Low-magnesium, trans-cleavage activity by type III, tertiary stabilized hammerhead ribozymes with stem 1 discontinuities
© Burke and Greathouse; licensee BioMed Central Ltd. 2005
Received: 04 June 2005
Accepted: 12 August 2005
Published: 12 August 2005
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.
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°C and 0.5 mM at 37°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.
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.
Self-cleaving hammerhead ribozymes contain three base-paired 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 . It is therefore important to define the ribozyme topologies and sequences that confer low magnesium activity.
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) . 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 trans-cleaving ribozyme with discontinuous stem 1
Kinetic parameters for discontinuous ribozymes derived from sTRSV
[MgCl 2 ], mM
net init rate, min -1
Two variants of RzC were built to assess the contribution of the tertiary interaction modules to the observed magnesium dependence. Ribozymes RzCΔ1 and RzCΔ2 are identical to RzC outside of stem 1. In ribozyme RzCΔ1, the seven nucleotide terminal loop sequence 5' UGUGCUUU 3' is replaced with a stable tetraloop 5' UUCG 3', while in ribozyme RzCΔ2, the loop and terminal three base pairs are removed altogether. When reactions were monitored in 10 mM MgCl2, the deletions had little effect on initial rates, as all three ribozymes cleaved substrate C with initial rates of 2–3 min-1. Decreasing the MgCl2 concentration to 2.0 mM, however, produced a clear difference in initial cleavage rates, which were greatest for RzC and slowest for RzCΔ2. In 0.5 mM MgCl2, the initial rate of RzC was 3 times greater than that of RzCΔ1 (0.3 vs. 0.09 min-1), and no substrate cleavage at all was detected for RzCΔ2 (Figure 3 and Table 1). Interestingly, the rates observed for RzCΔ1 were closer to those of RzC than to those of RzCΔ2. It is possible that the intermediate activity seen for RzCΔ1 at low magnesium may be due to weak tertiary stabilization using alternative interactions between loop 2 and the stable tetraloop at the end of loop 1, although this possibility was not further explored.
The enhanced activity of RzC relative to RzCΔ1 and RzCΔ2 at submillimolar MgCl2 support the underlying hypothesis that the TSM stabilizes productive RNA folding. The fraction of the pre-annealed ribozyme-substrate complex that participates in the rapid phase of the cleavage reaction (fa) decreases only slightly for RzC at sub-millimolar MgCl2, while it drops precipitously for RzCΔ1 and RzCΔ2, suggesting that that fraction of the ribozyme that folds into a productive conformation is greater for RzC than for the other two ribozymes. The fraction of the ribozyme-substrate complex that can access the active conformation is given by f∞. (This estimation is actually a lower limit, as it assumes that the back reaction is negligible; a reasonable assumption given that the three-nucleotide 3' cleavage product is expected to dissociate rapidly, minimizing the possibility of re-ligation.) For reactions in which cleavage is observed, the value of f∞ is not strongly dependent upon MgCl2 concentration, although it is slightly lower for RzCΔ2 than for the other two. Together, these results indicate that the TSM sequence elements from the sTRSV hammerhead are providing similar tertiary stabilization in RzC.
Substrate affinity unaffected by TSM deletions
To rule out the possibility that the rate differences among RzC, RzCΔ1 and RzCΔ2 might be due to differential substrate-binding affinity, multiple turnover cleavage kinetics were measured as a function of substrate concentration using 1 nM ribozyme and excess substrate (10 to 200 nM). These conditions yielded between 4 and 28 nM cleaved product, indicating between four and twenty-eight turnovers. The slope of an Eadie-Hofstee plot of the rate data gives the apparent affinity of the ribozyme-substrate interaction as the Michaelis-Menten constant, Km. When multiple-turnover reaction kinetics were monitored in 10 mM MgCl2, all three ribozymes gave comparable Km values (~40 nM for RzC vs. ~70 nM for RzCΔ1 and RzCΔ2). Thus, substrate affinity is dominated by base paring within stem 3, and is not substantially affected by either the tertiary docking interactions or by the stacking between stems la and lb. Because the single-turnover reactions were performed using 10-fold excess ribozyme (1000 nM) over substrate (100 nM) at concentrations that are more than an order of magnitude above the estimated Km values (40–70 nM), the substrates are assumed to have been fully bound to ribozyme. Differences in the magnesium sensitivities among RzC, RzCΔ1 and RzCΔ2 are therefore not due to relative occupancy of the enzyme, but rather to conformational differences arising from differential tertiary stabilization. It was not possible to compare affinities of the three ribozymes at 0.5 mM MgCl2, as only RzC was active under these conditions. The slight curvature in its Eadie-Hofstee plot could be interpreted as experimental noise, or as reflecting higher affinity at low concentrations of substrate than at high concentrations.
