Engineered allosteric ribozymes that respond to specific divalent metal ions

In vitro selection was used to isolate five classes of allosteric hammerhead ribozymes that are triggered by binding to certain divalent metal ion effectors. Each of these ribozyme classes are similarly activated by Mn2+, Fe2+, Co2+, Ni2+, Zn2+ and Cd2+, but their allosteric binding sites reject other divalent metals such as Mg2+, Ca2+ and Sr2+. Through a more comprehensive survey of cations, it was determined that some metal ions (Be2+, Fe3+, Al3+, Ru2+ and Dy2+) are extraordinarily disruptive to the RNA structure and function. Two classes of RNAs examined in greater detail make use of conserved nucleotides within the large internal bulges to form critical structures for allosteric function. One of these classes exhibits a metal-dependent increase in rate constant that indicates a requirement for the binding of two cation effectors. Additional findings suggest that, although complex allosteric functions can be exhibited by small RNAs, larger RNA molecules will probably be required to form binding pockets that are uniquely selective for individual cation effectors.