Engineering of small ribozymes acting on RNA: What is needed to make a new function work with an existing catalyst?

The engineering of nucleic acids has been a longstanding objective in research, with the field gaining significant attention following the discovery of ribozymes in the early 1980s. Numerous nucleic acid catalysts have been developed to catalyze a wide range of reactions, and the structures of ribozymes have been modified to allow allosteric regulation by an external cofactor. All these constructs hold considerable promise for applications in biosensors for medical and environmental diagnostics, as well as in molecular tools for regulating cellular processes. In addition to the development of nucleic acid enzymes through in vitro selection, rational design offers a robust strategy for engineering ribozymes with customized properties. The structures and mechanisms of numerous nucleic acid catalysts have been thoroughly elucidated, making structural modulation a viable approach for designing their functional properties. Rational design necessitates the consideration of several parameters, and a range of tools is available to guide sequence design. In this review, we discuss sequence, structural, and functional design, primarily using the example of the hairpin ribozyme, to highlight the challenges and opportunities of rational nucleic acid enzyme engineering.