Synthesis of spin-labeled riboswitch RNAs using convertible nucleosides and DNA-catalyzed RNA ligation.

Chemically stable nitroxide radicals that can be monitored by electron paramagnetic resonance (EPR) spectroscopy can provide information on structural and dynamic properties of functional RNA such as riboswitches. The convertible nucleoside approach is used to install 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and 2,2,5,5-tetramethylpyrrolidin-1-oxyl (proxyl) labels at the exocyclic N(4)-amino group of cytidine and 2'-O-methylcytidine nucleotides in RNA. To obtain site-specifically labeled long riboswitch RNAs beyond the limit of solid-phase synthesis, we report the ligation of spin-labeled RNA using an in vitro selected deoxyribozyme as catalyst, and demonstrate the synthesis of TEMPO-labeled 53 nt SAM-III and 118 nt SAM-I riboswitch domains (SAM=S-adenosylmethionine).