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  • Title: Design of Ribocomputing Devices for Complex Cellular Logic.
    Author: McCutcheon G, Chaudhary S, Hong S, Park D, Kim J, Green AA.
    Journal: Methods Mol Biol; 2022; 2518():65-86. PubMed ID: 35666439.
    Abstract:
    The ability to control cell function is a critical goal for synthetic biology and motivates the development of ever-improving methods for precise regulation of gene expression. RNA-based systems represent powerful tools for this purpose since they can take full advantage of the predictable and programmable base pairing properties of RNA to control gene expression. This chapter is focused on the computational design of RNA-only biological circuits that can execute complex Boolean logic expressions in living cells. These ribocomputing devices use toehold switches as building blocks for circuit construction, integrating sensing, computation, and signal generation functions within a gate RNA transcript that regulates expression of a gene of interest. The gate RNA in turn assesses the assembly state of networks of interacting input RNAs to execute AND, OR, and NOT operations with high dynamic range in E. coli. Harnessing in silico tools for device design facilitates scaling of the circuits to complex logic expressions, including four-input AND, six-input OR, and disjunctive normal form expressions with up to 12 inputs. This molecular architecture provides an intuitive and modular strategy for devising logic systems that can be readily engineered using RNA sequence design software and applied in vivo and in vitro. In this chapter, we describe the process for designing ribocomputing devices from the generation of orthogonal toehold switch libraries through to their use as building blocks for AND, OR, and NOT circuitry.
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