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Chemo‐Enzymatic Synthesis of Position‐Specifically Modified RNA for Biophysical Studies including Light Control and NMR Spectroscopy
Authors:Sara Keyhani  Thomas Goldau  Anja Blümler  Prof?Dr Alexander Heckel  Prof?Dr Harald Schwalbe
Institution:1. Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt am Main, Frankfurt/Main, Germany;2. Institute for Organic Chemistry and Chemical Biology, Goethe University Frankfurt am Main, Frankfurt/Main, Germany
Abstract:The investigation of non‐coding RNAs requires RNAs containing modifications at every possible position within the oligonucleotide. Here, we present the chemo‐enzymatic RNA synthesis containing photoactivatable or 13C,15N‐labelled nucleosides. All four ribonucleotides containing ortho‐nitrophenylethyl (NPE) photocages, photoswitchable azobenzene C‐nucleotides and 13C,15N‐labelled nucleotides were incorporated position‐specifically in high yields. We applied this approach for the synthesis of light‐inducible 2′dG‐sensing riboswitch variants and detected ligand‐induced structural reorganization upon irradiation by NMR spectroscopy. This chemo‐enzymatic method opens the possibility to incorporate a wide range of modifications at any desired position of RNAs of any lengths beyond the limits of solid‐phase synthesis.
Keywords:chemo-enzymatic  light-control  NMR spectroscopy  photocaged RNA  RNA
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