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

Abstract

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.

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.

Figure 1

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]).

Figure 2

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, RzCAΔ1 and RzCΔ2 are boxed.

Figure 3

Magnesium dependence of substrate cleavage by discontinuous ribozymes with varying degrees of tertiary stabilization. Traces shown are for cleavage at 37°C in 10 mM (diamonds) or 0.5 mM (triangles) MgCl₂. Data were fit to double exponential kinetic model as described in Methods, using best fit parameters given in Table 1.

Figure 5

Summary of magnesium dependence of initial rates. Initial rates observed in 10 (gray bars), 5 (diagonally hatched bars), 2 (white bars) and 0.5 (black bars) mM MgCl₂ are normalized to the rate observed in 5 mM MgCl₂. The down arrow for RzCΔ2 indicates that no cleavage was observed under these conditions. Symbols above the bars indicate magnesium sensitivity: "++," moderate magnesium dependence indicative of tertiary stabilization; "+/-," intermediate magnesium dependence indicative of modest tertiary stabilization; "-," strong magnesium sensitivity indicative of there being no tertiary stabilization.

Figure 4

Eadie-Hofstee plots for determining substrate binding affinities. Reaction velocity (v_o) in micromoles•min^-1 is normalized to total ribozyme concentration (1 nM). Indicated on each plot are the ribozyme species, the concentration of MgCl₂ in millimolar (in parentheses) and the calculated Km value (negative of the slope). Gray trendlines for RzC at 0.5 mM MgCl₂ subdivide the data set into regions of steep and shallow slopes with Km values of 90 nM and 12 nM.