectrometry

 

Sulfur modifications have been discovered on both DNA and RNA. Sulfur substitution of oxygen atoms at nucleobase or backbone locations in the nucleic acid framework led to a wide variety of sulfur-modified nucleosides and nucleotides. While the discovery, regulation and functions of DNA phosphorothioate (PS) modification, where one of the non-bridging oxygen atoms is replaced by sulfur on the DNA backbone, are important topics, this review focuses on the sulfur modification in natural cellular RNAs and therapeutic nucleic acids. The sulfur modifications on RNAs exhibit diversity in terms of modification locations and cellular functions, but the various sulfur modifications share common biosynthetic strategies across RNA species, cell types and all domains of life. The first section reviews the post-transcriptional sulfur modifications on nucleobase with emphasis on thiouridine on tRNA and phosphorothioate modification on RNA backbones, as well as the functions of the sulfur modifications on different species of cellular RNAs. The second section reviews the biosynthesis of different types of sulfur modifications and summarizes the general strategy for the biosynthesis of sulfur-containing RNA residues. One of the main goals of investigating the sulfur modifications is to enrich the genomic drug development pipeline and enhance our understandings of the rapidly growing nucleic acid-based gene therapy. The last section of the review focuses on the current drug development strategies employing sulfur substitution of oxygen atoms in therapeutic RNAs. 

Abstract The N4-methylation of cytidine (m4C and m42C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m4C and m42C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m4C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m42C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m42C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N4-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.