Reaction conditions for thermal hydrolysis of the FMN riboswitch aptamer were systematically analyzed. (A) Temperature dependence of the thermal hydrolysis reaction at pH 9, as monitored by change in UV absorbance at 260 nm with time. The hydrolysis time course for 2′–3′ cCMP at pH 9 and 95°C is plotted on the same graph. (B) PAGE analysis of basic hydrolysis reaction products at different time points. Thermal hydrolysis of 32P body-labeled FMN riboswitch aptamer performed at pH 9 and 95°C. (C) The pH dependence of the thermal hydrolysis reaction at 95°C, as monitored by change in UV absorbance at 260 nm with time. (D) PAGE analysis of neutral hydrolysis reaction products at different time points. Thermal hydrolysis of 32P body-labeled FMN riboswitch aptamer was performed at pH 7 and 95°C. Under the same conditions, hydrolysis of α-32P-labeled GTP generates 32P-labeled GMP.
