Directed evolution of RNA and protein-based molecular switches

Biological systems use ligand-dependent proteins and nucleic acids as molecular switches to transduce signals into appropriate cellular responses. Artificial molecular switches are of particular interest because they provide control of biological function with small molecules chosen by the researcher. We created two ligand-dependent switches using directed evolution techniques. From random RNA libraries expressed in yeast, we evolved RNA-based transcriptional activation domains that are comparable in potency to the strongest natural activation domains. Site-directed mutagenesis, sequence alignment, and computational secondary structural prediction elucidated the functional elements of these evolved RNAs.; We fused a small molecule-binding RNA aptamer to the evolved RNA-based transcriptional activation domain and designed a conformational shift in which a helical element required for function was stabilized upon ligand binding. Selection and screening in S. cerevisiae optimized a linker region, generating an RNA that is ten-fold more active in the presence of the small-molecule ligand. The method of generating aptamers to a protein target and appending a small molecule-dependent aptamer may serve as a general approach to creating ligand-dependent regulators of biological function.; In a second series of experiments, we evolved a protein-based molecular switch that regulates protein function at the post-translational level. Insertion of self-splicing protein elements (inteins) into a protein blocks the target protein's function until splicing occurs. We developed selections and screens in E. coli and S. cerevisiae that link intein splicing to cellular survival or fluorescence. Random cassette mutagenesis of the sequence around aromatic to alanine mutations failed to create ligand-dependent intein mutants in 108-member libraries. We therefore inserted a natural ligand-binding domain (from the estrogen receptor) into an intein sequence to confer small molecule-binding activity. Simple insertion destroyed protein-splicing activity. Several rounds of selection and screening identified intein-ER mutants with increased activity and dependence on 4-hydroxytamoxifen. Insertion of an evolved intein into four proteins in living cells revealed that ligand-dependent activation is general, rapid, dose-dependent, and post-translational. These small molecule-dependent inteins may therefore serve as a general tool for controlling the activity of an arbitrary protein of interest in vivo without requiring the discovery of specific small-molecule inhibitors for every protein target.