Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Patent
1997-05-05
2000-08-22
Degen, Nancy
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
43525233, 4353201, 435325, 435419, 536 231, 536 245, 536 251, C07H 2100, C07H 2102, C12N 121, C12N 1564
Patent
active
061070785
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The discovery that RNA molecules can function as enzymes revolutionized the understanding of chemistry in biological systems. It has now been demonstrated that RNA molecules can catalyze chemical transformations on themselves as well as on other RNA molecules [Castanotto, 1992]. The term ribozyme has been given to these RNA molecules. It has now become apparent that ribozymes play an important role in the biochemical function of many organisms.
Several types of ribozymes have been identified in living organisms. The first ribozyme to show catalytic turnover was RNA of ribonucleases P. Ribonucleases P (RNase P) cleaves precursor tRNAs (pre-tRNAs) at their 5' ends to give the mature 5'-termini of tRNAs. In Escherichia coli and Bacillus subtilis, the RNase P holoenzyme is composed of one basic protein subunit of approximate M.sub.r 14,000 (119 amino acids) and one single stranded RNA molecule of 377 and 401 nucleotides, respectively [Baer, 1990; Altman 1987; Waugh, 1989; Pace, 1990; Nichols, 1988]. The second ribozyme to show turnover was the L-19 intervening sequence (IVS) from tetrahymena. The 413 nucleotide intervening sequence (IVS) in the nuclear rRNA precursor from Tetrahymena thermophila can be excised and the two exons ligated in the complete absence of any protein [Kruger, 1982; Cech, 1981]. Unique to the Tetrahymena thermophila self-splicing reaction is the requirement of a guanosine or 5' guanosine nucleotide cofactor. The hammerhead self-cleavage reactions constitutes a third class of ribozymes. A number of plant pathogenic RNAs [Symons, 1989; Symons, 1990; Bruening, 1989; Bruening 1990], one animal viral RNA [Taylor, 1990] and a transcript from satellite II of DNA of the newt [Epstein, 1987; Epstein 1989] and from a Neurospora DNA plasmid [Saville, 1990] undergo a site specific self-cleavage reaction in vitro to produce cleavage fragments with a 2',3'-cyclic phosphate and a 5'-hydroxyl group. This self-cleavage reaction is non-hydrolytic, unlike RNases P RNA cleavage of pre-tRNAs, where the internucleotide bond undergoes a phosphoryl transfer reaction in the presence of Mg.sup.++ or other divalent cations. Metal cations may be essential to RNA catalysis [Pyle, 1993]. Other reactions documented to date show that ribozymes can catalyze the cleavage of DNA [Robertson, 1990; Herschlag 1990], the replication of RNA strands [Green, 1992], the opening of 2'-3'-cyclic phosphate rings [Pan, 1992], as well as react with phosphate monoesters [Zaug, 1986] and carbon centers [Noller, 1992; Piccirilli, 1992]. Finally, ribozymes with new kinds of catalytic reactivity are being created through techniques of in vitro selection and evolution [Joyce, 1992].
The ability to specifically target and cleave a designated RNA sequence has led to much interest in the potential application of hammerhead ribozymes as gene therapy agents or drugs. One component to the success of treating a disease or targeting a specific RNA or DNA strand (substrate) to be cleaved is the optimization of the ribozyme/substrate complex. Optimization may be performed in vitro whereby a ribozyme is modified until it achieves maximum chemical activity on a target RNA or DNA molecule. While much success has been achieved in vitro in targeting and cleaving a number of designated RNA sequences (Saxena and Ackerman, 1990; Lamb and Hayes, 1991; Evans, et al., 1992; Mazzolini, et al., 1992; Homann, et al., 1993), the translation of this success into ribozyme action in the whole cell has been limited. This is particularly the case for plant systems, with only one example of ribozyme induced cleavage presently reported (Steinecke, et al., 1992). Thus, it is not entirely clear how the success in vitro would correlate to the success in vivo.
Previous reports have demonstrated that high levels of ribozyme expression are required to achieve reduced accumulation of target sequence in vivo [Cameron and Jennings, 1989; Cotten and Birnsteil, 1989; Sioud and Drilca, 1991; L'Huillier, et al., 1992; Perriman et al., 1993]. Additionally,
REFERENCES:
patent: 5254678 (1993-10-01), Haseloff et al.
Hampel et al. Hairpin catalytic RNA model: Evidence for helices and sequence requirement for substrate RNA. Nucleic Acids Res. 18(2): 299-304, Feb. 1990.
Cotten et al., (1989) "Ribozyme mediated destruction of RNA in vivo." The EMBO Journal, vol. 8, No. 12, pp. 3861-3866.
Haseloff et al., (1988) "Simple RNA enzymes with new and highly specific endoribonuclease activities." Nature, vol. 334, pp. 585-591.
Keese Paul
Perriman Rhonda
Stapper Marianne
Degen Nancy
Gene Shears Pty Limited
Larson Thomas G.
White John P.
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