A Cytidine Phosphoramidite with Protected Nitroxide Spin Label: Synthesis of a Full-Length TAR RNA and Investigation by In-Line Probing and EPR Spectroscopy

Chemistry : a European Journal
Timo WeinrichMichael W Göbel

Abstract

EPR studies on RNA are complicated by three major obstacles related to the chemical nature of nitroxide spin labels: Decomposition while oligonucleotides are chemically synthesized, further decay during enzymatic strand ligation, and undetected changes in conformational equilibria due to the steric demand of the label. Herein possible solutions for all three problems are presented: A 2-nitrobenzyloxymethyl protective group for nitroxides that is stable under all conditions of chemical RNA synthesis and can be removed photochemically. By careful selection of ligation sites and splint oligonucleotides, high yields were achieved in the assembly of a full-length HIV-1 TAR RNA labeled with two protected nitroxide groups. PELDOR measurements on spin-labeled TAR in the absence and presence of arginine amide indicated arrest of interhelical motions on ligand binding. Finally, even minor changes in conformation due to the presence of spin labels are detected with high sensitivity by in-line probing.

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Citations

Apr 22, 2018·FEBS Letters·Ilektra-Chara GiassaLukas Trantirek
Dec 7, 2018·Organic & Biomolecular Chemistry·Christof DomnickStephanie Kath-Schorr
May 16, 2019·Chembiochem : a European Journal of Chemical Biology·Anandi KugeleMalte Drescher
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Jan 26, 2020·Angewandte Chemie·Christof DomnickStephanie Kath-Schorr
Aug 29, 2020·Nucleic Acids Research·Christine WuebbenOlav Schiemann
Dec 11, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Christine WuebbenOlav Schiemann
Jul 29, 2020·International Journal of Molecular Sciences·Yukiko KamiyaHiroyuki Asanuma
Mar 1, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Felix ZellmannMichael W Göbel
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Jul 16, 2019·Current Opinion in Chemical Biology·Marcel Hollenstein
May 28, 2021·Chembiochem : a European Journal of Chemical Biology·Hannah DepmeierStephanie Kath-Schorr
Dec 14, 2021·Biophysical Journal·Burkhard EndewardXianyang Fang

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