Human RNase H and compositions and uses thereof

Details Human RNase H and compositions and uses thereof Human RNase H and compositions and uses thereof

2000-10-06

2002-04-23

Wang, Andrew (Department: 1635)

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

Organic compounds

Carbohydrates or derivatives

C536S023100

Reexamination Certificate

active

06376661

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a human Type 2 RNase H which has now been cloned, expressed and purified to electrophoretic homogeneity and human RNase H and compositions and uses thereof.
BACKGROUND OF THE INVENTION
RNase H hydrolyzes RNA in RNA-DNA hybrids. This enzyme was first identified in calf thymus but has subsequently been described in a variety of organisms (Stein, H. and Hausen, P.,
Science,
1969, 166, 393-395; Hausen, P. and Stein, H.,
Eur. J. Biochem.,
1970, 14, 278-283). RNase H activity appears to be ubiquitous in eukaryotes and bacteria (Itaya, M. and Kondo K.
Nucleic Acids Res.,
1991, 19, 4443-4449; Itaya et al.,
Mol. Gen. Genet.,
1991 227, 438-445; Kanaya, S., and Itaya, M.,
J. Biol. Chem.,
1992, 267, 10184-10192; Busen, W.,
J. Biol. Chem.,
1980, 255, 9434-9443; Rong, Y. W. and Carl, P. L., 1990,
Biochemistry
29, 383-389; Eder et al.,
Biochimie,
1993 75, 123-126). Although RNase Hs constitute a family of proteins of varying molecular weight, nucleolytic activity and substrate requirements appear to be similar for the various isotypes. For example, all RNase Hs studied to date function as endonucleases, exhibiting limited sequence specificity and requiring divalent cations (e.g., Mg
2+
, Mn
2+
) to produce cleavage products with 5′ phosphate and 3′ hydroxyl termini (Crouch, R. J., and Dirksen, M. L.,
Nuclease,
Linn, S, M., & Roberts, R. J., Eds., Cold Spring Harbor Laboratory Press, Plainview, N.Y. 1982, 211-241).
In addition to playing a natural role in DNA replication, RNase H has also been shown to be capable of cleaving the RNA component of certain oligonucleotide-RNA duplexes. While many mechanisms have been proposed for oligonucleotide mediated destabilization of target RNAs, the primary mechanism by which antisense oligonucleotides are believed to cause a reduction in target RNA levels is through this RNase H action. Monia et al.,
J. Biol. Chem.,
1993, 266:13, 14514-14522. In vitro assays have demonstrated that oligonucleotides that are not substrates for RNase H can inhibit protein translation (Blake et al.,
Biochemistry,
1985, 24, 6139-4145) and that oligonucleotides inhibit protein translation in rabbit reticulocyte extracts that exhibit low RNase H activity. However, more efficient inhibition was found in systems that supported RNase H activity (Walder, R. Y. and Walder, J. A.,
Proc. Nat'l Acad. Sci. USA,
1988, 85, 5011-5015; Gagnor et al.,
Nucleic Acid Res.,
1987, 15, 10419-10436; Cazenave et al.,
Nucleic Acid Res.,
1989, 17, 4255-4273; and Dash et al.,
Proc. Nat'l Acad. Sci. USA,
1987, 84, 7896-7900.
Oligonucleotides commonly described as “antisense oligonucleotides” comprise nucleotide sequences sufficient in identity and number to effect specific hybridization with a particular nucleic acid. This nucleic acid or the protein(s) it encodes is generally referred to as the “target.” Oligonucleotides are generally designed to bind either directly to mRNA transcribed from, or to a selected DNA portion of, a preselected gene target, thereby modulating the amount of protein translated from the mRNA or the amount of mRNA transcribed from the gene, respectively. Antisense oligonucleotides may be used as research tools, diagnostic aids, and therapeutic agents.
“Targeting” an oligonucleotide to the associated nucleic acid, in the context of this invention, also refers to a multistep process which usually begins with the identification of the nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a foreign nucleic acid from an infectious agent. The targeting process also includes determination of a site or sites within this gene for the oligonucleotide interaction to occur such that the desired effect, either detection or modulation of expression of the protein, will result.
