Catalytic nucleic acid and methods of use

Details Catalytic nucleic acid and methods of use Catalytic nucleic acid and methods of use

1999-02-25

2002-05-14

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091210, C435S091300, C435S091310, C536S023100, C536S025100

Reexamination Certificate

active

06387617

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns molecules and methods for detection of specific agents in a medium.
BACKGROUND OF THE INVENTION
Ribozymes are typically RNA molecules which have enzyme-like catalytic activities usually associated with cleavage, splicing or ligation of nucleic acid sequences. The typical substrates for ribozymes catalytic activities are RNA molecules, although ribozymes may catalyze reactions in which DNA molecules (or maybe even proteins) serve as substrates.
Ribozymes which are active intracellularly work in cis, catalyzing only a single turnover, and are usually self-modified during the reaction. However, ribozymes can be engineered to act in trans, in a truly catalytic manner, with a turnover greater than one and without being self-modified. Two distinct regions can be identified in a ribozyme: the binding region which gives the ribozyme its specificity through hybridization to a specific nucleic acid sequence (and possibly also to specific proteins), and a catalytic region which gives the ribozyme the activity of cleavage, ligation or splicing.
It has recently been proposed to use ribozymes in order to treat diseases or genetic disorders by cleaving a target RNA, such as viral RNA or messenger RNA transcribed from genes that should be turned off. This was proposed as an alternative to blockage of the RNA transcript by the use of antisense sequences. Owing to the catalytic nature of the ribozyme, a single ribozyme molecule cleaves many molecules of target RNA and therefore therapeutic activity is achieved in relatively lower concentrations than those required in an antisense treatment (WO 96/23569).
The use of ribozymes for diagnostic purposes has been only seldomly mentioned. WO 94/13833 describes a method for detecting nucleic acid molecules in a solution by tailoring a specific ribozyme molecule having two regions, one complementary to the nucleic acid sequence to be detected, and the other complementary to a co-target molecule bearing a detectable label. The ribozyme is able to specifically and reversibly bind both to a selected target nucleic acid sequence and to the labeled co-target. When both the target and the co-target are bound, the ribozyme undergoes a conformational change which renders it active and able to cleave the label of the co-target, and the free label can then be detected. Upon cleavage of the co-target, the ribozyme is able to re-associate with an additional co-target, cleaving more label and producing more detectable signals.
WO 94/13791 concerns a regulatable ribozyme molecule which upon binding to a ligand alters its activity on a target RNA sequence. Again, as in WO 94/13833, binding to the target causes a conformational change in the ribozymes which renders it active. An example for such a change, is in the presence in the ribozyme of a redundant sequence which masks the core region of the ribozyme. Only upon binding of the target sequence to said redundant sequence, the core becomes unmasked, and thus active.
U.S. Pat. No. 5,472,840 (Stefano, J. E.) discloses a nucleic acid sequence comprising the MDV-1 motif (capable of autocatalytic replication in the presence of the enzyme of the Q-beta replicase) which becomes active only when it forms a specific structure, comprising the sequence GAAA, with a target nucleic acid molecule. The ribozyme of Stefano is prepared by modifying an existing ribozyme and features only the activity of cleavage in the presence of a very restricted range of target molecules.
Lizardi, M. P. and Helena Porta (
Biotechnology
13:161-164 (1995)) disclose an allosteric hammerhead ribozyme that is a priori inactive, and is triggered by specific interaction with a DNA allosteric effector that is complementary to a single-stranded loop in the RNA stranded ribozyme. This publication discloses a ribozyme which becomes active when a part of which becomes double-stranded.
The above publications disclose ribozymes which become catalytically active, either due to a conformational change or due to a hybridization reaction which render them double-stranded. These types of reactions may also occur spontaneously, for example, if a ribozyme is inactive due to the presence of a redundant sequence which masks its core region, this redundant sequence may either break, or open, even in the absence of the target, and thus the ribozyme will become catalytically active even without a target. Spontaneous reversion to an active state, of course renders the ribozymes impractical for diagnostic purposes.
Israel Patent Applications 112799 and 115772 (corresponding to PCT/US96/02380) disclose methods for the detection of catalytically active ribozymes in a medium, wherein typically the catalytically active ribozyme is used as a reporter for the presence of other biomolecules in a test sample. In accordance with the methods disclosed in these applications, catalytically active ribozyme, if present in an assayed medium, yields a reaction cascade in which more catalytically active ribozymes are produced in a positive-feedback manner in one of various embodiments specified in these applications. The catalytically active ribozyme may be produced, or activated only in the presence of an assayed molecule.
GENERAL DESCRIPTION OF THE INVENTION
In the following, the term “nucleozyme” will be used to denote an oligonucleotide or a complex formed between an oligonucleotide and a nucleic acid sequence or between an oligonucleotide and another molecule e.g. an oligonucleotide, a protein or a polypeptide, etc., which possesses a catalytic activity. An example of a nucleozyme is a ribozyme.
The term “proto-nucleozyme” will be used to denote a nucleic acid molecule or a complex of two or more such molecules, which has a priori no catalytic activity but which becomes catalytically active upon formation of a complex with a co-factor. A proto-nucleozyme is in fact a nucleozyme with a missing component, which missing component is completed by the co-factor. The complex between the proto-nucleozyme and the co-factor may also at times be referred to as “catalytic complex” and is in fact a nucleozyme since it has catalytic activity. The proto-nucleozyme may consist of deoxynucleotides (dNTP's), ribo-nucleotides (rNTP's), as well as other nucleotides such as 2′-O-methyl nucleotides, or any combinations of these.
The term “catalytic activity” is meant to encompass all possible catalytic activities of nucleozymes, including cleavage, ligation, splicing-out (cleaving both ends of a short nucleic acid sequence to remove it from a longer sequence and ligating the ends of the cut), splicing-in (cleaving open a nucleic acid sequence, inserting another short nucleic acid sequence and ligating the ends of the cut), rearrangement, as well as additional catalytic activities such as phosphorylation, kinase like activity, addition or removal of other chemical moieties, biotinilation, gap filling of missing nucleotides, polymerization, etc.
The term “co-factor” will be used to denote a molecule or a moiety within a molecule (e.g. a certain DNA sequence within a larger DNA molecule), which complexes with the proto-nucleozyme to yield a catalytic complex, namely an active nucleozyme. The co-factor completes a missing portion of the proto-nucleozyme so that it can become catalytically active, and turn into a nucleozyme.
The present invention relates to novel proto-nucleozymes and their use. The proto-nucleozymes of the invention have substantially no catalytic activity, as they miss a critical component, which is essential for the catalytic activity, said missing component is completed by the co-factor. In other words, in order to become catalytically active, the proto-nucleozymes need to complex with the co-factor, so as to form a catalytic complex (the nucleozyme) which can exert a catalytic activity. The term “having substantially no catalytic activity” is meant to denote that the proto-nucleozyme possesses either no catalytic activity or possesses a catalytic activity which is very much lower (typically by several orders of magnitude

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