Combinations of the polyamine spermine and magnesium ions synergize to dramatically enhance cleavage of the hairpin ribozyme. Certain synthetic basic tripeptides stimulate hairpin cleavage significantly at limiting magnesium ion concentration, notably the tripeptide of l-diaminobutyric acid (Dab). Of a range of novel synthetic spermine–amino acid conjugates, l-Dab-spermine (but not d-Dab nor other amino acid conjugates) was more effective than spermine itself.

The hairpin ribozyme achieves catalytic cleavage through interaction of essential nucleotides located in two distinct helical domains that include internal loops. Initial docking of the two domains is ion dependent and appears to be followed by a structural rearrangement that allows the ribozyme to achieve a catalytically active state that can undergo cleavage. The proposed structural rearrangement may also be ion dependent and is now of increased importance due to recent evidence that docking is not rate limiting and that metal ions are unlikely to be involved in the chemical cleavage step. An initial structural model of the docked hairpin ribozyme included a proposal 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, respectively. We have used a chemical functional group substitution technique to study whether this proposed ribose zipper is likely to be present in the active, conformationally rearranged ribozyme that is fit for cleavage. We have chemically synthesized a series of individually modified hairpin ribozymes containing 2'-analogues of nucleosides, that include 2'-deoxy and 2'-deoxy-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 position A(10), as well as some double substitutions, and we studied their cleavage rates under both single- and multiple-turnover conditions. We conclude that 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) if it rearranges to form a base-triple with A(9) and the essential residue neighboring the cleavage site G(+1), as recently proposed by another laboratory. Our results provide further support for conformational rearrangement as an important step in hairpin ribozyme cleavage.

The cleavage reaction of the hairpin ribozyme is facilitated by divalent metal ions, such as Mg2+, or by non-metallic polycations, such as the polyamine spermine. We show substantial enhancement of cleavage with combinations of metallic and nonmetallic ions. In addition we elucidate the locations of some ion binding sites by Fenton chemistry.

This chapter introduces synthetic Ribonucleic acid (RNA) modification and cross-linking approaches towards the structure of the hairpin ribozyme. New crystallographic and nuclear magnetic resonance (NMR) techniques have provided the first models of biologically interesting RNAs and RNA-protein interactions. One approach to probe RNA structure and function, which involves the chemical synthesis of model RNAs carrying individual functional group modifications at key locations, is thought to be involved in RNA catalysis and in interaction with proteins. These techniques were applied initially to the hammerhead ribozyme and to the HIV-1 Tat protein-TAR RNA interaction. The functional group modification techniques are applied to the two essential loop domains of the hairpin ribozyme. These methods are helpful in determining which functional groups are involved in structurally or functionally important hydrogen bonding or other ionic interactions. The chemical reaction involved in hairpin ribozyme action is a reversible tran-esterification resulting in phosphodiester cleavage to generate 5'-hydroxyl and 2'3'-cyclic phosphate moieties.

The hairpin ribozyme is a small catalytic RNA that achieves an active configuration by docking of its two helical domains in an antiparallel fashion. Both docking and subsequent cleavage are dependent on the presence of divalent metal ions, such as magnesium, but there is no evidence to date for direct participation of such ions in the chemical cleavage step. We show that aminoglycoside antibiotics inhibit cleavage of the hairpin ribozyme in the presence of metal ions with the most effective being 5-epi-sisomicin and neomycin B. In contrast, in the absence of metal ions, a number of aminoglycoside antibiotics at 10 mM concentration promote hairpin cleavage with rates only 13-20-fold lower than the magnesium-dependent reaction. We show that neomycin B competes with metal ions by ion replacement with the postively charged amino groups of the antibiotic. In addition, we show that the polyamine spermine at 10 mM promotes efficient hairpin cleavage with rates similar to the magnesium-dependent reaction. Low concentrations of either spermine or the shorter polyamine spermidine synergize with 5 mM magnesium ions to boost cleavage rates considerably. In contrast, at 500 microM magnesium ions, 4 mM spermine, but not spermidine, boosts the cleavage rate. The results have significance both in understanding the role of ions in hairpin ribozyme cleavage and in potential therapeutic applications in mammalian cells.

