Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1993-08-09
1997-09-30
LeGuyader, John L.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 231, 536 232, 435 6, 435 9131, 4351721, 514 44, C07H 2104, C12Q 168, A61K 4800
Patent
active
056726953
DESCRIPTION:
BRIEF SUMMARY
Certain naturally occuring ribonucleic acids (RNAs) are subject to self-cleavage. The first reported example is the cleavage of the ribosomal RNA precursor of the protozoan Tetrahymena (for a review see Cech, Ann.Rev. Biochem. 59 (1990), 543-568) which requires guanosine as cofactor. A number of examples of RNA cleavage have been subsequently discovered in viroid, virusoid and satellite RNAs (for reviews see Sheldon et al. in Nucleic Acids and Molecular Biology (1990) Vol. 4, pg. 227-242, ed. F. Eckstein and D. M. J. Lilley, Springer Verlag Berlin Heidelberg; Symons, TIBS 14 (1989), 445-450). These cleavages involve site-specific breakage of a phosphodiester bond in the presence of a divalent cation such as Mg.sup.2+, generating a 5'-hydroxyl and a 2',3'-cyclic phosphodiester terminus. Sequence analysis around the site of self-cleavage of several of such RNAs has led to the identification of a common structural feature essential for cleavage which was named a "hammerhead" structure (Hutchins et al., Nucleic Acids Res. 14 (1986) 3627-3640). This structure consists of three helices and 13 conserved nucleotides (framed in below scheme) which form a three dimensional structure amenable to cleavage at one particular position. The self-catalyzed cleavage is normally an intramolecular process, i.e. a single RNA molecule contains all the functions necessary for cleavage. However, Uhlenbeck (Nature 328 (1987), 596-600) has demonstrated that this hammerhead structure does not have to be embodied in one strand but can be made up of two strands. These two strands combine to form the hammerhead structure which leads to phosphodiester bond cleavage (indicated by an arrow) in one of the strands (strand S) whereas the other (strand E) remains unaltered and can participate in many cleavage reactions. This strand meets the definitions of an enzyme and is called a ribozyme. Whereas the framed sequences (below scheme) are conserved the others may vary provided that the structure of base paired and the single stranded regions remains intact. ##STR1##
The cleavage reaction after the trinucleotide GUC has been studied in detail (Ruffner et al., Gene 82 (1989), 31-41; Fedor and Uhlenbeck, Proc.Natl.Acad.Sci. USA 87 (1990), 1668-1672). Ribozymes with new specificities have also been constructed (Haseloff and Gerlach, Nature 334 (1988), 585-591) indicating that cleavage can for example also take place after the sequences GUA, GUU, CUC, AUC and UUC.
Further examples for RNA enzymes are the hairpin RNA (Hampel et al., Nucleic Acids Res. 18 (1990), 299-304), as well as RNA containing proteins such as the telomerase (Greider and Blackburn, Nature 337 (1989), 331-337) and the RNase P (Baer et al., in Nucleic Acids and Molecular Biology (1988), Vol. 3, pp. 231-250, ed. F. Eckstein and D. M. J. Lilley, Springer Verlag, Berlin/Heidelberg).
Ribozymes are potentially of interest for use as therapeutic agents (for review see Rossi and Sarver, TIBTECH 8 (1990), 179-183). A possible strategy would be to destroy an RNA necessary for the expression of both foreign genes such as vital genes and particular endogenous genes. This requires the construction of a RNA molecule which is able to form a hammerhead or a hairpin structure with the target RNA and to cleave this at a predetermined position. A first application to the inhibition of the HIV-1 virus by this strategy has been reported (Sarver et al., Science 247 (1990), 1222-1224). Other examples of the action of targeted hammerhead ribozymes in vivo are Cammeron and Jennings (Proc.Natl.Acad. Sci. USA 86 (1986), 9139-9143) and in vitro Cotten et al. (Mol.Cell.Biol. 9 (1989), 4479-4487).
Further, other useful catalytic properties of ribozymes are known, e.g. dephosphorylase and nucleotidyl transferase activities (see Patent Application W088/04300). Therein RNA enzymes are disclosed which are capable of dephosphorylating oligonucleotide substrates with high sequence specifity, which distinguishes them from known protein enzymes. RNA molecules also can act as RNA polymerases, differing from protein e
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Benseler Fritz
Eckstein Fritz
Heidenreich Olaf
Olsen David B.
Pieken Wolfgang
LeGuyader John L.
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.v.
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