Asymmetric hammerhead ribozymes

Details Asymmetric hammerhead ribozymes Asymmetric hammerhead ribozymes

1998-09-18

2001-05-29

Schwartzman, Robert A. (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S006120, C435S091310, C435S375000, C536S024300, C536S024310, C536S024320, C536S024500

Reexamination Certificate

active

06238917

ABSTRACT:

Hammerhead ribozymes were discovered as the self-cleaving motifs in a number of small circular plant pathogenic RNAs (Buzayan et al. 1986; Hutchins et al. 1986; Symonds, 1992). Uhlenbeck (Uhlenbeck, 1987) showed that a ribozyme was able to act in a bimolecular fashion, and as a true enzyme on a very specific substrate with defined secondary structure and stringent sequence requirements. Haseloff and Gerlach (Haseloff, et al. 1988) described the division of the hammerhead into a form in which the conserved nucleotides were located on the enzyme strand, the only sequence requirements for the substrate being GUC, just 5′ to the cleavage site, this sequence requirement was shown to be too restrictive and only the sequence UH just 5′ of the cleavage site was required for activity (Perriman, et al. 1992; Shimayama, et al.1995; Zoumadakis, et al. 1995) (H═A, U or C). Since 1988 this configuration has been the paradigm for the design of hammerhead ribozymes. The cleavage mechanism for the hammerhead ribozyme is usually analyzed according to the conventional enzyme mechanism, i.e. reversible substrate binding, the cleavage step, reversible product dissociation and the product is not consumed in the reaction (Hertel, et al. 1994). The cleavage step has been assumed to be essentially independent of the length and sequence of the helices I and III (Fedor, et al. 1992). The inventors (Hendry, et al. 1995) have made a number of observations which suggest that the cleavage rate constant may be dependent of the length and/or sequence of the helices I and III.
There is a great need for ribozymes that have high catalytic rates. The need for highly effective ribozymes is particularly important for ribozyme compounds with modified oligonucleotides because the cost of the starting materials. While prior workers have prepared hammerhead ribozymes varying the length of both the 5′ and 3′ hybridizing arms, helixes I and III, respectively, (Denman et al. 1995; Ellis, et al. 1993; Fedor et al. 1990; Gast et al. 1994; Homann et al. 1994; Zoumadakis, et al. 1995) this is the first disclosure of specific asymmetric hammerhead compounds showing dramatic rate increases.
SUMMARY OF THE INVENTION
This invention is directed to a compound having the formula:
as defined in the detailed description. The compound may be covalently linked to a delivery agent. The invention also includes a composition which comprises the compound in association with an acceptable carrier. The invention also includes a method of cleaving an RNA target sequence which comprises contacting a target sequence with the compound as described above. Further, a method of treating a disease in man or animals associated with a particular RNA which comprises administrating to the man or animal the compound. Further, the invention also includes a diagnostic reagent which comprises the compound.


REFERENCES:
patent: 5149796 (1992-09-01), Rossi et al.
patent: 5254678 (1993-10-01), Haseloff et al.
patent: 5334711 (1994-08-01), Sproat
patent: 93/23057 (1993-11-01), None
patent: 93/23569 (1993-11-01), None
Denman et al. “Different activity of trans-acting hammerhead ribozymes targeted to b-amyloid peptide precursor mRNA by altering the symmetry of helicies I and III” Arch. Biochem. Biophys. 323 (1): 71-78, Oct. 20, 1995.

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