Low-magnesium, trans-cleavage activity by type III, tertiary stabilized hammerhead ribozymes with stem 1 discontinuities.
BACKGROUND
Low concentrations of free magnesium in the intracellular environment can present critical limitations for hammerhead ribozymes, especially for those that are designed for intermolecular (trans) cleavage of a host or pathogen RNA. Tertiary stabilizing motifs (TSM's) from natural and artificial ribozymes with a "type I" topology have been exploited to stabilize trans-cleaving hammerheads. Ribozymes with "type II" or "type III" topologies might seem incompatible with conversion to trans-cleavage designs, because opening the loop at the end of stem 1 or stem 2 to accommodate substrate binding is expected to disrupt the TSM and eliminate tertiary stabilization.


RESULTS
Stem 1, together with single-stranded segments capping or internal to this stem, contains both the substrate-binding and tertiary stabilization functions. This stem was made discontinuous within the sTRSV hammerhead ribozyme, thereby separating the two functions into discrete structural segments. The resulting ribozyme, designated "RzC," cleaved its 13 nucleotide target substrate at MgCl2 concentrations as low as 0.2 mM at 25 degrees C and 0.5 mM at 37 degrees C. Under multiple-turnover conditions, nearly thirty turnovers were observed at the highest substrate:RzC ribozyme ratios. Similar stabilization was observed for several derivatives of RzC. Catalytic activity was diminished or eliminated at sub-millimolar MgCl2 concentrations for ribozymes with weakened or deleted tertiary interactions. Eadie-Hofstee analysis revealed that the stabilized and non-stabilized ribozymes bind their substrates with equivalent affinities, suggesting that differences in observed activity are not the result of diminished binding. Some of the stabilized and non-stabilized ribozymes appear to fold into a heterogeneous collection of conformers, only a subset of which are catalytically active.


CONCLUSIONS
Hammerhead ribozymes with the "type III" topology can be converted to a tertiary, trans-cleavage design. Separating the stabilization and substrate recognition functions of stem 1 increases cleavage activity at physiological concentrations of divalent magnesium while retaining recognition of exogenous targets. Trans-cleaving ribozymes that exploit the tertiary stabilizing motifs of all natural hammerhead topologies can therefore be used in intracellular applications.
Burke
Donald H
DH
Department of Chemistry, Indiana University, Bloomington, IN 47405-7102, USA. BurkeDH@missouri.edu
Greathouse
S Travis
ST
D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004795:Enzyme Stability; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects / Q000502:physiology
D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D008274:Magnesium