RATIONAL DESIGN OF ALLOSTERIC RIBOZYMES

Background: Efficient operation of cellular processes relies on the st rict control that each cell exerts over its metabolic pathways. Some p rotein enzymes are subject to allosteric regulation, in which binding sites located apart from the enzyme's active site can specifically rec ognize effector molecules and alter the catalytic rate of the enzyme v ia conformational changes. Although RNA also performs chemical reactio ns, no ribozymes are known to operate as true allosteric enzymes in bi ological systems. It has recently been established that small-molecule receptors can readily be made of RNA, as demonstrated by the in vitro selection of various RNA aptamers that can specifically bind correspo nding ligand molecules. We set out to examine whether the catalytic ac tivity of an existing ribozyme could be brought under the control of a n effector molecule by designing conjoined aptamer-ribozyme complexes. Results: By joining an ATP-binding RNA to a self-cleaving ribozyme, w e have created the first example of an allosteric ribozyme that has a catalytic rate that can be controlled by ATP. A 180-fold reduction in rate is observed upon addition of either adenosine or ATP, but no inhi bition is detected in the presence of dATP or other nucleoside triphos phates. Mutations in the aptamer domain that are expected to eliminate ATP binding or that increase the distance between aptamer and ribozym e domains result in a loss of ATP-specific allosteric control. Using a similar design approach, allosteric hammerhead ribozymes that are act ivated in the presence of ATP were created and another ribozyme that c an be controlled by theophylline was created. Conclusions: The catalyt ic features of these conjoined aptamer-ribozyme constructs demonstrate that catalytic RNAs can also be subject to allosteric regulation - a key feature of certain protein enzymes. Moreover, by using simple rati onal design strategies, it is now possible to engineer new catalytic p olynucleotides which have rates that can be tightly and specifically c ontrolled by small effector molecules.