Modified nucleotides can be incorporated site specifically into RNA by the use of total chemical synthesis as well as by use of a variety of recombinant RNA techniques. The range of nucleotide analogues includes modifications to base, sugar, and phosphate for structure–function analysis and for cross-linking studies as well as to answer specific mechanistic questions in RNA catalysis. We describe how RNA containing site-specific modifications are prepared, concentrating in particular on routes involving chemically synthesized oligoribonucleotides, and give examples of their application in studies of the hammerhead and hairpin ribozymes. © 1998 John Wiley & Sons, Inc. Biopoly 48: 39–55, 1998

Abstract Many structural and biochemical projects on RNA and RNA:protein interactions have benefited from recent significant improvements in methods of chemical and enzymatic synthesis of oligoribonucleotides(1-6). These methods can also now be used to introduce site-specifically into RNA a range of nucleotide analogues containing modified functional groups or attachment sites for cross-linking (7-10). lt is not unusual in a new project involving RNA for all the three oligoribonucleotide synthesis techniques described in this book to be used together, namely chemical synthesis (this chapter), in vitro transcription (Chapter 2), and ligation (Chapter 3).

The hairpin ribozyme is one of a number of small catalytic RNAs that are excellent paradigms for RNA structure-function analysis and have potential also as therapeutic agents. This review outlines current understanding of the structure of the hairpin ribozyme and its basis for catalytic activity. Included also is a discussion of the functional group requirements for cleavage and the first steps being taken to understanding its folding. Finally, recent developments are highlighted in engineering the hairpin ribozyme for intracellular use as a potential gene therapy agent.

The hairpin ribozyme is a small catalytic RNA composed of two helical domains containing a small and a large internal loop and, thus, constitutes a valuable paradigm for the study of RNA structure and catalysis. We have carried out molecular modelling of the hairpin ribozyme to learn how the two domains (A and B) might fold and approach each other. To help distinguish alternative inter-domain orientations, we have chemically synthesized hairpin ribozymes containing 2'-2' disulphide linkages of known spacing (12 or 16 A) between defined ribose residues in the internal loop regions of each domain. The abilities of cross-linked ribozymes to carry out RNA cleavage under single turnover conditions were compared to the corresponding disulphide-reduced, untethered ribozymes. Ribozymes were classed in three categories according to whether their cleavage rates were marginally, moderately, or strongly affected by cross-linking. This rank order of activity guided the docking of the two domains in the molecular modelling process. The proposed three-dimensional model of the hairpin ribozyme incorporates three different crystallographically determined structural motifs: in domain A, the 5'-GAR-3'-motif of the hammerhead ribozyme, in domain B, the J4/5 motif of group I ribozymes, and connecting the two domains, a "ribose zipper", another group I ribozyme feature, formed between the hydroxyl groups of residues A10, G11 of domain A and C25, A24 of domain B. This latter feature might be key to the selection and precise orientation of the inter-domain docking necessary for the specific phosphodiester cleavage. The model provides an important basis for further studies of hairpin ribozyme structure and function.

A general procedure for the preparation of S-nucleosidyl S-aryl disulfides from the corresponding thioesters is described. This procedure has been used for the preparation of a ribonucleoside disulphide (8d), a key intermediate for the synthesis of oligoribonucleotides containing 3′-S-phosphorothiolate linkages. The regioselectivity of the Arbusov reaction of 8d with phosphites has been examined. The X-ray structure of 3′-deoxy-3′-S-(2-nitrophenyldisulfanyl)thymidine (9b) is also reported.

The synthesis of an RNA dinucleotide (IspU) containing a 3'-S-phosphorothiolate linkage is described. The compound is prepared from 9-(3-deoxy-3-iodo-beta-D-xylofuranosyl)hyperanthine with installation of the phosphorothiolate group via an Arbusov reaction and protection of the ribose 2'-hydroxyl as a silyl ether. IspU is found to be a substrate for several enzymes including T4 polynucleotide kinase, snake venom phosphodiesterase, and ribonuclease T-2. Base-catalyzed cleavage of the dinucleotide is accelerated (similar to 2000-fold) relative to that of the phosphate-linked compound IpU. Product characterization and kinetic analysis show that IspU is cleaved through the same mechanism as IpU. The observed rate acceleration is argued to reflect stabilization of the anionic transition state by the polarizable sulfur atom.

The hairpin ribozyme is a small self-cleaving INA of unknown three-dimensional structure that can be engineered ibr RNA cleavage in trans and has potential as a therapeutic agent. We are developing a chemical crosslinking approach to study the inter-domain distances in the catalytically active structure oi a S-stranded hairpin ribozyme. The substrate RNA was crosslinked to ribozyme strand B through tethered disulfide formation iia the 2'-positions of specific nucleoside residues located within the two internal loops or domains A and B, respectively. Preliminary results suggest that the catalytic activity is retained in the crosslinked ribozymes.

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