In the typical GTase reaction, the nucleophilic attack on the aphosphate of GTP by the enzyme during the first step of the reaction results in the formation of a covalent intermediate in which GMP is linked via a phosphoamide bond to a lysine residue of the enzyme [2] . Interestingly, it has previously been demonstrated that the nucleotide analog ribavirin triphosphate can be used as a substrate by the GTase to form an enzyme-RMP covalent intermediate [25] . Although MPA is not a nucleoside analog per se, we were still interested to monitor its ability to potentially be used as a substrate by the enzyme. The appearance of a slower migrating protein species is traditionally observed upon electrophoresis through a polyacrylamide gel when an RNA capping enzyme is incubated with GTP [25] . This slower migrating species corresponds to the enzyme with covalently bound GMP. We therefore incubated the enzyme in the presence of GTP or MPA and the polypeptide was analyzed by capillary electrophoresis. The appearance of a slower migrating protein species was observed repeatedly when the protein was incubated with GTP (Fig. 3A) . However, this was not observed when the enzyme was incubated with MPA (Fig. 3A) . We therefore conclude that MPA is not a substrate for the GTase. Accordingly, the transfer of MPA to an acceptor RNA could not be detected when the enzyme was incubated in the presence of MPA (Fig. 3B ). This was tested by incubating the enzyme with an RNA substrate (81 nt) synthesized in the presence of [alpha-32 P]GTP. This RNA substrate harbored a radiolabelled diphosphate 59-end (59 ppG-RNA 39, where the boldface indicates the radiolabelled moiety) and 27 internally labelled guanosine residues. The RNA was then incubated with the GTase in the presence of GTP or MPA. The products of the reaction were extracted with phenol/chloroform and the RNA acceptor molecules were recovered by ethanol precipitation. Aliquots of the RNA samples were then digested with nuclease P1 and alkaline phosphatase and analyzed by polyethyleneiminecellulose thin layer chromatography (Fig. 3B) . The transfer of radiolabeled GMP to RNA was confirmed by demonstrating the release of a GpppG structure following digestion of the RNA samples with both nuclease P1 and alkaline phosphatase. However, the transfer of MPA to the acceptor RNA could not be detected when the enzyme was incubated in the presence of MPA (Fig. 3B) . Overall, these results demonstrate that MPA is not a substrate for the GTase, and that it cannot be transferred to an acceptor RNA.