Combining protein and DNA engineering to create bioswitches

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Project Summary and Relevance The goal of this project is to develop mechanisms by which ordinary proteins can be turned into ligand- activated conformational switches. When naturally-occurring proteins of this type are discovered, their engineering can result in technologies that transform biology. For example, CRISPR-associated protein catalytic activity is switched on by binding of guide RNA, and calmodulin undergoes a large conformational change upon ligating calcium. Developing these proteins into DNA manipulation tools and fluorescent calcium sensors, respectively have revolutionized gene editing and the study of calcium signaling. The current proposal asks the question, “what else is possible if other proteins and enzymes can be made to switch on/off by binding of DNA, RNA, or other ligands?”. The proposed project takes a combined biophysical, computational, and cellular approach to develop a general mechanism for linking protein function to ligand binding. Three families of protein switches will be created. The first is a biosensor that plugs into existing DNA tools (such as aptamers and toehold-mediated strand displacement hairpins) without any modification to the sensor, to detect a DNA or RNA sequence of choice. The output is ratiometric (blue/green) luminescence that can be detected by cell phone camera. The second family employs fibronectin 3 ‘monobodies’ as the input domains and fluorescent proteins as the output domains to provide a ratiometric FRET response, or large increase in fluorescence intensity, when encountering an intracellular target. In the third switch design, the enzymatic activity of a bacterial RNase is turned on by cytomegalovirus (CMV) RNA to kill CMV-infected human cells. This last aim addresses the pressing need of preventing transplant-related CMV disease. Relevance. This study will open the biological activity of the human proteome to potential regulation by binding of nucleic acids, proteins, and small molecules. The modular design allows mixing and matching of different proteins to generate molecules with functionalities not found in nature. Examples include biosensors for pathogens and disease biomarkers, and an enzyme that kills virally-infected human cells while leaving uninfected cells unharmed.