(5h) How to Make An Information Processing Device out of RNA

RNA is a rich and versatile building block for the construction of information processing devices. These devices detect the levels of specified intracellular biomolecules and control cellular behavior accordingly. With few constraints on the identity of the recognized biomolecule or the targeted gene, RNA-based information processing devices can be rapidly implemented toward various applications in medicine and biotechnology. However, current examples serve as single instances of successful design that do not immediately lend to the construction of novel devices. My thesis work has focused on delineating design principles for the construction of information processing devices out of RNA and applying these principles to the design of devices that operate through RNA interference (RNAi).

Ligand-responsive RNAi substrates RNAi is an endogenous pathway present in humans and other eukaryotes that mediates targeted gene silencing and has garnered recent interest as a revolutionary biological research tool and a powerful therapeutic strategy. While RNAi has left an indelible mark on the scientific community, exerting greater control by engineering ligand responsiveness would advance the applicability of this already impressive pathway. Toward this goal, we developed functional composition frameworks for two distinct types of RNAi substrates, small hairpin RNAs and microRNAs. Both of our frameworks enabled facile replacement of the biomolecule sensory and gene targeting domains, thus mediating rapid implementation as biosensors or autonomous control devices. Experimental and computational characterization studies provided a comprehensive understanding of device behavior, thereby facilitating forward design.

Design principles for riboswitch design Riboswitches are genetic regulatory elements that serve as a dominant class of RNA-based information processing devices. Various experimental characterization studies of riboswitches have shown that kinetic factors such as ligand binding and RNA folding have a tremendous impact on device performance, although these factors have not been comprehensively evaluated or considered when formulating design principles for device construction. We explored the contribution of kinetics factors to riboswitch performance in silico, where model predictions match experimental observations, including results from our ligand-responsive RNAi substrates. From our modeling results, we developed a universal set of design principles that guide riboswitch assembly and performance tuning. Current work is focused on applying these principles to the redesign of existing riboswitches for improved performance.

Harnessing the many faces of RNA Beyond my thesis work, I am interested in understanding how nature utilizes RNA and translating this knowledge into the improved design of biological systems. Nature has devised ingenious ways to interface RNA with natural regulatory architectures as exemplified by bacterial small RNAs, riboswitches, alternatively spliced RNAs, and microRNAs. These RNAs are involved in many fundamental cellular processes including stochastic noise dampening, metabolism, stress response, development, and carcinogenesis, although characterization of these RNAs is still ongoing. I plan to investigate these processes from a systems design perspective and directly apply these insights to the fields of synthetic biology, metabolic engineering, and disease therapeutics.