Systems biology relies on perturbations to endogenous genetic networks in order to study the effects of network topology and dynamics on the system response over short and long time scales. These perturbations often involve mutation or deletion of designated nodes (genes), and subsequent characterization of system response is often performed under different environmental conditions. An alternative approach is to introduce new connectivities that would provide unique insights into global network behavior and a means to expand upon the network's existing regulatory capabilities. Additional benefits would be provided if the dynamics of the synthetic connections can be specified and tightly controlled. Research within synthetic biology has produced an assortment of complex regulatory networks that have been integrated into endogenous networks. These synthetic networks function almost entirely at the transcriptional level, which restricts how easily they can be implemented as linkages between any given nodes within an endogenous network. Post-transcriptional control can overcome this restriction, especially with the discovery of effective gene silencing mechanisms such as RNA interference (RNAi). The RNAi pathway has been used to orchestrate the sequence-specific degradation of target transcripts, thereby regulating expression levels of the target genes. Practically any gene can be targeted through this mechanism, suggesting that RNAi may serve as a foundation for introducing network linkages between any given nodes.

To this end we have engineered shRNAs (activators of RNAi) so that their activity is regulated by a specified ligand. The resulting molecules, termed shRNA switches, are comprised of an shRNA domain, an aptamer domain, and a linker domain. shRNA switches can adopt two conformations that differ in their ability to be processed by the RNAi machinery. The active conformation includes a formed shRNA domain and a disrupted aptamer domain, whereas the inactive conformation includes a disrupted shRNA domain and a formed aptamer domain. Ligand binding to the aptamer domain stabilizes the inactive conformation, thereby modulating the relative expression level of the target gene. Ligand responsiveness is mediated by the aptamer domain, which is distinct from the shRNA domain and modular; namely, it can be replaced with different aptameric sequences to enable gene regulation through potentially any biomolecule within the cell. Based on the seemingly limitless number of endogenous targets afforded by aptamers and RNAi, shRNA switches have the potential to serve as linkages between any nodes in an endogenous network.

We will discuss our work investigating the mechanism behind ligand regulation of shRNA switch activity and the potential that these molecules hold as ligand-responsive gene regulatory systems as demonstrated within a model system of mammalian cells. Through our work, we have delineated a set of rules that govern the behavior of each shRNA switch and provide a significant link between sequence and function. A simple mathematical model based upon the described mechanism was also developed that accurately represents experimental data and can serve as a predictive tool. We will show that a combination of rational design and mathematical modeling guides the forward engineering of new shRNA switches in a variety of applications, including intelligent therapeutics and the study of large-scale genetic networks.

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