ABSTRACT The advent of protein design in recent years has brought us within reach of developing a “nanoscale programing language,” in which molecules serve as operands with their conformational states functioning as logical gates. Combining these operands into larger molecules and molecular complexes through protein engineering will allow us to write and execute “code” using nanoscale computing agents (NCAs). NCAs offer an orthogonal and complementary means for controlling cellular phenotypes. In the past 12 years, our group has developed technology toward this end, by engineering proteins that can be controlled by light and small molecules. We designed functional prototypes that have already offered valuable insights in the cellular motility field. The focus of the parent R35 is to develop NCAs for the targeted regulation of cellular activity. The main goals of this supplementary proposal are: (1) Extend the repertoire of inputs for regulation of proteins. We plan to utilize/design proteins that respond to pH and temperature via conformational change in order to modulate the activities of target proteins. (2) Build a multi-input NCA prototype. In order to facilitate the creation of higher order allosterically regulated protein systems and to conduct complex computations in cells, we have been working on building novel protein regulatory tools responding to diverse molecular cues, such as temperature and pH. Using such regulatory tools, we want to build a multi-input NCA prototype. In this direction, currently, we have successfully designed two-input protein systems in two candidate proteins using chemogenetic and optogenetic tools. Using the designed two-input systems we have controlled specific cellular phenotypes. Using various regulatory tools we plan to build a multi-input NCA prototype. Addressing these challenges will provide a significant leap in technology for programming living cells. One of the important aspects in our study is to test the NCAs using in vitro techniques. Here, we propose to test the NCAs in vitro using MicroScale Thermophoresis. We request supplemental funding for MicroScale Thermophoresis equipment to perform in vitro interaction analysis. The addition of MicroScale Thermophoresis equipment is vital to our ability to design new input sensors and validate the designed NCAs.