Figure 2.

HPLC analysis of hydrolytic susceptibility of m⁷GpppG, m⁷Gpppm⁷G, 7-methylguanosine diphosphate (m⁷GDP), and 2,2,7-trimethylguanosine diphosphate (m₃^2,2,7GDP) to recombinant DcpS. All experiments were performed using condition set (2): 20 °C in 50 mM Tris HCl buffer containing 200 mM KCl, 0.5 mM EDTA and 1 mM DTT (final pH 7.6) with 0.2 μM DcpS. Initial concentrations of cap analogs were 15 μM. The HPLC peak with a retention time of 2.5 min corresponds to the reaction buffer.

(A) DcpS enzymes efficiently hydrolyze m⁷GpppG. Complete conversion of the substrate to m⁷GMP and GDP products occurs within less than 3 minutes.

(B) and (D) m⁷GDP and m₃^2,2,7GDP, respectively, are not hydrolyzed following 24 h of incubation with DcpS. The HPLC peak with a retention time of 10.8 min in the m₃^2,2,7GDP profile corresponds to a substrate contaminant.

(C) Hydrolysis of m⁷Gpppm⁷G is significantly slower than that of m⁷GpppG. After 30 min, a small amount of the substrate is still observed in all cases. Chromatographic peaks corresponding to the products, m⁷GDP and m⁷GMP, retain their intensity after the complete degradation of m⁷Gpppm⁷G (24 h of investigation), confirming the resistance of m⁷GDP to enzymatic cleavage.