Allosteric ribozymes are engineered RNAs that operate
as molecular switches whose rates of catalytic activity
are modulated by the binding of specific effector molecules.
New RNA molecular switches can be created by using “allosteric
selection,” a molecular engineering process that
combines modular rational design and in vitro evolution
strategies. In this report, we describe the characterization
of 3′,5′-cyclic nucleotide monophosphate (cNMP)-dependent
hammerhead ribozymes that were created using allosteric
selection (Koizumi et al., Nat Struct Biol, 1999,
6:1062–1071). Artificial phylogeny data
generated by random mutagenesis and reselection of existing
cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that
each is comprised of distinct effector-binding and catalytic
domains. In addition, patterns of nucleotide covariation
and direct mutational analysis both support distinct secondary-structure
organizations for the effector-binding domains. Guided
by these structural models, we were able to disintegrate
each allosteric ribozyme into separate ligand-binding and
catalytic modules. Examinations of the independent effector-binding
domains reveal that each retains its corresponding cNMP-binding
function. These results validate the use of allosteric
selection and modular engineering as a means of simultaneously
generating new nucleic acid structures that selectively
bind ligands. Furthermore, we demonstrate that the binding
affinity of an allosteric ribozyme can be improved through
random mutagenesis and allosteric selection under conditions
that favor tighter binding. This “affinity maturation”
effect is expected to be a valuable attribute of allosteric
selection as future endeavors seek to apply engineered
allosteric ribozymes as biosensor components and as controllable
genetic switches.