Identification and Partial Purification of Human Double Strand RNase Activity A NOVEL TERMINATING MECHANISM FOR OLIGORIBONUCLEOTIDE ANTISENSE DRUGS

Abstract We have identified a double strand RNase (dsRNase) activity that can serve as a novel mechanism for chimeric antisense oligonucleotides comprised of 2′-methoxy 5′ and 3′ “wings” on either side of an oligoribonucleotide gap. Antisense molecules targeted to the point mutation in codon 12 of Harvey Ras (Ha-Ras) mRNA resulted in a dose-dependent reduction in Ha-Ras RNA. Reduction in Ha-Ras RNA was dependent on the oligoribonucleotide gap size with the minimum gap size being four nucleotides. An antisense oligonucleotide of the same composition, but containing four mismatches, was inactive. When chimeric antisense oligonucleotides were prehybridized with 17-mer oligoribonucleotides, extracts prepared 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 strand nucleases. The dsRNase activity resulted in cleavage products with 5′-phosphate and 3′-hydroxyl termini. Partial purification of dsRNase from rat liver cytosolic and nuclear fractions was effected. The cytosolic 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 of detergents and several organic solvents.