Chemo-Enzymatic Synthesis of Position-Specifically Modified RNA for Biophysical Studies including Light Control and...

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 ¹³ C,¹⁵ N-labelled nucleosides. All four ribonucleotides containing ortho-nitrophenylethyl (NPE) photocages, photoswitchable azobenzene C-nucleotides and ¹³ C,¹⁵ N-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.