Title: De novo designed Toehold Repressors

Paul Carlson, Julius Lucks

Abstract: 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 for engineering gene expression at our disposal. Moreover, 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 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 recent work demonstrating the successful forward design of RNA-mediated translational activators called toehold switches, we sought to use a similar approach to create de novo designed RNA translational repressors. Using the Nucleic Acid Package (NUPACK), we successfully inverted the structural logic of toehold activators to create toehold repressors capable of attenuating gene expression up to 98% (50-fold repression). In this design, the structure of the toehold repressor frees the ribosome binding site (RBS) and start codon, allowing translation to proceed in a default ON state. Binding of a designed trans-acting antisense RNA leads to structural rearrangement and formation of a hairpin, which occludes the RBS and start codon, preventing translation in the OFF state. This approach shows promise for creating a large library of independent regulators with large dynamic range.