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1. Non‐Chromatographic Purification of Synthetic RNA Using Bio‐Orthogonal Chemistry

   https://doi.org/10.1002/cpz1.247  

   He, Muhan; Wu, Xunshen; Mao, Song; Haruehanroengra, Phensinee; Khan, Irfan; Sheng, Jia; Royzen, Maksim (September 2021, Current Protocols)

   Abstract Solid‐phase synthesis of RNA oligonucleotides over 100 nt in length remains challenging due to the complexity of purification of the target strands from the failure sequences. This article describes a non‐chromatographic procedure that will enable routine solid‐phase synthesis and purification of long RNA strands. The optimized five‐step process is based on bio‐orthogonal inverse electron demand Diels‐Alder chemistry betweentrans‐cyclooctene (TCO) and tetrazine (Tz), and entails solid‐phase synthesis of RNA on a photo‐labile support. The target oligonucleotide strands are selectively tagged with Tz while on‐support. After photocleavage from the solid support, the target oligonucleotide strands can be captured and purified from the failure sequences using immobilized TCO. The approach can be applied for purification of 76‐nt long tRNA and 101‐nt long sgRNA for CRISPR experiments. Purity of the isolated oligonucleotides should be evaluated using gel electrophoresis, while functional fidelity of the sgRNA should be confirmed using CRISPR‐Cas9 experiments. © 2021 Wiley Periodicals LLC. Basic Protocol: Five‐step non‐chromatographic purification of synthetic RNA oligonucleotides Support Protocol 1: Synthesis of the components that are required for the non‐chromatographic purification of long RNA oligonucleotides. Support Protocol 2: Solid‐phase RNA synthesis 

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2. Detection and Quantification of RNA Phosphorothioate Modifications Using Mass Spectrometry

   https://doi.org/10.1002/cpnc.113  

   Wu, Ying; Zheng, Ya Ying; Lin, Qishan; Sheng, Jia (August 2020, Current Protocols in Nucleic Acid Chemistry)

   Abstract 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 

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3. Synthesis of 5‐Cyanomethyluridine (cnm ⁵ U) and 5‐Cyanouridine (cn ⁵ U) Phosphoramidites and Their Incorporation into RNA Oligonucleotides

   https://doi.org/10.1002/cpnc.114  

   Mao, Song; Tsai, Hsu‐Chun; Sheng, Jia (August 2020, Current Protocols in Nucleic Acid Chemistry)

   Abstract 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 

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4. Sensitive RNA Synthesis Using Fluoride-Cleavable Groups for Linking and Amino Protection

   https://doi.org/10.26434/chemrxiv-2024-mh72n  

   Apostle, Alexander; Perera, Manoj; Middleton, Daniel; Fang, Shiyue (November 2024, RSC and ACS)

   A chemical method suitable for the synthesis of RNAs containing modifications such as N4-acetylcytidine (ac4C) that are unstable under the basic and nucleophilic conditions used by standard RNA synthesis methods is described. The method uses the 4-((t-butyldimethylsilyl)oxy)-2-methoxybutanoyl (SoM) group for the protection of exo-amino groups of nucleobases and the 4-((t-butyldimethylsilyl)oxy)-2-((aminophosphaneyl)oxy)butanoyl (SoA) group as the linker for solid phase synthesis. RNA cleavage and amino deprotection are achieved using fluoride under the same conditions used for the removal of the 2′-OH silyl protecting groups. Using the method, a wide range of electrophilic and base-sensitive groups including those that play structural and regulatory roles in biological systems and those that are artificially designed for various purposes are expected to be able to be incorporated into any position of any RNA sequences. As a proof of concept, a 26-mer RNA containing the highly sensitive ac4C epitranscriptomic modification was successfully synthesized and purified with RP HPLC. MALDI MS analysis indicated that the ac4C modification is completely stable under the fluoride deprotection conditions. The sensitive RNA synthesis method is expected to be able to overcome the long lasting obstacle of accessing various modified sensitive RNAs to projects in areas such as epitranscriptomics, molecular biology and the development of nucleic acid therapeutics. 

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5. Developing a solid-phase method for the enzymatic synthesis of heparan sulfate and chondroitin sulfate backbones

   https://doi.org/10.1093/glycob/cwad093  

   Stancanelli, Eduardo; Liu, Wei; Su, Guowei; Padagala, Vijay; Liu, Jian (February 2024, Glycobiology)

   Abstract Despite the recent progress on the solution-phase enzymatic synthesis of heparan sulfate (HS) and chondroitin sulfate (CS), solid-phase enzymatic synthesis has not been fully investigated. Here, we describe the solid-phase enzymatic synthesis of HS and CS backbone oligosaccharides using specialized linkers. We demonstrate the use of immobilized HS linker to synthesize CS, and the use of immobilized CS linker to synthesize HS. The linkers were then digested with chondroitin ABCase and heparin lyases, respectively, to obtain the products. Our findings uncover a potential approach for accelerating the synthesis of structurally homogeneous HS and CS oligosaccharides. 

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