The breaking of RNA strands by 2'-O-transphosphorylation is a ubiquitous reaction in biology, and enzymes that catalyze this reaction play key roles in RNA metabolism. The mechanisms of 2'-O-transphosphorylation in solution are relatively well studied, but complex and can involve different transition states depending on how the reaction is catalyzed. Because of this complexity and the lack of experimental information on transition-state structure, pinning down the chemical details of enzyme-catalyzed RNA strand cleavage has been difficult. Kinetic isotope effects (KIEs) provide information about changes in bonding as a reaction proceeds from ground state to transition state, and therefore they provide a powerful tool for revealing mechanistic detail. Application of kinetic isotope analyses to RNA 2'-O-transphosphorylation faces three fundamental challenges: synthesis of RNA substrate isotopomers with