Skip to main content
. 2021 Sep 21;66:53–60. doi: 10.1016/j.nbt.2021.09.002

Fig. 5.

Fig. 5

SARS-CoV-2 Toehold switch performance. (A) Illustration of a CFPS-based biosensor utilizing toehold switch riboregulators and NanoLuc reporter. Top: Depiction of the toehold switch sequence components and secondary structure without the presence of the target viral RNA. When unbound, the hairpin loop remains “closed” and sequesters the ribosome binding site (RBS) repressing translation. Within the CFPS reaction, toehold switch RNA is actively transcribed from DNA template. Bottom: Binding of the target viral RNA sequence to the toehold domain “opens” the switch releasing the RBS for gene translation resulting in the synthesis of the bioluminescent reporter NanoLuc in the CFPS system. (B) Performance of toehold switches designed, constructed and tested in this work as measured by the ratio of relative bioluminescence of CFPS toehold switch reaction in the presence of 6 μM SARS-CoV-2 target RNA sequence to CFPS toehold switch reaction without target RNA. Error bars represent one standard deviation for n = 2 liquid-based CFPS reactions. (C) CFPS performance of Toehold Switch B in the presence of the indicated concentration of SARS-CoV-2 target RNA. “No DNA” indicates bioluminescence of negative control to which no switch plasmid was added. Error bars represent one standard deviation for n = 2 liquid-based CFPS reactions. (D) Detection of 40 nM SARS-CoV-2 target RNA in human saliva at 30 % (v/v) in CFPS enabled by the addition of murine RNase Inhibitor (mRI). “No DNA” indicates bioluminescence of negative control to which no switch plasmid was added. Error bars represent one standard deviation for n = 2 liquid-based CFPS reactions.