Figure 2. Development of a 2’ O-Methyl RNA Antidote that Reverses E07 Binding.

(A) Regions of the E07 sequence targeted by 15-base DNA antidote candidates. (B) A screening assay to identify the DNA antidote that best blocks E07 binding revealed a number of potent antidotes, including DNA Antidote 9 (A9). A431 cells were stained with AF488-E07 (or control aptamer AF488-C36) +/− a 50-fold excess of each of the DNA antidote candidates and analyzed by flow cytometry. The mean fluorescence intensity (MFI) of every sample was divided by the MFI of cells treated with AF488-E07 alone. Note that all antidotes are abbreviated as “A” and then the antidote number, such that Antidote 9 = A9. (C) Schematic of the E07 predicted structure with the antidote A9 target region illustrated via green dots. (D–E) After staining cells with 100nM of AF488-E07 at 4°C, cells were exposed to antidote at 37°C before returning the cells to 4°C for washing and flow cytometry analysis. Shown are representative flow cytometry analyses. (D) Cells were exposed to a 250-fold excess of antidote for either 2 min or 10 min at 37°C. Both DNA-based A9 and its 2’OMe RNA analog mA9, but not a scrambled-sequence control 2’OMe RNA antidote (sA9), enhanced removal of cell-bound AF488-E07 over time as analyzed by flow cytometry. (E) Cells were exposed to increasing concentrations of DNA A9 or 2’OMe mA9 antidotes for 10 min at 37°C. Both the DNA A9 and 2’OMe RNA mA9 antidotes were similarly effective at removing cell-bound E07 after incubation at 37°C for 10 min at concentrations ≥ 5uM (≥ 50-fold excess of antidote), but the 2’OMe RNA version was more potent at lower concentrations. (n=3; representative flow data of ≥ 10⁴ gated events shown) See also Figure S1A, Figure S2 and Figure S3.