Extended Data Fig. 2: Comparison of sequence constraints for toehold switches and LIRAs.

a, The toehold switch contains a 9- to 12-nt stem sequence immediately after the start codon that is defined by the sequence of the input RNA. This clamp region cannot encode an in-frame stop codon, which in turn imposes sequence constraints on the input RNA sequence. The clamp also adds three to four N-terminal residues to the output protein. b, LIRAs employ a 6-bp clamp domain downstream of the start codon to achieve low translational leakage with high ON-state signals. This clamp domain is not correlated with the input RNA sequence but does incorporate two additional residues into the N-terminus of the output protein. c, Experimental study of the effect of different clamp sequences on OFF-state GFP fluorescence of four LIRA H01 variants with different clamp sequences. OFF-state expression is unaffected by clamp sequence. d, Effect of different clamp sequences on the ON-state GFP fluorescence for LIRA H01 variants. e, ON/OFF fluorescence of LIRA H01 clamp variants. Significant variations in ON-state GFP and ON/OFF levels do occur, but all systems show ON/OFF levels greater than 50-fold. These results demonstrate that LIRAs can accommodate changes in clamp sequence and output protein N-terminal residues, while displaying good performance. n = 3 biological replicates, bars represent the geometric mean ± s.d. for c and d and the arithmetic mean ± s.d. for e.