Characterizing the Dynamics of the Leader–Linker Interaction in the Glycine Riboswitch with Site-Directed Spin Labeling

[Image: see text] Site-directed spin labeling with continuous wave electron paramagnetic resonance (EPR) spectroscopy was utilized to characterize dynamic features of the kink–turn motif formed through a leader–linker interaction in the Vibrio cholerae glycine riboswitch. Efficient incorporation of spin-labels into select sites within the phosphate backbone of the leader–linker region proceeded via splinted ligation of chemically synthesized spin-labeled oligonucleotides to in vitro transcribed larger RNA fragments. The resultant nitroxide EPR line shapes have spectral characteristics consistent with a kink–turn motif and reveal differential backbone dynamics that are modulated by the presence of magnesium, potassium, and glycine.