Design of nuclease resistant protein kinase C alpha DNA enzymes with potential therapeutic application

For the therapeutic application of catalytic nucleic acids it is desirable to have small, stable and inexpensive compounds that are active at physiolo gical Mg2+ concentrations. We have explored the possibility of using the ve rsatile 10-23 DNA catalytic core to suppress the expression of the protein kinase C alpha (PKC alpha) isoform in malignant cells. By introducing eithe r a 3'-3'-inverted thymidine nucleotide or site-specific phosphorothioate m odification into a PKCa DNA enzyme, we have designed stable catalysts that retained a significant in vitro cleavage activity. Ln particular, a DNA enz yme containing phosphorothioate analogues in the antisense arms and in the pyrimidine residues of the catalytic core was found to be remarkably stable in 50 % human serum (t(1/2) > 90 hours) and inhibited in vitro cell growth by up to 90 % at nanomolar concentrations. The inhibition of PKC alpha gen e expression is sequence-specific, as a DNA enzyme with reversed antisense arms was found to be ineffective. Epifluorescence microscopic analysis of c ells transfected with a 5' fluorescein isothiocyanate-conjugated DNA enzyme showed that the DNA enzyme molecules are mainly localised in the nuclei. M ost of the DNA enzyme-treated cells were killed by apoptosis. The ability o f the described PKC alpha DNA enzymes to trigger apoptosis (apoptozymes) in malignant cells illustrates their therapeutic potential. Furthermore, such agents can be a valuable tool for probing gene function. (C) 2000 Academic Press.