Nucleic acid catalysts with endonuclease activity

Details Nucleic acid catalysts with endonuclease activity Nucleic acid catalysts with endonuclease activity

2000-01-07

2003-12-02

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

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

Method of regulating cell metabolism or physiology

C435S006120, C435S091100, C435S091310, C435S320100, C435S325000, C536S023100, C536S023200, C536S024500

Reexamination Certificate

active

06656731

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to nucleic acid molecules with catalytic activity and derivatives thereof.
The following is a brief description of enzymatic nucleic acid molecules. This summary is not meant to be complete but is provided only for understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
Enzymatic nucleic acid molecules (ribozymes) are nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be used, for example, to target virtually any RNA transcript (Zaug et al., 324
, Nature
429 1986; Cech, 260
JAMA
3030, 1988; and Jefferies et al., 17
Nucleic Acids Research
1371, 1989).
Because of their sequence-specificity, trans-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for human disease (Usman & McSwiggen, 1995
Ann. Rep. Med. Chem
. 30, 285-294; Christoffersen and Marr, 1995
J. Med. Chem
. 38, 2023-2037). Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.
There are seven basic varieties of naturally occurring enzymatic RNAs. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
In addition, several in vitro selection (evolution) strategies (Orgel, 1979
, Proc. R. Soc. London
, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of phosphodiester linkages and amide linkages, (Joyce, 1989
, Gene
, 82, 83-87; Beaudry et al., 1992
, Science
257, 635-641; Joyce, 1992
, Scientific American
267, 90-97; Breaker et al., 1994
, TIBTECH
12, 268; Bartel et al., 1993
, Science
261:1411-1418; Szostak, 1993
, TIBS
17, 89-93; Kumar et al, 1995
, FASEB J
., 9, 1183; Breaker, 1996
, Curr. Op. Biotech
., 7, 442).
The enzymatic nature of a ribozyme is advantageous over other technologies, since the effective concentration of ribozyme necessary to effect a therapeutic treatment is generally lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically, Thus, a single ribozyme (enzymatic nucleic acid) molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.
The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA-cleaving ribozymes for the purpose of regulating gene expression. The hammerhead ribozyme functions with a catalytic rate (k
cat
) of ~1 min
−1
in the presence of saturating (10 mM) concentrations of Mg
2+
cofactor. However, the rate for this ribozyme in Mg
2+
concentrations that are closer to those found inside cells (0.5-2 mM) can be 10- to 100-fold slower. In contrast, the RNase P holoenzyme can catalyze pre-tRNA cleavage with a k
cat
of ~30 min
−1
under optimal assay conditions. An artificial ‘RNA ligase’ ribozyme has been shown to catalyze the corresponding self-modification reaction with a rate of ~100 min
−1
. In addition, it is known that certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 min
−1
. Finally, replacement of a specific residue within the catalytic core of the hammerhead with certain nucleotide analogues gives modified ribozymes that show as much as a 10-fold improvement in catalytic rate. These findings demonstrate that ribozymes can promote chemical transformations with catalytic rates that are significantly greater than those displayed in vitro by most natural self-cleaving ribozymes. It is then possible that the structures of certain self-cleaving ribozymes may be optimized to give maximal catalytic activity, or that entirely new RNA motifs can be made that display significantly faster rates for RNA phosphodiester cleavage.
An extensive array of site-directed mutagenesis studies have been conducted with the hammerhead ribozyme to probe relationships between nucleotide sequence and catalytic activity. These systematic studies have made clear that most nucleotides in the conserved core of the hammerhead ribozyme cannot be mutated without significant loss of catalytic activity. In contrast, a combinatorial strategy that simultaneously screens a large pool of mutagenized ribozymes for RNAs that retain catalytic activity could be used more efficiently to define immutable sequences and to identify new ribozyme variants. Although similar in vitro selection experiments have been conducted with the hammerhead ribozyme (Nakamaye & Eckstein, 1994
, Biochemistry
33, 1271; Long & Uhlenbeck, 1994
, Proc. Natl. Acad. Sci
., 91, 6977; Ishizaka et al., 1995
, BBRC
214, 403; Vaish et al., 1997
, Biochemistry
36, 6495), none of these efforts have successfully screened full-sized hammerhead ribozymes for all possible combinations of sequence variants that encompass the entire catalytic core.
The hammerhead ribozyme is one of the smallest ribozymes known and has thus attracted much attention for the study of structure-function relationships in catalytic. RNAs as well as for its potential for the sequence-specific inhibition of gene expression (Usman et al., supra). The hammerhead cleaves RNA sequence-specifically adjacent to the general triplet sequence NUX, where N is any nucleotide and X can be A, U or C. Cleavage behind a guanosine such as in GUG is very slow (4.3×10
−5
min
−1
) compared to the triplet substrate GUC (1 min
−1
) (Baidya et al., 1997
, Biochemistry
36, 1108). Although the X-ray structure of this ribozyme has been solved and a mechanism proposed (Pley et al., 1994
Nature
, 372, 68; Scott et al., 1996
, Science
274, 2065), the question of what determines its specificity for the NUX sequence is still largely unresolved. One way of obtaining an insight into this problem might be to compare sequences of hammerhead ribozymes with different triplet cleaving specificities. In previous publications, it was demonstrated, using in vitro selection, that the native hammerhead sequence necessary to cleave a typical cleavage triplet, NUX, can not be altered (Nakamaye & Eckstei

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