title: Chemical synthesis of RNA with site-specific methylphosphonate modifications.
abstract: Methylphosphonate(mP)-modified RNA serves as valuable probe to evaluate biomolecular interactions between the nucleic acid backbone and binding partners, such as proteins or small molecules. Here, we describe an efficient workflow for the synthesis of RNA with a single mP modification in diastereomerically pure form. While the automated assembly of mP-modified RNA is straightforward, its deprotection under basic conditions is challenging; a carefully optimized step-by-step procedure is provided. In addition, we demonstrate purification and separation strategies for the RP and SP-configurated RNA diastereomers using a combination of anion-exchange and reversed-phase HPLC, and comment on troubleshooting if their separation appears difficult. Furthermore, we demonstrate the stereochemical assignment of short RP and SP mP-modified RNA diastereomers based on 2D ROESY NMR spectroscopy and we report on the impact of the mP modification on thermal and thermodynamic stabilities of RNA-DNA hybrid and RNA-RNA duplexes.
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences, CMBI, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck, Austria.
authors.forename: Sara
authors.initials: S
authors.lastname: Flür
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences, CMBI, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck, Austria. Electronic address: ronald.micura@uibk.ac.at.
authors.forename: Ronald
authors.initials: R
authors.lastname: Micura
mesh_terms: D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis; D009943:Organophosphorus Compounds / Q000737:chemistry*; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
chemical_list: D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; C032627:methylphosphonic acid; D012313:RNA
keywords: Methylphosphonate; RNA solid-phase synthesis; Stereochemical assignment; Thermodynamic base pairing stability