Trans-cleaving hammerheads with discontinuous or extended stem I and with tertiary stabilizing motifs (TSMs) have been tested previously against short RNA substrates in vitro at low Mg2+ concentration. However, the potential of these ribozymes for targeting longer and structured RNAs in vitro and in vivo has not been examined. Here, we report the in vitro cleavage of short RNAs and of a 464-nt highly structured RNA from potato spindle tuber viroid (PSTVd) by hammerheads with discontinuous and extended formats at submillimolar Mg2+. Under these conditions, hammerheads derived from eggplant latent viroid and peach latent mosaic viroid (PLMVd) with discontinuous and extended formats, respectively, where the most active. Furthermore, a PLMVd-derived hammerhead with natural TSMs showed activity in vivo against the same long substrate and interfered with systemic PSTVd infection, thus reinforcing the idea that this class of ribozymes has potential to control pathogenic RNA replicons. ; Ministerio de Ciencia e Innovacio' n (BFU2008-03154/ BMC to R.F.) ; Generalidad Valenciana (ACOMP/2010/ 278 to R.F.) ; Ministerio de Educación y Ciencia (Predoctoral fellowship to A.C.) ; Consejo Superior de Investigaciones Científicas (Postdoctoral I3P contract to S.G.) of Spain ; Funding for open access charge: Ministerio de Ciencia e Innovación (BFU2008-03154/ BMC to R.F.) ; Flores Pedauye, R.; Gago Zachert, SP.; Carbonell, A. (2011). Trans-cleaving hammerhead ribozymes with tertiary stabilizing motifs: in vitro and in vivo activity against a structured viroid RNA. NUCLEIC ACIDS RESEARCH. 39:2432-2444. doi:10.1093/nar/gkq1051. ; Senia ; 2432 ; 2444 ; 39