Pulsed electron double resonance (PELDOR) spectroscopy reveals a prearranged tertiary structure of the 27 nucleotides long engineered neomycin-responsive riboswitch. Measured distances between spin labels at positions U4-U14, U4-U15. U14-U26, and U15-U26 were unchanged upon neomycin binding which implies that the global stern-loop architecture is preserved in the absence and presence of the ligand. On the basis of our results. we infer that low-temperature PELDOR data Unambiguously demonstrate the existence of an enthalpically favorable set of RNA conformations ready to bind the ligand without major global rearrangement.