Poster abstracts
Poster number 43 submitted by Amin Espah Borujeni
The Riboswitch Calculator: automated physics-based design of synthetic riboswitches from diverse RNA aptamers
Amin Espah Borujeni (Chemical Engineering, Penn State University), Dennis M. Mishler (Molecular Biosciences, University of Texas at Austin), Jingzhi Wang (Chemistry, Emory University), Walker Huso (Chemical Engineering, Penn State University), Justin P. Gallivan (Chemistry, Emory University), Howard M. Salis (Chemical Engineering and Biological Engineering, Penn State University)
Abstract:
Riboswitches are RNA-based biosensors that regulate gene expression by employing an RNA aptamer that senses chemicals, changes its shape, and alters mRNA’s transcription or translation rate. Riboswitches can be applied in various disciplines such as in medical diagnosis, metabolic engineering, environmental remediation, and human health. Although hundreds of RNA aptamers have been generated to bind important ligands, only a few of these aptamers were converted into riboswitches, mainly due to the need for expert design or high-throughput screening, both of which require experience and human intuition. To overcome this challenge, we have developed a biophysical model and an automated design method to convert aptamers into translation-regulating riboswitches. This biophysical model predicts the sequence-structure-function relationship for the riboswitches directly from their aptamer’s specificities, aptamer’s surrounding sequences, and ligand and mRNA concentrations. To validate our intuition-free approach, we designed 52 riboswitches from the theophylline, TMR, and fluoride aptamers and measured their activation in 136 in vivo and in vitro conditions. Our automated design method reliably generated functional riboswitches with high activation ratios, up to 383-fold. We demonstrated the capability of our method in designing riboswitches that use aptamers with pseudoknot structures (by testing fluoride aptamer) and for ligands that must be sensed at very low concentrations (by testing TMR aptamer). We also elucidated the key reasons why some riboswitches fail to regulate gene expression, and thus developed quantitative criteria to predict whether a riboswitch is thermodynamically driven or kinetically trapped. We next showed that increasing mRNA concentration has an opposite effect on the riboswitch function in in vivo versus in vitro conditions. We related this counter-intuitive observation to the mRNA’s excluded volume effect within the crowded cellular environments versus diluted in vitro systems. Finally, we incorporated our design method into a user-friendly web interface, called the Riboswitch Calculator, enabling the scientific community and other non-experts to easily design and build riboswitches for their own applications.
Keywords: Synthetic riboswitch, Biophysics, Automated design