Investigation of the proposed interdomain ribose zipper in hairpin ribozyme cleavage using 2 '-modified nucleosides

The hairpin ribozyme achieves catalytic cleavage through interaction of ess ential nucleotides located in two distinct helical domains that include int ernal loops. Initial docking of the two domains is ion dependent and appear s to be followed by a structural rearrangement that allows the ribozyme to achieve a catalytically active state that can undergo cleavage. The propose d structural rearrangement may also be ion dependent and is now of increase d importance due to recent evidence that docking is not rate limiting and t hat metal ions are unlikely to be involved in the chemical cleavage step. A n initial structural model of the docked hairpin ribozyme included a propos al for a ribose zipper motif that involves two pairs of hydroxyl groups at A(10) and G(11) in domain A pairing with C-25 and A(24) in domain B, respec tively, We have used a chemical functional group substitution technique to study whether this proposed ribose zipper is likely to be present in the ac tive, conformationally rearranged ribozyme that is fit for cleavage. We hav e chemically synthesized a series of individually modified hairpin ribozyme s containing 2'-analogues of nucleosides, that include 2'-deoxy and 2'-deox y-2'-fluoro at each of the four nucleoside positions, 2'-amino-2'-deoxy, 2' -deoxy-2'-thio, and 2'-arabino at position C-25, and 2'-oxyamino at positio n A(10), as well as some double substitutions, and we studied their cleavag e rates under both single- and multiple-turnover conditions. We conclude th at at least some of the hydrogen-bonding interactions in the ribose zipper motif, either as originally proposed or in a recently suggested structural variation, are unlikely to be present in the active rearranged form of the ribozyme that undergoes cleavage. Instead, we provide strong evidence for a very precise conformational positioning for the residue C-25 in the active hairpin. A precise conformational requirement would be expected for C-25 i f it rearranges to form a base-triple with As and the essential residue nei ghboring the cleavage site G(+1), as recently proposed by another laborator y, Our results provide further support for conformational rearrangement as an important step in hairpin ribozyme cleavage.