The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2'-fluoro, 2'-O-methyl, 2'-O-propyl and 2'-O-pentyl nucleotides. Duplexes formed between 2' modified oligoribonucleotides and RNA have typical A-form geometry as observed by circular dichroism spectroscopy. Modifications, with the exception of 2'-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2'-fluoro:2'fluoro > 2'-O-propyl:2'-O-propyl > 2'-O-methyl:2'-O- methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2'-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2' modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoribonucleotides and not for 2'-deoxy oligoribonucleotides.

The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-␣ expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2 modifications have been incorporated into the protein kinase C-␣ targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5°C per modification) and an increase in resistance toward both 3-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-␣ in mediating the phorbol ester-induced changes in c-fos, c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-␣ protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos or junB. These results demonstrate that 2-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-␣ expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides.

Metabolism of 2'-deoxyphosphorothioate oligonucleotides ISIS 11061 and ISIS 11637 was examined with capillary gel electrophoresis (CGE) and on-line HPLC electrospray mass spectrometry (HPLC/ES-MS). Oligonucleotides were isolated from plasma, liver, and kidneys of rats injected with ISIS 11061 and ISIS 11637. Metabolites found in plasma were consistent with 3'-exonuclease activity. Metabolites isolated from liver and kidney were consistent with 3'- and/or 5'-exonuclease activity. HPLC/ES-MS analysis of ISIS 11061 isolated from kidney indicated extensive degradation from the 3' terminus, but metabolites consistent with 5' degradation and combinations of 3' and 5' truncations also were observed. ISIS 11061 isolated from liver showed less extensive degradation. The 5' truncated metabolites represented the predominant species in contrast to the kidney sample. Metabolites with masses consistent with combinations of 3' and 5' truncations were also observed in liver. The metabolic profiles generated by CGE analysis of these samples agreed qualitatively with mass spectrometric results. HPLC/ES-MS enabled the simultaneous determination of degradation products that are the same length but differ in composition. CGE could discriminate species that differed by one nucleotide in length. HPLC/ES-MS was shown to be a useful tool to study the complex metabolism of antisense oligonucleotides in vivo.

We have previously described the characterization of a 20mer phosphorothioate oligodeoxynucleotide (ISIS 4189) which inhibits murine protein kinase C-alpha (PKC-alpha) gene expression, both in vitro and in vivo. In an effort to increase the antisense activity of this oligonucleotide, 2'-O-propyl modifications have been incorporated into the 5'- and 3'-ends of the oligonucleotide, with the eight central bases left as phosphorothioate oligodeoxynucleotides. Hybridization analysis demonstrated that these modifications increased affinity by approximately 8 and 6 degrees C per oligonucleotide for the phosphodiester (ISIS 7815) and phosphorothioate (ISIS 7817) respectively when hybridized to an RNA complement. In addition, 2'-O-propyl incorporation greatly enhanced the nuclease resistance of the oligonucleotides to snake venom phosphodiesterase or intracellular nucleases in vivo. The increase in affinity and nuclease stability of ISIS 7817 resulted in a 5-fold increase in the ability of the oligonucleotide to inhibit PKC-alpha gene expression in murine C127 cells, as compared with the parent phosphorothioate oligodeoxynucleotide. Thus an RNase H-dependent phosphorothioate oligodeoxynucleotide can be modified as a 2'-O-propyl 'chimeric' oligonucleotide to provide a significant increase in antisense activity in cell culture.

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