Creating Libraries of Orthogonal RNA Toehold Repressors

RNA regulators of gene expression are becoming increasingly important components of the synthetic biology toolbox. RNA is a versatile and designable molecule, and is one of the most powerful substrates at our disposal for engineering gene expression. The ability to propagate information directly via RNA-RNA interactions creates the basis for the bottom-up design of genetic networks to implement logic or carry out complex patterns of gene expression. An important class of such regulators is RNA-mediated repressors, which repress downstream gene expression in response to a trans-acting antisense RNA. Rational mutagenesis and sequence mining have been successful in creating libraries of orthogonal (independently-acting) RNA repressors that function on the transcriptional and translational levels. However, the construction of large networks will require additional regulators with enhanced dynamic range and reduced crosstalk. Inspired by work demonstrating the successful de novo design of RNA-mediated translational activators called toehold switches (Cell, 159, 925-939, 2014), we sought to use a similar approach to create de novo designed RNA translational repressors. In this setup, the default structural state frees the ribosome binding site (RBS) and start codon, allowing translation to proceed in an ON state. Binding of a designed trans-acting antisense RNA leads to structural rearrangement and occlusion of the RBS and start codon, preventing translation in the OFF state. Using two different approaches, we successfully designed toehold repressors with the Nucleic Acid Package (NUPACK) capable of attenuating gene expression up to 98% (50-fold repression). We further demonstrate the ability to construct libraries of orthogonal repressors with potential applications ranging from nucleic acid-based diagnostics to metabolic engineering.