Effects of sequence context on cleavage in sub-millimolar MgCl2
Generalization of discontinuous hammerhead ribozyme design
[MgCl 2 ], mM
init rate, min -1
Importance of stem 1b stability
The lengths of stems 1 and 2 critically determine the positioning of the interacting nucleotides at the ends of each stem. For a given stabilized hammerhead ribozyme, variants with shorter or longer stems 1 are not expected to retain tertiary stabilization – their tertiary interaction elements would be nearer to (or farther from) the core, and the nucleotides in loop 1 would be rotated by approximately 30° around the helical axis for each base pair change in helix length. The sTRSV and RzC hammerhead ribozymes have 6 total base pairs in stem 1 (three each in stems la and 1b). In contrast, the hammerhead ribozyme from peach latent mosaic viroid (PLMVd) has only five base pairs in its stem 1. In this case a three-nucleotide loop 1 (UAA) interacts with a hexaloop (UAGAGU) in loop 2 [2, 4]. Ribozyme RzD was designed to determine whether hammerhead ribozymes with 5 nucleotides in stem 1 could be converted from cis-cleavage to trans-cleavage by following the design strategy used to generate ribozyme RzC. Specifically, stem 1 was divided into a three base pair stem la (to promote intermolecular substrate binding) and a two base pair stem 1b (to preserve the original PLMVd tertiary interaction). Ribozyme RzD showed markedly sharper dependence on divalent magnesium than had been observed for RzC, with initial cleavage rates declining ~300-fold as MgCl2 was decreased from 5 mM to 0.5 mM. The 2 bp of stem 1b thus appear to be insufficiently stable to support the discontinuous design.
Conformational heterogeneity and generalizability of the design
It is common for hammerhead ribozymes to fold into heterogeneous populations in which a subset cleaves rapidly while the remaining RNAs convert over time into active fold which then cleave. This pattern is evident in the biphasic kinetics seen with several of the constructs described here (Figures 3 and 5; Tables 1 and 2). The tertiary stabilized, type 1 hammerhead ribozyme from the intestinal fluke parasite, Schistosoma mansoni (SMα1), also exhibits multiphasic kinetics indicative of conformational heterogeneity . We observed similar results with the tertiary stabilized type 2 hammerhead ribozyme from the Dolichopoda cave cricket, and found that this heterogeneity was largely eliminated by changing a few nucleotides within stems 1 and 3 (M. Roychowdhury & D. Burke, in preparation). While conformational heterogeneity affects initial rates and the chemical interpretation of catalytic mechanism, it is of minimal relevance to modulation of gene expression by intracellularly expressed ribozymes so long as the target RNA can be cleaved fast enough to exert the desired biological effect. The discontinuous design used in ribozyme RzC yields rapid, multiple-turnover cleavage at physiological concentration of divalent magnesium, thereby demonstrating its potential utility for use inside cells.
While this manuscript was in preparation, Weinberg and Rossi described a slightly different design for hammerheads with discontinuous stems 1, in which additional base pairs are allowed to form between ribozyme and substrate by introducing a new stem at the discontinuity . Although those authors only evaluated single-turnover cleavage under conditions of high divalent magnesium (10 mM), the results described here suggest that the Weinberg and Rossi design may also allow cleavage at sub-millimolar concentrations of magnesium.
The "Discontinuous Stem 1" design described here takes advantage of natural stabilizing tertiary interactions in approximately native structural contexts to enable trans-cleavage of model substrates at physiological concentrations of MgCl2. The design was particularly effective for ribozymes derived from sTRSV hammerhead ribozyme, and markedly less effective for a ribozyme derived from PLMVd. Hammerhead ribozyme RzC cleaved its 13 nucleotide target substrate effectively at MgCl2 concentrations as low as 0.2 mM at 25°C and 0.5 mM at 37°C. Catalytic activity was reduced or eliminated at sub-millimolar MgCl2 concentrations for ribozymes in which tertiary interactions are diminished (RzCΔ1) or removed (RzCΔ2). Two additional ribozymes that altered the internal guide sequence, including one targeted to the human ras oncogene, were active in low concentrations of MgCl2, demonstrating that this design could be adapted for use against other targets. We envision that trans-cleaving ribozymes that exploit the tertiary stabilizing motifs of sTRSV or other type II or type III hammerheads could be used in gene therapies or other intracellular applications.
Ribozymes, RNA substrates, and oligonucleotides
RNA substrates were synthesized by Dharmacon (Lafayette, CO) and DNA oligonucleotides by Integrated DNA Technologies (Coralville, IA). Ribozymes were synthesized by transcription in vitro from synthetic DNA templates, then radiolabeled and purified as described .
Determination of single-turnover kinetic parameters and initial rates
tertiary stabilizing motif
satellite RNA of the Tobacco Ring-Spot Virus
peach latent mosaic viroid.
The authors wish to thank Peter Unrau (Simon Frasier University), Manami Roychowdhury-Saha and Vanvimon Saksmerprome for comments on the manuscript, and Sugata Roychowdhury for insightful discussions early in the project. This work was supported by undergraduate research grants to S.T.G. from the Earl G. Sturdevant, Harry G. Day, Indiana University Honors College and the HHMI scholarship funds, and by grants to D.H.B. from the NIH under award AI45344 and from the David and Lucille Packard Interdisciplinary Science program.
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