Low-magnesium, trans-cleavage activity by type III, tertiary stabilized hammerhead ribozymes with stem 1 discontinuities

BMC Biochemistry Low-magnesium, trans-cleavage activity by type III, tertiary stabilized hammerhead ribozymes with stem 1 discontinuities Donald H Burke 0 1 S Travis Greathouse 1 0 Department of Molecular Microbiology and Immunology, 471h Life Sciences Center, University of Missouri-Columbia, School of Medicine , 1201 Rollins Dr., Columbia, MO 65212-7310 U.S.A 1 Department of Chemistry, Indiana University , Bloomington, IN 47405-7102 U.S.A 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 transcleaving 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 25C and 0.5 mM at 37C. Under multipleturnover 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. Conclusion: 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. - Background Self-cleaving hammerhead ribozymes contain three basepaired stems joined by a highly conserved core. Tertiary stabilizing motifs (TSM) of diverse morphologies between single-stranded elements at the ends of, or within, stems 1 and 2 increase cleavage activity at physiological concentrations of divalent magnesium ions in vitro and in cells [1-8]. This discovery has propelled a resurgence of interest in metal ion binding by hammerhead ribozymes [9,10] and in the use of intracellularly expressed ribozymes as gene-knockdown agents. Low magnesium concentrations in the intracellular environment can be a critical limitation for hammerhead ribozymes. Although the total intracellular concentration of divalent magnesium is approximately 3.5 to 8.5 mM, analysis of 31P chemical shift indicates that free Mg2+ ranges from 0.2 to 1.2 mM and is generally between 0.4 to 0.8 mM depending on tissue type and physiological state [11-15]. Consistent with this view, the intracellular kinetic behavior of a hairpin ribozyme is more closely approximated by in vitro assays carried out at 2.0 mM MgCl2 than at 10 mM MgCl2 [16]. It is therefore important to define the ribozyme topologies and sequences that confer low magnesium activity. Hammerheads are classified as being of type I, II or III according to whether the 5' and 3' termini reside within stem 1, 2 or 3, respectively (Figure 1A). The distinct connectivity patterns make these three types topologically non-equivalent. Tertiary-stabilized type I hammerheads, such as the SMl ribozyme from Schistosoma mansoni, are readily adapted for trans-cleavage by opening the loop at the end of stem 3. In the SMl ribozyme, however, nucleotides from both the substrate and ribozyme strands contribute to establishing stable tertiary interactions, significantly limiting the range of substrates that can be targeted for cleavage at physiological concentrations of Mg2+. We recently described hammerhead ribozyme RzB, which was derived from in vitro selections from a library of type I self-cleaving hammerheads. RzB carries an artificial TSM that is nearly independent of the sequence of the RNA fragment to be cleaved, freeing the experimental design from constraints encountered in ribozymes based on SMl [4]. FAi.gTuyrpee1s I, II and III hammerhead ribozymes A. Types I, II and III hammerhead ribozymes. Peripheral regions shown as dotted lines contain the tertiary stabilizing motifs and can be of arbitrary sizes. Stems 1, 2 and 3 are indicated. B. The type III hammerhead ribozyme from sTRSV, showing tertiary interactions predicted from comparative sequence analysis, mutational data and computational modeling [2] (pairwise interactions depicted according to ref [22]). C G U U U U C C G G U G U A G G 1 C C A C G 5' RzC 1 RzC 2 RzC-ras G A G C G 5' G C 3' G C U U U AGAG G C U A U 1a RFibgouzryem2es described in this study Ribozymes described in this study. Stems la, Ib, 2 and 3 are indicated. Substrate strands are shaded. Nucleotides involved in establishing the roles of tertiary stabilization in RzC, RzCA 1 and RzC 2 are boxed. There have not been reports of using the type II or type III topology for low-magnesium trans-cleavage. Opening the loop at the end of stem 1 in these ribozymes to accommodate substrate binding is expected to disrupt the TSM and eliminate tertiary stabilization. We reasoned that type II and type III hammerhead ribozymes could nevertheless be used for trans-cleavage at physiological magnesium concentrations if the substrate binding function of stem 1 could be separated from the tertiary stabilizing function of the TSM carried within loop 1. To this end, we constructed trans-cleaving versions of the type III self-cleaving hammerhead ribozyme from the Tobacco Ring-Spot Virus satellite RNA (sTRSV) [17]. The functional separation was achieved by placing both the 5' end of the ribozyme and the 3' end of the cleavage substrate within stem 1. Ribozymes with a discontinuous stem 1 exhibited tertiary stabilization at physiological magnesium. We further demonstrate that this stabilization extends to cleavage of a human ras oncogene mRNA fragment. Results & Discussion Low-magnesium activity of (...truncated)