Library design and array experiment. (A,B) Conversion from the 11ntR to the IC3R (red) or Vc2R (orange) involves four or five mutations and 16 or 32 sequence variants, respectively. These variants can be arranged in 24 or 120 single-mutant pathways (Supplemental Fig. S2). (C) Schematic of the array experiment (Buenrostro et al. 2014; She et al. 2017; Denny et al. 2018). A library of chip piece hairpin sequences is sequenced and transcribed on an Illumina MiSeq flowcell. The chip piece library contains 20 scaffold sequences (pink) and 44 TLR variants (blue) spanning mutational pathways from the 11ntR to the IC3R and Vc2R, resulting in a total of 880 unique sequences. The TLR variants (blue) and a GGAA tetraloop (black) in the chip piece bind to a GAAA or GUAA TL (green) and an R1 TLR (black) in a flow piece hairpin that is flowed onto the MiSeq flowcell. The two tertiary contacts facilitate binding of the flow piece to the immobilized chip piece hairpins. Titrating a fluorescently labeled flow piece RNA allows for the fitting of isothermal binding isotherms. (D) Example binding isotherms for three TLR sequences: the wt 11ntR (blue; n = 68 clusters), an 11ntR mutant (green; n = 46 clusters) and the wt IC3R (red; n = 78 clusters). Points represent the median fluorescence across all sequence clusters on the flowcell, with error bars depicting the 95% confidence interval (CI) obtained via bootstrap analysis. The fit isotherms are depicted as lines going through the data points, with the 95% error interval shaded.
