Because the ability to effectively detect and respond to subtle chemical cues is so crucial to biological function, evolution has invented many diverse, intricate mechanisms for the robust, precise sensing of molecular stimuli. The ability to systematically recreate such mechanisms in artificial systems would likewise be useful in many biotechnologies, for example in biosensors, synthetic biology, and targeted drug delivery. In response, the work I present here focuses on the rational, quantitative recreation of biological mechanisms of sensing and actuation in artificial biomolecular systems.

The first aim of my thesis work, detailed in Chapters 2 and 3, centered on the rational engineering of allosteric cooperativity into normally non-cooperative artificial receptors. This mechanism, which occurs when multiple copies of identical target molecule bind to a receptor in an "all-or-nothing" fashion, increases the order of the binding curve, narrowing the transition window between bound and unbound, and enhancing the receptors' sensitivity to small changes in target concentration. To achieve this effect requires that the first copy of target molecule to bind shift the receptor from a low-affinity to a high-affinity conformation, thus increasing its affinity for the binding of subsequent copies of target molecule. Chapter 2 centers on proof-of-concept efforts to engineering this mechanism into a particularly simple and well-understood model receptor, a DNA-binding molecular beacon. I follow this in Chapter 3 with the development of a disorder-based strategy suitable for the introduction of cooperativity into more complex receptors, including even those of unknown structure.

The second aim of my thesis work, detailed in Chapters 4 and 5, focused on the engineering of multicomponent, stimulus-responsive biomolecular systems from a different perspective, specifically on the development of a quantitative understanding of the physics of stimulus-responsive materials. Materials that assemble and dissolve in response to chemical stimuli are ubiquitous in Biology, for example in transport vesicles, viruses, and cell membranes. As with the imitation of allosteric cooperativity, the ability to rationally imitate the properties of these materials in an artificial, technological context would be useful in many applications. Although there exist many successful examples of such artificial, stimulus-responsive materials, design efforts thus far have been fairly qualitative, and systematic approaches to systematically control their properties do not exist. Part of the reason for this is that the relationship between properties such as network architecture and cooperativity, thermodynamic stability and molecular and micron scale behavior are not well understood, and there is a lack of simple, quantitative techniques for measuring the response of these materials. In response, I developed simple, straightforward techniques to measure the dissolution of a model hydrogel simultaneously at both the molecular and micron length scales, which I describe in Chapter 4. In Chapter 5, I employed these methods to explore the relationship between the thermodynamic stability and response kinetics of a model hydrogel, demonstrating the ability to quantitatively control the materials response kinetics using simple strategies previously applied for the quantitative tuning of solution-phase biomolecular switches.