Utilizing nucleic strand displacement reactions for sensing and computation in living cells

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The overall goal of the project is the utilization of so-called “strand displacement processes” (i.e., dynamic replacement of RNA strands in an RNA structure) to perform simple computations in living cells, and especially mammalian cells. This will allow the readout of the cellular state via the presence of specific RNA molecules and will enable responding to it by changing transcription levels or even by modification of the genome of the cell. Our project will build upon our previous work on strand-displacing guide RNAs (SD gRNAs) for the CRISPR associated nuclease Cas12a, which were already shown to work in vitro and inside bacterial cells. Building up more powerful RNA “circuits” in the complex environment of living cells poses considerable challenges, some of which we aim to address in this project.We first aim at establishing design rules for efficient strand displacement circuits in mammalian cells. In order to perform such processes in mammalian cells efficiently, we will explore various RNA design parameters that are expected to influence hybridization and strand displacement kinetics. We further aim at the development of a set of orthogonal components, which facilitate multi-input circuits, and finally use the design rules to generate cascaded circuits. We also aim at understanding additional influences on the kinetics of strand displacement processes that might play a role in the complex environment of a cell. To this end we will experimentally model several aspects of the cellular environment in a cell-free context. In particular, we will study the influence of large pools of competing DNA or RNA species on strand displacement kinetics, but also other aspects such as macromolecular crowding. We then aim to extend existing modeling frameworks for nucleic acid-based reaction circuits to account for these effects and potentially predict the kinetics of strand displacement processes in vivo. Finally, we wish to apply our insights to use natural RNA as input for strand displacement circuits in mammalian cells. In order to utilize cellular RNA species, we will develop strategies that allow efficient sensing of long RNA molecules with their specific sequence constraints, which will include an evaluation of sequence domains available for hybridization and also competition with RNA-binding proteins. Further, we will study the impact of intracellular localization and concentration of the RNA molecules on the functionality of our circuits.