Strategies and technologies for engineering gene-regulatory RNA devices [electronic resource]

Engineered biological systems will provide solutions to diverse global challenges, enabling new and enhanced products for application in chemical processing, materials synthesis, sustainable technologies, and human health. Ability to manipulate and probe biological systems is limited by our ability to noninvasively access, process, report on, and respond to information encoded in the properties of molecules in living systems. Developing genetically encoded information processing and control technologies is critical to addressing and overcoming fundamental challenges in basic and applied biomedical research. RNA poses a strong candidate for a substrate in which to build genetic control devices. Examples of functional RNA molecules playing key roles in controlling the behavior of natural biological systems have grown over the past decade. The relative ease in modeling RNA molecules has enabled design of synthetic counterparts, act with diverse function as components including sensors, regulators, controllers (ligand-responsive RNA regulators), and scaffolds. These synthetic regulatory RNAs are providing new tools for temporal and spatial control in biological systems. A modular platform was described for the construction of RNA devices composed of distinct domains that encode sensing, transmission and actuation functions. The sensor domain is composed of an RNA aptamer, a nucleic acid structure evolved in vitro to bind with high affinity to a given ligand. The actuator domain is composed of a hammerhead ribozyme, which self-cleaves at a specific sequence under proper secondary and tertiary folding. The transmitter domain couples the sensor and actuator domains and communicates the ligand-bound conformational state of the sensor domain to the actuator domain by affecting the folded state of the actuator into either its ribozyme-active or -inactive conformation. The devices are placed in the 3' untranslated region of a target transcript, where self-cleavage inactivates the transcript, thereby lowering gene expression. This framework has been extended to the assembly of devices exhibiting higher-order information processing operations, including logic gates, signal filters and programmed cooperativity. As a demonstration of the broader significance of this class of devices, they have been successfully implemented as biological control systems to regulate signaling pathways and clinically-relevant phenotypes. However, it is critical to develop an improved understanding of the underlying molecular mechanisms and parameters guiding the activities of these devices in vivo in order to develop improved design strategies and associated regulatory activity to extend the utility of these genetic devices for a broader range of applications. To advance the RNA device design, we have developed and implemented novel methods to measure important parameters. We characterized the causal relationship between the in vitro device cleavage rate constant device parameter and in vivo gene- regulatory activity, and utilized this for efficient device performance characterization and design. We described a have novel, two-color, in vivo fluorescent activated cell sorting-based approach to identify sequences that yielded improved catalytic activities within the device platform and enabled efficient tailoring of device regulatory activities. We further developed a quantitative assay based on surface plasmon resonance technology for rapid measurements of device cleavage and ligand binding, the important parameters governing the underlying device mechanism. We incorporate this assay into the RNA device design cycle, pre-filtering candidate devices by in vitro cleavage and binding activity for subsequent in vivo testing. By this method we efficiently developed of new protein-responsive RNA devices in both yeast and mammalian hosts. Finally, we devised a platform utilizing next generation sequencing and fluorescent activated cell sorting for simultaneous measurements of in vitro cleavage and in vivo gene-regulatory activities of large RNA device libraries. Enabled by the large screening capacity, we develop a new RNA device architecture, with modularity instilled at the design level. By this approach we generate RNA devices without a programmed conformational change that results in improved device gene- regulatory performance. Taken together, these new technologies for characterizing important device performance characters and resultant gene-regulatory activity provide a comprehensive framework for designing, testing and implementing RNA genetic controllers for engineering biological systems.

Keywords: Bioengineering.
Advisor: Smolke Christina D. primary advisor. Thesis advisor
Das Rhiju
Swartz James R. advisor. Thesis advisor