Tools4Cells: Real-Time Measurement of Endogenous Biomolecules

About This Grant

A key measurement in modern biology research is measuring changes in the levels of specific proteins in live cells. However, many of the methods by which proteins are detected, such as Western blots, are hopelessly antiquated and provide only a small snapshot of information. This project envisions a new method for biologists to measure changes in live cells. Specifically, the method is designed to measure the rates of changes, the duration of the effects, and the return to a normal state, all in one experiment. These measurements are enabled by a new type of “turn-on biosensor” which can monitor protein levels in real time. Prototypes for this new type of biosensor will be developed using computational design, machine learning, and directed evolution. The initial application will be monitoring levels of beta-catenin, a master regulator of cell growth and specialization. Beta-catenin levels are known to change in response to chemical signals released by nearby cells, making it an ideal system for exploring the design and applications of the new turn-on biosensors. The new biosensors described in this Tools4Cells project not only provide much richer biological information than the current state-of-the-art, they will have broader impact on the field as simpler, more quantitative, less expensive, and more sustainable. Fluorescent biosensors have been pursued for decades, but nearly all current strategies rely on split binding domains or domains with large conformational changes upon analyte binding. However, there are very few such domains, limiting applications to relatively few analytes. Also, current biosensors require extended washout steps and other manipulations, and their sensitivity is severely limited by high background from the “always-on” fluorescent proteins and dyes. This project develops turn-on biosensors (TOBs) which fluoresce only when the analyte is bound. TOBs use a modular binding protein to recognize the analyte, which ensures generalizability. For initial applications, the binding protein will be a nanobody to allow selective recognition of a specific target protein. The nanobody is fused to a variant of the self-labeling protein HaloTag, which allows covalent installation of a synthetic, environment-sensitive dye. TOBs will be engineered using computational methods and directed evolution to minimize dye fluorescence in the absence of analyte and to maximize fluorescence in the presence of analyte. TOBs that report levels of the transcription factor beta-catenin will be developed and tested in cell culture models of beta-catenin activation and inhibition. These critical pathways are known to be mediated by changes in the cellular levels of beta-catenin, but this method will demonstrate real-time measurements of these changes. Once developed, TOBs will be readily adaptable and useful to a large proportion of the cell biology community. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.