RNA hydrolysis

Details RNA hydrolysis RNA hydrolysis

1992-09-16

2001-07-10

Marx, Irene (Department: 1651)

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

Organic compounds

Carbohydrates or derivatives

C546S082000, C546S259000, C546S257000, C536S025300

Reexamination Certificate

active

06258941

ABSTRACT:

FIELD OF INVENTION
This invention relates to sequence-directed RNA hydrolysis under physiologically relevant conditions and particularly to metal complexes covalently linked to oligodeoxynucleotides as sequence-directed RNA hydrolysis agents.
BACKGROUND OF THE INVENTION
Recently hydrolysis of phosphate esters has received extensive investigation as reported in the art due to the relevance of this chemistry to biological systems, and specifically transition metal complexes have been examined as phosphate ester hydrolysis catalysts in order to model the reactions catalyzed by the ATPase and phosphatase classes of enzymes. Such reported studies have generally employed activated p-nitrophenyl phosphate esters or phosphate anhydrides (ATP) as substrates (R. D. Cornelius,
Inorg. Chem
. 1980, 19, 1286-1290; P. R. Norman et al,
J. Am. Chem. Soc
. 1982, 104, 2356-2361 and F. Tafesse et al,
Inorg. Chem
. 1985, 24, 2593-2594). It has been reported that tetramine complexes of Co(III) are capable of promoting the hydrolysis of adenosine 3′,5′-monophosphate (cAMP) (J. Chin et al,
Can. J. Chem
. 1987, 65, 1882-1884) and adenosine monophosphate (AMP) (J. Chin et al,
J. Am. Chem. Soc
. 1989, 111, 4103-4105). Also, it is known that many divalent cations are capable of catalyzing the hydrolysis of RNA (J. J. Butzow et al,
Biochemistry
1971, 10, 2016-2027 and J. J. Butzow et al,
Nature
1975, 254, 358-359). Additionally, zinc ion in the presence of imidazole buffers has been shown to catalyze the hydrolysis of the RNA dimer 3′,5′-UpU at 80° C. (R. Breslow et al,
Proc. Natl. Acad. Sci
. 1989, 86, 1746-1750).
C. A. Stein et al,
Cancer Research
May, 1988, 48, 2659-2668 gives a detailed review on the application of antisense oligodeoxynucleotides as modulators of gene expression and concludes by proposing a more subtle and effective approach would be to attach a chemical group to the oligomer that can result in localized catalytic hydrolysis of RNA. This technique would be more specific than the use of a radical-producing group such as iron EDTA. Stein et al theorizes that a suitable RNA hydrolysis group would be an imidazole group, which is known to be involved in phosphodiester hydrolysis in the active site of ribonuclease enzymes.
University Patents, Inc. in PCT International Patent Application published under number WO 88/04300 on Jun. 16, 1988 discloses RNA enzymes or ribozymes, acting as endoribonucleases, as catalyzing the cleavage of RNA molecules with a sequence specificity of cleavage greater than that of known ribonucleases and approaching that of the DNA restriction endonucleases, thus serving as RNA sequence-specific endoribonucleases. Ribozymes are entirely or partly comprised of RNA itself, and therefore are chemically and enzymatically highly unstable relative to Applicants' DNA-based compounds. Such instability detracts from the practical applicability of RNA hydrolysis agents.
C. B. Chen et al,
J. Am. Chem. Soc
. 1988, 110, 6570-6572 describes that 1,10-phenanthroline-copper(II) is effective for targeted cleavage of both RNA and DNA and thus is useful for sequence-specific cleavage of RNA. This teaching is directed to oxidative cleavage of RNA by metal complexes linked to DNA at a temperature of 65° C. as opposed to the hydrolytic cleavage of RNA under physiologically relevant conditions required by Applicants' invention. The ancillary reagents, in the quantities required to drive the Chen et al oxidative degradation of RNA, are not compatible with living cells; furthermore, the 1,10-phenanthrolinecopper-oligodeoxynucleotide conjugate employed is itself degraded oxidatively under the conditions of oxidative RNA cleavage (the rate of oxidative cleavage by the 1,10-phenanthrolinecopper system is similar for both RNA and DNA).
P. G. Schultz and coworkers in a series of articles (D. R. Corey et al,
J. Am. Chem. Soc
. 1988, 110, 1614-1615; R. Zuckerman et al,
J. Am. Chem. Soc
. 1988, 110, 6592-6594 and R. Zuckerman et al,
Proc. Natl. Acad. Sci. USA
1989, 86, 1766-1770) have described the preparation of site-selective DNA and RNA hydrolysis agents comprised of an enzyme (staphylococcal nuclease, ribonuclease S, or mutants of these parent enzymes) covalently linked to oligodeoxynucleotides. In one report (D. R. Corey et al,
Biochemistry
1989, 28, 8277-8286), the location of the linker arm and its length were varied, which resulted in changes in catalytic efficiency and site of cleavage.
Considerable art has been developed on cleavage of RNA utilizing enzymes and ribozymes. At present the art is void of a teaching using metal complexes which cleave RNA hydrolytically at a physiologically relevant pH and temperature as synthetic analogs for enzymes or ribozymes to obtain sequence-directed hydrolysis of RNA. Sequence-directed RNA hydrolysis is highly desirable today in order to prepare catalytic antisense oligodeoxynucleotides useful as a means for inhibiting the expression of specific genes. Such RNA hydrolysis is necessary to provide a basis for catalytic antisense drug development.
STATEMENT OF THE INVENTION
This invention is directed to the hydrolytic cleavage of RNA at physiologically relevant conditions. The underlying basis of this invention is the use of metal complexes which perform as synthetic analogs for enzymes or ribozymes in the hydrolysis of RNA. Conjugate as used herein means a compound comprised of a metal complex covalently linked to a nucleoside or nucleotide. Oligodeoxynucleotide conjugate as used herein means a compound comprised of a metal complex covalently linked to an oligodeoxynucleotide.
A first aspect of this invention is directed to the discovery of metal complexes useful for promoting RNA hydrolysis. A second aspect of this invention is directed to a conjugate which is active for RNA hydrolysis comprised of a metal complex covalently linked to a nucleoside or nucleotide. A third aspect of this invention is directed to the sequence-directed hydrolytic cleavage of RNA by a metal complex covalently linked to an oligodeoxynucleotide. The oligodeoxynucleotide provides molecular recognition via Watson Crick base pairing to the target RNA sequence.
Accordingly, a major object of this invention is to provide for the hydrolysis of RNA at physiologically relevant conditions. Other objects of this invention include (1) the discovery of metal complexes which are effective for the hydrolysis of RNA, (2) the preparation of conjugates which retain RNA cleavage behavior, (3) the preparation of oligodeoxynucleotide conjugates effective for the sequence-directed hydrolysis of RNA under physiologically relevant conditions. Other objects and advantages of this invention will become apparent upon further study of this disclosure and the appended claims.