RNase HI from
E.coli
is the best-characterized member of the RNase H family. The 3-dimensional structure of
E.coli
RNase HI has been determined by x-ray crystallography, and the key amino acids involved in binding and catalysis have been identified by site-directed mutagenesis (Nakamura et al.,
Proc. Natl. Acad. Sci. USA,
1991, 88, 11535-11539; Katayanagi et al.,
Nature,
1990, 347, 306-309; Yang et al.,
Science,
1990, 249, 1398-1405; Kanaya et al.,
J. Biol. Chem.,
1991, 266, 11621-11627). The enzyme has two distinct structural domains. The major domain consists of four &agr; helices and one large &bgr; sheet composed of three antiparallel &bgr; strands. The Mg
2+
binding site is located on the &bgr; sheet and consists of three amino acids, Asp-10, Glu-48, and Gly-11 (Katayanagi et al.,
Proteins: Struct., Funct., Genet.,
1993, 17, 337-346). This structural motif of the Mg
2+
binding site surrounded by &bgr; strands is similar to that in DNase I (Suck, D., and Oefner, C.,
Nature,
1986, 321, 620-625). The minor domain is believed to constitute the predominant binding region of the enzyme and is composed of an a helix terminating with a loop. The loop region is composed of a cluster of positively charged amino acids that are believed to bind electrostatistically to the minor groove of the DNA/RNA heteroduplex substrate. Although the conformation of the RNA/DNA substrate can vary, from A-form to B-form depending on the sequence composition, in general RNA/DNA heteroduplexes adopt an A-like geometry (Pardi et al.,
Biochemistry,
1981, 20, 3986-3996; Hall, K. B., and Mclaughlin, L. W.,
Biochemistry,
1991, 30, 10606-10613; Lane et al.,
Eur. J. Biochem.,
1993, 215, 297-306). The entire binding interaction appears to comprise a single helical turn of the substrate duplex. Recently the binding characteristics, substrate requirements, cleavage products and effects of various chemical modifications of the substrates on the kinetic characteristics of
E.coli
RNase HI have been studied in more detail (Crooke, S. T. et al.,
Biochem. J.,
1995, 312, 599-608; Lima, W. F. and Crooke, S. T.,
Biochemistry,
1997, 36, 390-398; Lima, W. F. et al.,
J. Biol. Chem.,
1997, 272, 18191-18199; Tidd, D. M. and Worenius, H. M.,
Br. J. Cancer,
1989, 60, 343; Tidd, D. M. et al.,
Anti
-
Cancer Drug Des.,
1988, 3, 117.
In addition to RNase HI, a second
E.coli
RNase H, RNase HII has been cloned and characterized (Itaya, M.,
Proc. Natl. Acad. Sci. USA,
1990, 87, 8587-8591). It is comprised of 213 amino acids while RNase HI is 155 amino acids long.
E. coli
RNase HIM displays only 17% homology with
E.coli
RNase HI. An RNase H cloned from
S. typhimurium
differed from
E.coli
RNase HI in only 11 positions and was 155 amino acids in length (Itaya, M. and Kondo K.,
Nucleic Acids Res.,
1991, 19, 4443-4449; Itaya et al.,
Mol. Gen. Genet.,
1991, 227, 438-445). An enzyme cloned from
S. cerevisae
was 30% homologous to
E.coli
RNase HI (Itaya, M. and Kondo K.,
Nucleic Acids Res.,
1991, 19, 4443-4449; Itaya et al.,
Mol. Gen. Genet.,
1991, 227, 438-445). Thus, to date, no enzyme cloned from a species other than
E. coli
has displayed substantial homology to
E.coli
RNase H II.
Proteins that display RNase H activity have also been cloned and purified from a number of viruses, other bacteria and yeast (Wintersberger,
U. Pharmac. Ther.,
1990, 48, 259-280). In many cases, proteins with RNase H activity appear to be fusion proteins in which RNase H is fused to the amino or carboxy end of another enzyme, often a DNA or RNA polymerase. The RNase H domain has been consistently found to be highly homologous to
E.coli
RNase HI, but because the other domains vary substantially, the molecular weights and other characteristics of the fusion proteins vary widely.
In higher eukaryotes two classes of RNase H have been defined based on differences in molecular weight, effects of divalent cations, sensitivity to sulfhydryl agents and immunological cross-reactivity (Busen et al.,
Eur. J. Biochem.,
1977, 74, 203-208). RNase H Type 1 enzymes are re

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