Ribozyme Redesign From an Existing RNA Scaffold Enables Multifunctional Hydrolysis for Molecular Release

ABSTRACT The discovery of novel ribozymes is crucial for both advancing RNA technologies and illuminating potential RNA world scenarios. Currently, catalytic evolution of ribozymes primarily relies on the slow, random process of natural or artificial selection. In this study, we show that a distinct catalytic feature can be deliberately imposed onto a preexisting ribozyme scaffold through ribozyme redesign with a minimal alteration rather than molecular evolution. We demonstrate a single‐nucleotide mutation repurposes an alkyl‐transfer ribozyme into a multifunctional hydrolysis ribozyme, while preserving its overall catalytic scaffold. This hydrolase ribozyme operates analogously to a canonical protein enzyme, transforming various ester and ether substrates into the release of carboxylic acids, alcohols, and amines. Structural and kinetic analyses uncover a water‐mediated mechanism for ester/ether bond cleavage. Application of this hydrolase ribozyme in living cells enables controlled activation and release of diverse payloads on demand, such as fluorescent and drug molecules, for cell‐specific discrimination. This study highlights the previously underappreciated multifunctionalities within RNA catalytic architectures, broadening the scope of developing versatile ribozymes for diversified applications.