REFERENCES:
patent: 4267335 (1981-05-01), McGill
patent: 4711955 (1987-12-01), Ward
patent: 4795700 (1989-01-01), Dervan et al.
patent: 4837312 (1989-06-01), Dervan et al.
patent: 4859777 (1989-08-01), Toner
patent: 4873333 (1989-10-01), Stapersma
patent: 4933455 (1990-06-01), Stapersma
patent: WO 88/04300 (1988-06-01), None
patent: WO 89/05853 (1989-06-01), None
patent: 0003381 (1990-04-01), None
Cornelius, R. D., Inorg. Chem., 1980, 19, pp. 1286-1290.
Norman, P. R. et al, J. Am. Chem. Soc., 1982, 104, pp. 2356-2361.
Tafesse, F., Inorg. Chem., 1985, 24, 2593-2594.
Chin, J. et al, Can. J. Chem., 1987, 65, 1882-1884.
Chin, J. et al, J. Am. Chem. Soc., 1989, 111, pp. 4103-4105.
Eichhorn, G., et al, Biochemistry, 1971, 10, pp. 2014-2017.
Butzow, J. J., et al, Nature, 1975, 254, pp. 358-359.
Breslow, R., et al, Proc. Natl. Acad. Sci., 1989, 86, pp. 1746-1750.
Stein, C. A., et al, Cancer Research, May, 1988, 48, pp. 2659-2668.
Chen, C. B., et al, J. Am. Chem. Soc., 1988, 110, pp. 6570-6572.
Zuckerman, R. N., et al, J. Am. Chem. Soc., 1988, 110, pp. 6592-6594.
Zuckerman, R. N., et al, Proc. Natl. Acad. Sci.USA, 1989, 86, pp. 1766-1770.
Corey, D. R., et al, Biochemistry, 1989, 28, pp. 8277-8286.
Zuckerman, R. N., et al, J. Am. Chem. Soc., 1988, 110, 1614-1615.
Chen et al. Proc. Natl. Ac

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