Multi-state design of kinetically-controlled RNA aptamer ribosensors

Metabolite-responsive RNA regulators with kinetically-controlled responses are widespread in nature. By comparison, very limited success has been achieved creating kinetic control mechanisms for synthetic RNA aptamer devices. Here, we show that kinetically-controlled RNA aptamer ribosensors can be engineered using a novel approach for multi-state, co-transcriptional folding design. The design approach was developed through investigation of 29 candidate p-aminophenylalanine-responsive ribosensors. We show that ribosensors can be transcribed in situ and used to analyze metabolic production directly from engineered microbial cultures, establishing a new class of cell-free biosensors. We found that kinetically-controlled ribosensors exhibited 5-10 fold greater ligand sensitivity than a thermodynamically-controlled device. And, we further demonstrated that a second aptamer, promiscuous for aromatic amino acid binding, could be assembled into kinetic ribosensors with 45-fold improvements in ligand selectivity. These results have broad implications for engineering RNA aptamer devices and overcoming thermodynamic constraints on molecular recognition through the design of kinetically-controlled responses.