Recently, Breaker and coworkers engineered hammerhead ribozymes
that rearrange from a catalytically inactive to an active
conformation upon allosteric binding of a specific ligand. To
monitor cleavage activity in real time, we have coupled a
donor–acceptor fluorophore pair to the termini of the
substrate RNA of such a hammerhead ribozyme, modified to cleave
in trans in the presence of the bronchodilator
theophylline. In the intact substrate, the fluorophores interact
by fluorescence resonance energy transfer (FRET). The specific
FRET signal breaks down as the effector ligand binds, the substrate
is cleaved, and the products dissociate, with a rate constant
dependent on the concentration of the ligand. Our biosensor
cleaves substrate at 0.46 min−1 in 1 mM
theophylline and 0.04 min−1 without effector,
and discriminates against caffeine, a structural relative of
theophylline. We have measured the theophylline-dependence profile
of this biosensor, showing that concentrations as low as 1 μM
can be distinguished from background. To probe the mechanism
of allosteric regulation, a single nucleotide in the communication
domain between the catalytic and ligand-binding domains was
mutated to destabilize the inactive conformation of the ribozyme.
As predicted, this mutant shows the same activity (0.3
min−1) in the presence and absence of
theophylline. Additionally, time-resolved FRET measurements
on the biosensor ribozyme in complex with a noncleavable substrate
analog reveal no significant changes in fluorophore distance
distribution upon binding of effector.