University of Minnesota
School of Physics & Astronomy

Biophysics Seminar

Thursday, April 5th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: James M. Carothers, Department of Bioengineering, University of Wisconsin
Subject:  Multi-state design of kinetically-controlled RNA aptamer ribosensors

Metabolite-responsive RNA regulators that react to changing conditions through molecular interactions are widespread in biology. In many of these systems, kinetic control mechanisms coordinate co-transcriptional RNA folding with metabolite binding and enable outputs that are highly-sensitive and highly-selective to target ligands. Although synthetic riboswitches exhibiting kinetic control have been identified by chance, it has not been possible to intentionally engineer kinetically-controlled RNA aptamer devices. Consequently, kinetic control mechanisms that could otherwise be exploited to overcome functional limits imposed by the thermodynamics of molecular recognition have remained beyond reach. We recently developed a novel approach for multi-state, co-transcriptional RNA folding design that has allowed us to engineer kinetically-controlled RNA aptamer ribosensors. In this architecture, in vitro selected RNA aptamers are coupled through a timer domain to a toehold-mediated strand displacement (TMSD) actuator such that co-transcriptional ligand-binding generates fluorescence from DNA gates through TMSD. We have shown 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. I will present these results and discuss the broader implications of this work for engineering RNA aptamer devices and overcoming thermodynamic constraints on molecular recognition through the design of kinetically-controlled responses.

Faculty Host: Vincent Noireaux

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