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.
Highlights
Ribonucleic acids with methylphosphonate modifications have attracted significant interest recently to probe ribosome function during elongation factor G (EF-G) catalyzed elongation by atomic mutagenesis and to study intracellular signaling by second messenger cyclic-di-GMP derivatives that are resistant to phosphodiesterases [1,2]
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
Ribonucleic acids with methylphosphonate modifications have attracted significant interest recently to probe ribosome function during EF-G catalyzed elongation by atomic mutagenesis and to study intracellular signaling by second messenger cyclic-di-GMP derivatives that are resistant to phosphodiesterases [1,2]
Summary
Ribonucleic acids with methylphosphonate (mP) modifications have attracted significant interest recently to probe ribosome function during EF-G catalyzed elongation by atomic mutagenesis and to study intracellular signaling by second messenger cyclic-di-GMP derivatives that are resistant to phosphodiesterases [1,2]. Thereby, the replacement of a nonbridging phosphate oxygen by a methyl group makes the nucleic acid backbone charge neutral. It erases the possibility for hydrogen bond formation since proton acceptor capability gets lost [3,4,5,6,7,8]. Access to methylphosphonates was limited to dimers or short oligomers of DNA or 2′-OCH3 RNA only [3,4,11], solution and solid-phase syntheses of mP containing nucleic acids were continuously improved by Engels et al [12], Agarwal et al [13], Lebedev et al [14], Heliński et al [15], Reynolds et al [4], and Schell et al [16]. Stable RNA with mP linkages usually goes along with a 2′-deoxy or 2′-methoxy ribose modification at the site of the methylphosphonate nucleoside [6,17]
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