Cleavage of tospovirus RNA by hammerhead ribozyme

초록

It has been reported that infection by TSWV virus was inhibited by the expression of nucleocapsid protein RNA in transgenic plants in a manner of antisense RNA. To improve that antisense RNA protection, ribozyme was applied. Two hammerhead ribozymes (RB226 & RB416) were constructed, and trans cleavage reactions were performed in vitro. Cis cleavage reactions were also carried out using the ribozyme-substrate fusion constructs. RB416 cleaved 35% of the initial substrate under the condition of 37 C, 10 mM MG++, 50:1 of ribozyme/substrate ratio and 12 h incubation period. But RB226 cleaved less than 10% of the initial substrate. Cis cleavage started as soon as Mg++ was added, but activity maintained only up to 70%. The cleavage activities of RB416 and RB226 with the facilitator oligonucleotides were increased up to 95% and 35%, respectively. But the secondary structure at the target sites was still the major limiting factor for the efficient cleavage of those ribozymes. These results indicated the feasibility of RN416 as a potential antiviral agent against TSWV infection.