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

Abstract

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 19F-labeled nucleotides. To showcase the applicability of such modified large RNAs, we incorporated a 19F-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 19F, we can detect conformational heterogeneity in the apo state of the riboswitch.