(152c) Engineering Ligand-Regulated Rnai Substrates as Novel Tools for Probing and Programming Cellular Systems

Information flow through cellular networks is responsible for regulating cellular function at both the single cell and multi-cellular systems levels. The ability to temporally and spatially monitor and regulate this information is critical to the understanding and programming of complex cellular behavior. Despite significant advances in the elucidation of the regulatory and metabolic networks that control cellular function, relatively little is understood about the dynamic fluctuations in protein expression, post-translational modifications, or metabolite concentrations associated with these networks. One of the key limitations to progress in this area is the lack of enabling technologies that allow for user-specified probing and programming of these cellular events.

RNA interference (RNAi) has emerged as a powerful tool for the sequence-specific control of gene expression. The associated RNAi pathway is present in practically all higher eukaryotes and is an essential component of cellular signaling pathways that pertain to development, cell cycle, and the control of carcinogenesis to name a few. RNAi substrates have been widely utilized as a research tool in the study of gene function and are being explored as molecular therapeutics. Many applications will require precise temporal and spatial regulation of RNAi in response to a variety of molecular inputs. Inducible promoter systems have been employed to achieve one level of regulation; however, such systems are limited to the natural inducer molecules or their derivatives as molecular inputs. In order to overcome this limitation, we are using recent advances in the mechanistic understanding of the RNAi pathway and nucleic acid design to generate new molecules for probing and programming cellular behavior. Based on the mechanisms of nucleic acid conformational dynamics that have been elucidated from natural and synthetic riboswitches and ligand-controlled riboregulators, we have engineered platforms for the construction of RNAi substrates that display regulated activity through the integration of modular RNA aptamer domains. Binding of a target ligand to the aptamer domain stabilizes an energetically unfavorable conformation that either is or is not processed by the RNAi machinery, depending on the platform design. The versatility and relative simplicity of nucleic acid design allows tuning of the response parameters of these molecular sensors that quantitatively link ligand concentration to RNAi-mediated knockdown. The relative ease with which aptamers can be selected to novel molecular inputs enables these platforms to function as tailor-made cellular sensors. In addition, these engineered substrates may be incorporated into synthetic genetic circuits. The construction of synthetic circuits that enable complex cellular behavior in both integrating multiple input/output signals and expanding response properties of cellular systems will be discussed. These circuits will be applied to both enhancing our understanding of natural genetic circuits and developing a new generation of 'intelligent' therapeutic molecules.