In vitro selection, and sensing applications of allosteric ribozymes (aptazymes)
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RNA is ubiquitous in biology, and can interact with other biomolecules based on their primary, secondary, or tertiary structures. The nature of many such interactions has been elucidated via crystal and NMR structures. The nature of RNA’s more dynamic interactions with other biomolecules is less well studied. These interactions have recently become increasingly relevant with the discovery of riboswitches, RNAs whose structure is allosterically controlled by cellular metabolites. Likewise, catalytic RNAs can also be allosterically controlled. Aptazymes are ribozymes which have been designed or engineered to be allosterically controlled by the binding of an effector molecule. We have utilized in vitro selection to discover L1 ligase aptazymes which are highly activated in the presence of the arginine-rich Rev peptide. This aptazyme is activated to a large degree by the entire Rev protein, but also by the arginine-rich Tat peptide. Reselection from a pool centered around the original Rev-dependent sequence has yielded a number of variants. Reselected variants were almost universally more specific than the parental sequence, and selections for altered specificity proved able to do very little. Further, we have discovered that the kinetic profile of this ribozyme is different than previously selected protein-dependent aptazymes. A relatively slow activation step alters the RNA structure from one with an affinity which is on par with nonspecific binding to a tightly bound complex which takes days to dissociate. This transition occurs via a pathway called tertiary structure nucleation. In addition to providing a greater understanding of allosteric control, aptazymes have utilitarian value as laboratory tools for detecting and responding to given analytes. For example, allosteric ribozymes could be used as genetic control elements. Towards this end, we have created an allosteric ribozyme based on the SunY group I intron in vitro, and are attempting to adapt it to function in vivo. Further utility can be achieved by incorporating functional biomolecules into optical, electrical, or micromechanical devices to extend their sensing capabilities. I conclude by reporting the use of both aptamers and aptazymes as recognition elements for biosensing devices.