RNA Switches for Synthetic Biology

Abstract

In addition to coding for protein sequences, RNA molecules encode a diverse set of gene-regulatory elements. RNA switches are one class of gene-regulatory elements that control protein expression in a manner that is dependent on the concentration of specific ligand molecules. These allosteric gene-regulatory elements have been shown as useful tools in engineering diverse cell types to display novel function. In particular, RNA switches have been used as genetically encoded biosensors and conditional controllers to direct cellular decisions based on the system's changing environment. A significant focus in the field has been the generation of novel RNA switches that are tailored for different biological systems. We review approaches that have been used to generate RNA switches, which leverage the unique physical properties of RNA and the myriad ways in which RNA can modulate gene expression.

Figure 1.

Diagrams showing the different components of a theoretical rationally designed conformational RNA switch. (A) An example aptamer (outlined) in both ligand-binding and non-ligand-binding conformations. (B) An example ribosome binding site (RBS) (outlined) in both ribosome-accessible and -inaccessible conformations. (C) The assembled RNA switch, in which a connecting sequence (outlined and with purple nucleotides) enables ligand binding to stabilize an RBS conformation that allows for ribosome binding.

Figure 2.

Reaction coordinate diagram showing the various RNA structure states that are modeled in Espah Borujeni et al. (2016), to produce a prediction of gene expression in the presence and absence of the ligand. ΔG_total reflects the difference in free energy between the minimal free energy conformation of the RNA molecule, bound to the ligand in the presence of excess ligand, and the free energy value of the ribosome-bound RNA molecule.

Figure 3.

Schematic of the FACS-Seq workflow. (A) A library of genetic elements controlling a fluorescent reporter is transformed into cells. (B) The cells are grown under certain conditions and sorted into separate bins based on the expression levels of the fluorescent reporter. (C) The bins are sequenced to obtain the counts of each genetic element sequence in the library. The counts per bin for each sequence are used to recreate a distribution of expression levels for that sequence.