DOI: 10.1101/501783Dec 19, 2018Paper

De-Novo-Designed Translational Repressors for Multi-Input Cellular Logic

BioRxiv : the Preprint Server for Biology
Jongmin KimAlexander A Green

Abstract

Synthetic biology aims to apply engineering principles toward the development of novel biological systems for biotechnology and medicine. Despite efforts to expand the set of high-performing parts for genetic circuits, achieving more complex circuit functions has often been limited by the idiosyncratic nature and crosstalk of commonly utilized parts. Here, we present a molecular programming strategy that implements RNA-based repression of translation using de-novo-designed RNAs to realize high-performance orthogonal parts with mRNA detection and multi-input logic capabilities. These synthetic post-transcriptional regulators, termed toehold repressors and three-way junction (3WJ) repressors, efficiently suppress translation in response to cognate trigger RNAs with nearly arbitrary sequences using thermodynamically and kinetically favorable linear-linear RNA interactions. Automated in silico optimization of thermodynamic parameters yields improved toehold repressors with up to 300-fold repression, while in-cell SHAPE-Seq measurements of 3WJ repressors confirm their designed switching mechanism in living cells. Leveraging the absence of sequence constraints, we identify eight- and 15-component sets of toehold and 3WJ repressors, r...Continue Reading

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