Transcriptional Interference in Toehold Switch-Based RNA Circuits

Gene regulation based on regulatory RNA is an important mechanism in cells and is increasingly used for regulatory circuits in synthetic biology. Toehold switches are rationally designed post-transcriptional riboregulators placed in the 5′ untranslated region of mRNA molecules. In the inactive state of a toehold switch, the ribosome-binding site is inaccessible to the ribosome. In the presence of a trigger RNA molecule, protein production is turned on. Using antisense RNA against trigger molecules (antitrigger RNA), gene expression can also be switched off again. We here study the utility of antisense transcription in this context, which enables a particularly compact circuit design. Our circuits utilize two inducible promoters that separately regulate trigger and antitrigger transcription, whereas their cognate toehold switch, regulating the expression of a reporter protein, is transcribed from a constitutive promoter. We explore various design options for the arrangement of the promoters and demonstrate that the resulting dynamic behavior is influenced by transcriptional interference (TI) effects depending on the promoter distance. Our experimental results are consistent with previous findings that enhanced local RNA polymerase concentrations due to active promoters in close proximity lead to an increase in transcriptional activity of the strongest promoter in the circuits. We observed that the range of this effect is larger when the participating promoters are stronger. Based on this insight, we combined two promoter arrangements for the realization of a genetic circuit comprised of two toehold switches, two triggers, and two antitriggers that function as a two-input two-output logic gate.