Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Abstract

Tertiary stabilizing motifs (TSMs) between terminal loops or internal bulges facilitate folding of natural hammerhead ribozymes (hRz) under physiological conditions. However, both substrate and enzyme strands contribute nucleotides to the TSMs of trans-cleaving hRz, complicating the design of hRz that exploit TSMs to target specific mRNA. To overcome this limitation, we used SELEX to identify new, artificial TSMs that are less sensitive to sequence context. Nucleotides in loop II or in a bulge within the ribozyme strand of stem I were randomized, while the interaction partner was held constant. All nucleotides of the substrate pair with the ribozyme, minimizing their possible recruitment into the TSM, as such recruitment could constrain choice of candidate target sequences. Six cycles of selection identified cis-acting ribozymes that were active in 100 µM MgCl 2 . The selected motifs partially recapitulate TSMs found in natural hRz, suggesting that the natural motifs are close to optimal for their respective contexts. Ribozyme "RzB" showed enhanced thermal stability by retaining trans-cleavage activity at 80°C in 10 mM MgCl 2 and at 70°C in 2 mM MgCl 2 . A variant of ribozyme "RzB" with a continuously paired stem 1 rapidly lost activity as temperature was increased. The selected motifs are modular, in that they permit trans-cleavage of several substrates in submillimolar MgCl 2 , including two substrates derived from the U5 genomic region of HIV-1. The new, artificial tertiary stabilized hRz are thus nearly independent of sequence context and enable for the first time the use of highly active hRz targeting almost any mRNA at physiologically relevant magnesium concentrations.

Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

“…1), carrying one of these artificial tertiary motifs exhibits exceptionally high rates in the range of 100-3000 min À1 in 1 mM divalent cations (Roychowdhury-Saha and Burke 2006). We observed an increase in thermal stability upon tertiary stabilization both for ribozyme RzB (Saksmerprome et al 2004) and for an unrelated kinase ribozyme, Kin.46 (Cho and Burke 2006 ] 1/2 % 0.16 mM) (Penedo et al 2004), in contrast to the two-stage folding process detected in minimal hammerhead ribozymes (Hammann and Lilley 2002;Penedo et al 2004). A second transition, which is spectroscopically silent but kinetically apparent, appears to take place at higher Mg 2+ concentrations, as the values of [Mg 2+ ] 1/2 for catalysis range from 3.9 to % 90 mM (Penedo et al 2004;Kim et al 2005;Roychowdhury-Saha and Burke 2006).…”

“…All cleavage reactions were performed at 37°C, pH 7.4. Reactions were carried out with a 2 mM ribozyme strand and a 50 nM substrate strand in 50 mM Tris d HCl and 10 mM EDTA, as described previously (Saksmerprome et al 2004). Briefly, each reaction was initiated by adding 5 mL of a cation solution to 70 mL of preannealed ribozyme-substrate solution at 37°C, pH 7.4.…”

“…Intracellular activity is typically modest or poor, largely due to low intracellular Mg 2+ concentrations. Several studies during the last several years highlight the importance of peripheral sequences in natural and engineered hammerhead ribozymes for their in vitro activity under physiological Mg 2+ conditions and for their activity inside cells (De la Pena et al 2003;Khvorova et al 2003;Saksmerprome et al 2004;Yen et al 2004). These peripheral elements pull the ribozyme into a compact structure that aligns the scissile phosphate for in-line displacement and that positions nucleobases G8 and G12 for acid-base chemistry (Martick and Scott 2006), as has been suggested by cross-linking studies and analysis of the pH dependence of cleavage kinetics (Han and Burke 2005;Heckman et al 2005;Lambert et al 2006a,b).…”

“…Artificial tertiary stabilizing motifs that function in the context of trans-cleaving versions of two natural ciscleaving molecules were recently identified using a selection-amplification approach (Saksmerprome et al 2004). A hammerhead ribozyme, designated RzB (Fig.…”

Topological rearrangement yields structural stabilization and interhelical distance constraints in the Kin.46 self-phosphorylating ribozyme

“…For example, dramatic reductions in the Mg 2+ concentration required for half-maximal activity ([Mg 2+ ] 1/2 ) have been observed by us (Saksmerprome et al 2004;Greathouse and Burke 2005;Roychowdhury-Saha and Burke 2006) and by others (Khvorova et al 2003;Canny et al 2004;Penedo et al 2004) for various hammerhead ribozymes upon inclusion of tertiary interactions between stems I and II. Tertiary contacts account for thermal stability in RNase P from thermophyllic bacteria (Baird et al 2006) and increase thermal stability in the hammerhead ribozyme RzB (Saksmerprome et al 2004). Mutants of Group I self-splicing introns that were selected for compact folds also demonstrated greater thermal stability than the less compact versions (Juneau and Cech 1999).…”