Computational design and experimental validation of fast oligonucleotide-sensing allosteric ribozymes with predefined...

Ribozymes can be engineered to function as molecular switches that exhibit different Boolean logic functions. Here, we describe two novel algorithmic pipelines for the design of YES and NOT Boolean-function oligonucleotide-sensing allosteric ribozymes bearing a predefined oligonucleotide-binding site sequence, capable of targeting known sequence RNA molecules. The algorithms generate allosteric riboswitches based on the extended hammerhead ribozyme by computing RNA thermodynamic properties and using random search. A computationally designed YES Boolean logic allosteric ribozyme was synthesized and tested in vitro. The tested ribozyme exhibits at least 1000-fold activation, demonstrating high ligand specificity and function. These algorithmic approaches provide a rapid and inexpensive way to create allosteric ribozymes targeting a known RNA sequence for constructing gene control elements and nucleic acid detection systems.