IDENTIFICATION AND PARTIAL-PURIFICATION OF HUMAN DOUBLE-STRAND RNASE ACTIVITY - A NOVEL TERMINATING MECHANISM FOR OLIGORIBONUCLEOTIDE ANTISENSE DRUGS

We have identified a double strand RNase (dsRNase) activity that can s erve as a novel mechanism for chimeric antisense oligonucleotides comp rised of 2'-methoxy 5' and 3' ''wings'' on either side of an oligoribo nucleotide gap. Antisense molecules targeted to the point mutation in codon 12 of Harvey Ras (Ha-Ras) mRNA resulted in a dose-dependent redu ction in Ha-Ras RNA, Reduction in Ha-Ras RNA was dependent on the olig oribonucleotide gap size with the minimum gap size being four nucleoti des, An antisense oligonucleotide of the same composition, but contain ing four mismatches, was inactive. When chimeric antisense oligonucleo tides were prehybridized with 17-mer oligoribonucleotides, extracts pr epared from T24 cells, cytosol, and nuclei resulted in cleavage in the oligoribonucleotide gap, Both strands were cleaved. Neither mammalian nor Escherichia coli RNase HI cleaved the duplex, nor did single stra nd nucleases. The dsRNase activity resulted in cleavage products with 5'-phosphate and 8'-hydroxyl termini. Partial purification of dsRNase from rat liver cytosolic and nuclear fractions was effected, The cytos olic enzyme was purified approximately 165-fold. It has an approximate molecular weight of 50,000-65,000, a pH optimum of approximately 7.0, requires divalent cations, and is inactivated by approximately 300 mM NaCl. It is inactivated by heat, proteinase K, and also by a number o f detergents and several organic solvents.