Site-Specific Labeling of RNAs with Modified and ¹⁹ F-Labeled Nucleotides by Chemo-Enzymatic Synthesis

More than 170 post-transcriptional modifications of RNAs have currently been identified. Detailed biophysical investigations of these modifications have been limited since large RNAs containing these post-transcriptional modifications are difficult to produce. Further, adequate readout of spectroscopic fingerprints are important, necessitating additional labeling procedures beyond the naturally occurring RNA modifications. Here, we report the chemo-enzymatic synthesis of RNA modifications and several structurally similar fluorine-modified analogs further optimizing a recently developed methodology.^[1] This chemo-enzymatic method allows synthesis of also large RNAs. We were able to incorporate 16 modified nucleotides and 6 ¹⁹ F-labeled nucleotides. To showcase the applicability of such modified large RNAs, we incorporated a ¹⁹ F-labeled cytidine into the aptamer domain of the 2'dG sensing riboswitch (2'dG-sw) from Mesoplasma florum, enabling characterizing RNA fold, ligand binding and kinetics. Thanks to the large chemical shift dispersion of ¹⁹ F, we can detect conformational heterogeneity in the apo state of the riboswitch.