(A,C) RT-qPCR measurements of ribozyme cleavage for gates produced in IVT. In A, reverse transcription was conducted at 37°C. In C, reverse transcription was conducted at 50°C. ΔΔCt (Equation 2; Materials and Methods) and corresponding fraction uncleaved RNA are labeled on the right and left axes, respectively. Error bars indicate standard deviation from three technical replicates. Replicate 1 and replicate 2 indicate replicates from two IVT reactions and RNA purifications conducted on different days. (B) Denaturing gel electrophoresis results for Rh gates after a 10 min incubation at 37°C in RT-qPCR reaction mixture with or without BO. Prior to incubation, the RNAs were purified from IVT reactions and annealed with or without BO (Materials and Methods). G1h* RNA incubated at 50°C was similarly protected by the BO (Supplemental Fig. S21). Amplification curves and primer efficiency plots for (A) and (C) are presented in Supplemental Sections 2.4, 2.5.
