Enzymatic DNA molecules

Details Enzymatic DNA molecules Enzymatic DNA molecules

1997-08-25

2001-12-04

Schwartzman, Robert A. (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S091200, C536S023100, C536S024500

Reexamination Certificate

active

06326174

ABSTRACT:

TECHNICAL FIELD
The present invention relates to nucleic acid enzymes or catalytic (enzymatic) DNA molecules that are capable of cleaving other nucleic acid molecules, particularly RNA. The present invention also relates to compositions containing the disclosed enzymatic DNA molecules and to methods of making and using such enzymes and compositions.
BACKGROUND
The need for catalysts that operate outside of their native context or which catalyze reactions that are not represented in nature has resulted in the development of “enzyme engineering” technology. The usual route taken in enzyme engineering has been a “rational design” approach, relying upon the understanding of natural enzymes to aid in the construction of new enzymes. Unfortunately, the state of proficiency in the areas of protein structure and chemistry is insufficient to make the generation of novel biological catalysts routine.
Recently, a different approach for developing novel catalysts has been applied. This method involves the construction of a heterogeneous pool of macromolecules and the application of an in vitro selection procedure to isolate molecules from the pool that catalyze the desired reaction. Selecting catalysts from a pool of macromolecules is not dependent on a comprehensive understanding of their structural and chemical properties. Accordingly, this process has been dubbed “irrational design” (Brenner and Lerner,
PNAS USA
89: 5381-5383 (1992)).
Most efforts to date involving the rational design of enzymatic RNA molecules or ribozymes have not led to molecules with fundamentally new or improved catalytic function. However, the application of irrational design methods via a process we have described as “directed molecular evolution” or “in vitro evolution”, which is patterned after Darwinian evolution of organisms in nature, has the potential to lead to the production of DNA molecules that have desirable functional characteristics.
This technique has been applied with varying degrees of success to RNA molecules in solution (see, e.g., Mills, et al.,
PNAS USA
58: 217 (1967); Green, et al.,
Nature
347: 406 (1990); Chowrira, et al.,
Nature
354: 320 (1991); Joyce,
Gene
82: 83 (1989); Beaudry and Joyce,
Science
257: 635-641 (1992); Robertson and Joyce,
Nature
344: 467 (1990)), as well as to RNAs bound to a ligand that is attached to a solid support (Tuerk, et al.,
Science
249: 505 (1990); Ellington, et al.,
Nature
346: 818 (1990)). It has also been applied to peptides attached directly to a solid support (Lam, et al.,
Nature
354: 82 (1991)); and to peptide epitopes expressed within a viral coat protein (Scott, et al.,
Science
249: 386 (1990); Devlin, et al.,
Science
249: 249 (1990); Cwirla, et al.,
PNAS USA
87: 6378 (1990)).
It has been more than a decade since the discovery of catalytic RNA (Kruger, et al.,
Cell
31: 147-157 (198:7); Guerrier-Takada, et al.,
Cell
35: 849-857 (1983)). The list of known naturally-occurring ribozymes continues to grow (see Cech, in
The RNA World
, Gesteland & Atkins (eds.), pp. 239-269, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1993); Pyle,
Science
261: 709-714 (1993); Symons,
Curr. Opin. Struct. Biol.
4: 322-330 (1994)) and, in recent years, has been augmented by synthetic ribozymes obtained through in vitro evolution. (See, e.g., Joyce,
Curr. Opin. Struct. Biol.
4: 331-336 (1994); Breaker & Joyce,
Trends Biotech.
12: 268-275 (1994); Chapman & Szostak,
Curr. Opin. Struct. Biol.
4: 618-622 (1994).)
It seems reasonable to assume that DNA can have catalytic activity as well, considering that it contains most of the same functional groups as RNA. However, with the exception of certain viral genomes and replication intermediates, nearly all of the DNA in biological organisms occurs as a complete duplex, precluding it from adopting a complex secondary and tertiary structure. Thus it is not surprising that DNA enzymes have not been found in nature.
Until the advent of the present invention, the design, synthesis and use of catalytic DNA molecules with nucleotide-cleaving capabilities has not been disclosed or demonstrated. Therefore, the discoveries and inventions disclosed herein are particularly significant, in that they highlight the potential of in vitro evolution as a means of designing increasingly more efficient catalytic molecules, including enzymatic DNA molecules that cleave other nucleic acids, particularly RNA.
BRIEF SUMMARY OF THE INVENTION
The present invention thus contemplates a synthetic or engineered (i.e., non-naturally-occurring) catalytic DNA molecule (or enzymatic DNA molecule) capable of cleaving a substrate nucleic acid (NA) sequence at a defined cleavage site. The invention also contemplates an enzymatic DNA molecule having an endonuclease activity.
In one preferred variation, the endonuclease activity is specific for a nucleotide sequence defining a cleavage site comprising single-stranded nucleic acid in a substrate nucleic acid sequence. In another preferred variation, the cleavage site is double-stranded nucleic acid. Similarly, substrate nucleic acid sequences may be single-stranded, double-stranded, partially single- or double-stranded, looped, or any combination thereof.
In another contemplated embodiment, the substrate nucleic acid sequence includes one or more nucleotide analogues. In one variation, the substrate nucleic acid sequence is a portion of, or attached to, a larger molecule.
In various embodiments, the larger molecule is selected from the group consisting of RNA, modified RNA, DNA, modified DNA, nucleotide analogs, or composites thereof. In another example, the larger molecule comprises a composite of a nucleic acid sequence and a non-nucleic acid sequence.
In another embodiment, the invention contemplates that a substrate nucleic acid sequence includes one or more nucleotide analogs. A further variation contemplates that the single stranded nucleic acid comprises RNA, DNA, modified RNA, modified DNA, one or more nucleotide analogs, or any composite thereof. In one embodiment of the disclosed invention, the endonuclease activity comprises hydrolytic cleavage of a phosphoester bond at the cleavage site.
In various preferred embodiments, the catalytic DNA molecules of the present invention are single-stranded in whole or in part. These catalytic DNA molecules may preferably assume a variety of shapes consistent with their catalytic activity. Thus, in one variation, a catalytic DNA molecule of the present invention includes one or more hairpin loop structures. In yet another variation, a catalytic DNA molecule may assume a shape similar to that of “hammerhead” ribozymes. In still other embodiments, a catalytic DNA molecule may assume a conformation similar to that of
Tetrahymena thermophila
ribozymes, e.g., those derived from group I introns.
Similarly, preferred catalytic DNA molecules of the present invention are able to demonstrate site-specific endonuclease activity irrespective of the original orientation of the substrate molecule. Thus, in one preferred embodiment, an enzymatic DNA molecule of the present invention is able to cleave a substrate nucleic acid sequence that is separate from the enzymatic DNA molecule—i.e., it is not linked to the DNAzyme. In another preferred embodiment, an enzymatic DNA molecule is able to cleave an attached substrate nucleic acid sequence—i.e., it is able to perform a reaction similar to self-cleavage.
The invention also contemplates enzymatic DNA molecules (catalytic DNA molecules, deoxyribozymes or DNAzymes) having endonuclease activity, whereby the endonuclease activity requires the presence of a divalent cation. In various preferred, alternative embodiments, the divalent cation is selected from the group consisting of Pb
2+
, Mg
2+
, Mn
2+
, Zn
2+
, and Ca
2+
. Another variation contemplates that the endonuclease activity requires the presence of a monovalent cation. In such alternative embodiments, the monovalent cation is preferably selected from the group consisting of Na
+
and K
+
.
In vario

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