Nucleic acid catalysts with endonuclease activity

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

2001-02-08

2004-12-14

McGarry, Sean (Department: 1635)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S006120, C435S325000, C435S375000, C536S023100, C536S024100, C514S04400A

Reexamination Certificate

active

06831171

ABSTRACT:

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 (e.g., 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.
In general, enzymatic nucleic acids with RNA cleaving activity 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.
Several approaches such as 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 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, for example, 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 ribozymes such as the hammerhead ribozyme and the hairpin 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 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.
Tang et al, 1997
, RNA
3, 914, reported novel ribozyme sequences with endonuclease activity, where the authors used an in vitro selection approach to isolate these ribozymes.
Vaish et al., 1998 PNAS 95, 2158-2162, describes the in vitro selection of a hammerhead-like ribozyme with an extended range of cleavable triplets.
Breaker, International PCT publication No. WO 98/43993, describes the in vitro selection of hammerhead-like ribozymes with sequence variants encompassing the catalytic core.
The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the catalytic nucleic acid molecules of the instant invention.
SUMMARY OF THE INVENTION
This invention relates to nucleic acid molecules with catalytic activity, which are particularly useful for cleavage of RNA or DNA. The nucleic acid catalysts of the instant invention are distinct from other nucleic acid catalysts known in the art. The nucleic acid catalysts of the instant invention do not share sequence homology with other known ribozymes. Specifically, nucleic acid catalysts of the instant invention are capable of catalyzing an intermolecular or intramolecular endonuclease reaction.
In a preferred embodiment, the invention features a nucleic acid molecule with catalytic activity having either the formulae I and II:
 3′—X—Z—Y—5′  Formula II
In the above formulae, each N represents independently a nucleotide or a non-nucleotide linker, which may be same or different; X and Y are independently oligonucleotides of length sufficient to stably interact (e.g., by forming hydrogen bonds with complementary nucleotides in the target) with a target nucleic acid molecule (the target can be an RNA, DNA or RNA/DNA mixed polymers, including polymers that may include base, sugar, and/or phosphate nucleotide modifications; such modifications are preferably naturally occurring modifications), preferably, the length of X and Y are independently between 3-20 nucleotides long, e.g., specifically, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, and 20); X and Y may have the same lengths or may have different lengths; m, n, o, and p are integers independently greater than or equal to 1 and preferably less than about 100, specifically 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50; wherein if (N)
m
and (N)
n
and/or (N)
o
and (N)
p
are nucleotides, (N)m and (N)n and/or (N)
o
and (N)
p
are optionally able to interact by hydrogen bond interaction; preferably, (N)m and (N)n and/or (N)
o and (N)
p
independently form 1, 2, 3, 4, 5, 6, 7, 8, 9 base-paired stem structures; D is U, G or A; L
1
and L
2
are independently linkers, which may be the same or different and which may be present or absent (i.e., the molecule is assembled from two separate molecules), but when present, are nucleotide and/or non-nucleotide linkers, which may comprise a single-stranded and/or double-stranded region; _

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