RNase H-independent antisense activity of oligonucleotide N3'->P5' phosphoramidates

Abstract

Oligonucleotide N3'-->P5'phosphoramidates are a new and promising class of antisense agents. Here we report biological properties of phosphoramidate oligonucleotides targeted against the human T cell leukemia virus type-I Tax protein, the major transcriptional transactivator of this human retrovirus. Isosequential phosphorothioate oligodeoxynucleotides and uniformly modified and chimeric phosphoramidate oligodeoxynucleotides containing six central phosphodiester linkages are all quite stable in cell nuclei. The uniformly modified anti-tax phosphoramidate oligodeoxynucleotide does not activate nuclear RNase H, as was shown by RNase protection assay. In contrast, the chimeric phosphoramidate-phosphodiester oligodeoxynucleotide is an efficient activator of RNase H. The presence of one or two mismatched nucleotides in the phosphodiester portion of oligonucleotides affected this activation only negligibly. When introduced into tax-transformed fibroblasts ex vivo, only the uniformly modified anti-tax phosphoramidate oligodeoxynucleotide caused a sequence-dependent reduction in the Tax protein level. Neither the chimeric phosphoramidate nor the phosphorothioate oligodeoxynucleotides significantly reduced tax expression under similar experimental conditions.

Oligonucleotide N3′→P5′ Phosphoramidates and Thio‐Phoshoramidates as Potential Therapeutic Agents

“…Treatment of BV 173 cells, as well as primary bone marrow cells taken from patients with CML in chronic phase, with this phosphoramidate was associated with a significant reduction of targeted bcr-abl mRNA level, as determined using RT PCR analysis. The mRNA level reduction was observed despite apparent inability of this oligonucleotide phosphoramidate to induce RNase H-mediated cleavage of the heteroduplexes formed by the phosphoramidates with complementary RNA strands in vitro [31] [32]. A similar decrease of c-myb mRNA level was also observed as the result of treatment of HL-60 cells with antisense phosphoramidate 15-mer GTGCCGGGGTCTTCG oligonucleotide, but not with mismatched GTCCTGGGGTCGTCG control compound.…”

Modulation of gene expression by antisense and antigene oligodeoxynucleotides and small interfering RNA

“…It has a very high binding affinity to complementary DNA or RNA with nuclease resistance. Unlike PS ODNs, N3′→P5′ modified ODNs do not activate RNase H when bound to complementary RNA [48]. Despite inability to activate RNase H, N3′→P5′ phosphoramidate ODNs have shown more potent sequence-specific antisense activity than phosphorothioate derivatives in vitro [48] and in vivo [49].…”

“…Unlike PS ODNs, N3′→P5′ modified ODNs do not activate RNase H when bound to complementary RNA [48]. Despite inability to activate RNase H, N3′→P5′ phosphoramidate ODNs have shown more potent sequence-specific antisense activity than phosphorothioate derivatives in vitro [48] and in vivo [49]. Skorski et al (1997) [49] treated severe combined immunodeficient mice with equal doses of phosphoramidate ODNs, PS ODNs and mismatched ODNs.…”

Use of Antisense Vectors and Oligodeoxynucleotides in Neuro-Oncology

“…The more specific part of the mRNA targeted is often at the 5) end of the transcript, spanning the translation initiation codon [31,37]. mRNA inactivation occurs either through steric blocking of the ribosome complex, or by triggering mRNA cleavage by RNase H [6,13,33,41,[48][49][50][51]. RNase H sensitivity is dependent upon the backbone modification [41,51].…”

“…mRNA inactivation occurs either through steric blocking of the ribosome complex, or by triggering mRNA cleavage by RNase H [6,13,33,41,[48][49][50][51]. RNase H sensitivity is dependent upon the backbone modification [41,51]. Antisense ODNs can also interfere with gene expression by triple-helix formation, in which the ODN binds doublestranded DNA in the nucleus [13,17,40,[52][53][54][55].…”