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Engineering translation machinery to produce light-responsive protein-polymers

Periodic Reporting for period 3 - PolControl (Engineering translation machinery to produce light-responsive protein-polymers)

Reporting period: 2020-11-01 to 2022-04-30

Biological systems are extraordinarily complex yet organized on both the spatial and temporal levels, and if we are to study, perturb, engineer, or heal biological entities, we must be able to precisely and selectively manipulate key players in such systems. Thus, a central challenge in the basic and engineering sciences is the design of environmentally responsive agents that respond to our external cues on-demand, to allow us to control, interrogate, and manipulate biological systems. Among the potentially exploitable external stimuli, light is a particularly desirable actuator, as it enables high resolution in space and in time with minimal disturbance to biological systems.

Some of the key players in biological systems are called proteins. Proteins are produced from 20 natural elements called amino acids. These 20 building blocks have enabled much of the diversity and function of proteins and has allowed them to perform a multitude of biological roles. To manipulate such proteins and understand their function, we seek to expand biology beyond the standard set of 20 amino acids by encoding light-responsive synthetic amino acids. However, the addition of new chemical groups in proteins by incorporation of synthetic amino acids remains a laborious task that often fails due to inadequate engineering of the protein-making machinery. Thus, working within the confines of current technologies both hinders efficient incorporation of light-responsive synthetic amino acids, as well as the types of light-responsive groups that can be incorporated.

The overall objective of this study is to develop a technology for efficient incorporation of light-responsive synthetic amino acids and to apply this technology to engineer customized light-responsive formulations for applications in drug delivery, for cell and tissue engineering, and for analyzing and manipulating biological processes. To achieve this we are developing genome engineering tools to generate a platform for the selection of protein-making machinery that are capable of efficiently incorporating a variety of light-responsive chemical groups into proteins. These tools will allow us, and many other laboratories to design light-responsive proteins, and to elucidate their function and manipulate their activity in a various biological systems.
We developed a strategy for the selection of highly efficient protein-making components for the incorporation of a variety of synthetic amino acids, among them light-responsive synthetic amino acids. Using this strategy, we were able to demonstrate proof of concept for imparting light-responsive behavior by producing “smart” protein-based materials that change their solubility upon the application of light as an external stimulus. We are now expanding the number and type of light-sensitive synthetic amino acids that can be incorporated into proteins, utilizing the above-mentioned light-responsive materials to control intracellular aggregation of proteins, and designing additional light-responsive proteins and polymers.
We have developed new methodologies for the selection of efficient machinery for synthetic amino acid incorporation. Using this tool, we have identified several components that are able to incorporate a variety of light-responsive synthetic amino acids in multiple places inside a single protein. This previously unattainable capability has allowed us to generate light-responsive protein-based biomaterials. We expect to continue to select additional machinery for other light-responsive synthetic amino acids and to design additional families of light-responsive proteins and protein-based materials and apply them to control extra- and intra-cellular biological systems.