Miniribozymes active at low magnesium ion concentrations

Details Miniribozymes active at low magnesium ion concentrations Miniribozymes active at low magnesium ion concentrations

2001-06-22

2004-12-07

Epps-Ford, Janet L. (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S006120, C435S091310, C435S320100, C435S325000, C514S04400A, C536S023200, C536S024500

Reexamination Certificate

active

06828148

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to minimised ribozymes, herein referred to as “miniribozymes”, and in particular it relates to a class of miniribozymes which has been selected on the basis of the very high cleavage rates by the members of the class at low Mg
2+
concentration. This invention also extends to compositions comprising these miniribozymes, to transfer vectors and host cells as well as methods of cleaving a target RNA in a subject and other methods of use of these miniribozymes.
Throughout this application, various references are cited in brackets. The full citations may be found in the numerical list of references immediately following the Example(s). These publications are hereby incorporated by reference into the present application.
BACKGROUND OF THE INVENTION
There is currently much interest in the catalytic potential of RNA both as a candidate molecule for precellular evolution and as gene targeted therapeutics. To date there are in excess of 30 distinct RNA motifs known to perform catalysis in the absence of protein. These include ribozymes derived from natural RNAs and also those produced by in vitro selection (1). The reactive capacity of any polynucleotide is a function of the chemistry of the individual nucleotides and the secondary and tertiary structure of the polymer, both of which are affected by coordination with metal ions (2,3). Structure within an oligonucleotide can be viewed as a direct function of the sequence identity. The catalytic versatility of a nucleic acid is therefore amenable to combinatorial studies, such as in vitro selection, involving the artificial manipulation of a discrete sequence space in response to selective pressure.
The 2′ hydroxyl of ribonucleic acids is a crucial functional entity permitting nucleophilic attack by the deprotonated form on an adjacent bridging phosphate ester leading to hydrolysis (giving 5′ OH and 2′-3′ cyclic phosphate ends) via a pentacoordinate transition state (4-7). Enzymes of either nucleic acid or protein composition that catalyse reactions of phosphates are invariably metal ion dependent (8). Metals have been recruited by biology for this purpose presumably because phosphates are a favourable metal ion ligand, and because coordination with metals withdraws electron density from the phosphorus centre, making it more susceptible to nucleophilic attack. Metal ions may also assist the deprotonation of the attacking nucleophile and assist the stabilisation of the developing negative charge on the leaving group (8-10).
The hammerhead ribozyme was first identified as a self (cis) cleaving sequence found in a number of small, circular, RNA pathogens (virusoids and viroids) found in plants, and a satellite RNA found in newt (11). Its consensus structure consists of three helical regions which form at their junction, a conserved bed of 15 nucleotides. The bulk of the conserved nucleotides can be located on a single oligoribonucleotide constituting an enzymatic entity capable of cleaving multiple substrates (12,13). Ribozymes designed accordingly can be directed in trans against any RNA substrate containing an endogenous 5′ UH (where H=C, U, or A) (14-16). There is significant interest in these enzymes because they offer a means of specifically inactivating deleterious RNA, eg. viral or oncogenic mRNAs, and thereby ameliorating disease.
Hammerhead cleavage of an RNA phosphodiester bond exhibits divalent metal ion dependence (17) typical of this form of catalysis in nature. Generally the metal ion is proposed to act both structurally to augment and direct specific helical interactions, and as a catalytic co-factor functional in the chemical step of the reaction (2,9,18). Mg
2+
is known to perform both these roles effectively. Bassi et al (19-21) report that there is a two stage folding process leading to formation of the ground state, each with a specific Mg
2+
requirement. Once the ground state is obtained, Mg
2+
is thought to play a role in activating the 2′ OH nucleophile of C17, either by direct coordination, or by providing an appropriately positioned basic group (Mg—OH), to effect deprotonation, promoting nucleophilic attack on the adjacent phosphorus (22-24).
The role of helix II in the hammerhead ribozyme has been investigated in several deletion studies (25-28). The presence of Watson-Crick base pairing in this region is thought to stabilise the active conformation of the conserved nucleotides stacked above it (28). The role of helix II is therefore seemingly to limit the number of steric possibilities closer to the cleavage site. This effect is no doubt variable and will depend on the composition of nucleotides between positions A9 and G12. Crystal structures for the hammerhead ribozyme show proximity between helix I and helix II (29,30). These structures suggest a plausible interaction (perhaps Mg
2+
mediated) between helix I and helix II. Whilst this interaction is remote from the cleavage site, it affects the global architecture of the molecule, and thus the cleavage rate. It has been speculated that the interaction between helix I and II may in fact stabilise an inactive conformation (31). The truncation of helix I appears to amend this interaction and allows higher rates of catalysis to be observed (31). Minimisation strategies involving helix II therefore might offer an alternative means of circumventing or closing down this particular equilibrium pathway, and thereby improve the catalytic outcome.
Miniribozymes are derivatives of the hammerhead ribozyme where helix II has been replaced by a linker with a single Watson-Crick base pair (32). This minimisation strategy has created a novel structural format. Whilst the full length ribozyme has been subject to selection over evolutionary time, size constrained, trans cleaving ribozymes have not been exposed to selection in nature. Minimised ribozymes have been shown to cleave long RNAs more efficiently than full length hammerheads (27), and could therefore provide improved trans cleaving activity in a cellular environment.
The work leading to the present invention has included in vitro optimisation of the novel miniribozyme structure. In particular, in vitro selection was used to search an 18 nt RNA sequence space corresponding in size to a miniribozyme (FIG.
1
). The aims were primarily to identify all motifs within this size constrained domain, capable of supporting Mg
2+
dependent phosphodiester cleavage of a 29 mer RNA substrate containing a 13 nt segment of human IL-2 mRNA. Subsequently, the aim was to direct the active component of this population towards optimum catalytic efficiency at low concentrations of Mg
2+
(0.5-2 mM) such as occur intracellularly (33). This work has shown that the active population consisted almost entirely of molecules containing conserved nucleotides conforming to recognised hammerhead motifs. This set of molecules exhibited highly variable catalytic activity. It was an expectation that hammerhead-like molecules would form a subset of the active sequence space. An important goal was therefore to optimise the nucleotide composition between positions 9 and 12 amongst hammerhead-like molecules, within a context of size constraint, and therefore to evolve a linker between A9 and G12 which most efficiently favours equilibration of the active conformation.
SUMMARY OF THE INVENTION
This invention is directed to a selected class of miniribozymes, capable of hybridising with a target RNA to be cleaved, and exhibiting very high cleavage rates at low Mg
2+
concentration. These miniribozymes may be used both in vitro and in vivo, and their uses extend to both the diagnostic and therapeutic fields, that is, these miniribozymes may be used as diagnostic or therapeutic agents.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect, the present invention provides a compound of the formula IA (SEQ ID NO:1) or 1B (SEQ ID NO:2):
wherein each X represents a nucleotide which may be the same or different and may be substituted or mo

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