Automated design of highly diverse riboswitches

Abstract

Riboswitches that couple binding of ligands to recruitment of molecular machines offer sensors and control elements for RNA synthetic biology and medical biotechnology. Current approaches to riboswitch design enable significant changes in output activity in the presence vs. absence of input ligands. However, design of these riboswitches has so far required expert intuition and explicit specification of complete target secondary structures, both of which limit the structure-toggling mechanisms that have been explored. We present a fully automated method called RiboLogic for these design tasks and high-throughput experimental tests of 2,875 molecules using RNA-MaP (RNA on a massively parallel array) technology. RiboLogic designs explore an unprecedented diversity of structure-toggling mechanisms validated through experimental tests. These synthetic molecules consistently modulate their affinity to the MS2 bacteriophage coat protein upon binding of flavin mononucleotide, tryptophan, theophylline, and microRNA miR-208a, achieving activation ratios of up to 20 and significantly better performance than control designs. The data enable dissection of features of structure-toggling mechanisms that correlate with higher performance. The diversity of RiboLogic designs and their quantitative experimental characterization provides a rich resource for further improvement of riboswitch models and design methods.