A divalent cation-dependent variant of the ribozyme with stringent Ca selectivity co-opts a preexisting nonspecific metal ion-binding site.

Ribozymes use divalent cations for structural stabilization, as catalytic cofactors, or both. Because of the prominent role of Ca in intracellular signaling, engineered ribozymes with stringent Ca selectivity would be important in biotechnology. The wild-type ribozyme () requires glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. Previously, a ribozyme variant with three adenosine mutations () was identified, which dispenses with GlcN6P and instead uses, with little selectivity, divalent cations as cofactors for site-specific RNA cleavage. We now report a Ca-specific ribozyme () evolved from that is >10,000 times more active in Ca than Mg, is inactive in even 100 mM Mg, and is not responsive to GlcN6P. This stringent selectivity, reminiscent of the protein nuclease from , allows rapid and selective ribozyme inactivation using a Ca chelator such as EGTA. Because functions in physiologically relevant Ca concentrations, it can form the basis for intracellular sensors that couple Ca levels to RNA cleavage. Biochemical analysis of reveals that it has co-opted for selective Ca binding a nonspecific cation-binding site responsible for structural stabilization in and Fine-tuning of the selectivity of the cation site allows repurposing of this preexisting molecular feature.