Oligonucleotide N3 ' -> P5 ' phosphoramidates as potential therapeutic agents

Uniformly modified nucleic acids analogues, oligonucleotide N3' --> P5' pho sphoramidates, containing 3'-amino instead of 3'-hydroxyl nucleosides, were synthesized and studied. These compounds form very stable duplexes with co mplementary native phosphodiester DNA and exceptionally stable duplexes wit h RNA strands. Increases in duplex melting temperature, Delta T-m, relative ly to their phosphodiester counterparts, reaches 2.9-3.5 degrees C per modi fied nucleoside. Moreover, the phosphoramidate compounds form extremely sta ble triple stranded complexes with single or double stranded DNA oligomers under near physiological salt and pH conditions. Melting temperatures of th ese triplexes usually exceed that of the isosequential phosphodiester count erparts by up to 35 degrees C. For 11-15-mers 2'-deoxyphosphoramidates are structurally and functionally similar to the native RNA molecules and thus can be used as RNA decoys. They are resistant to enzymatic digestion by nuc leases both in vitro and in vivo. Oligonucleotide phosphoramidates apparent ly are cell permeable, and they have a good bioavailability and biodistribu tion, while being non-toxic in mice at therapeutically relevant doses. Dupl exes of the several studied phosphoramidates with complementary RNA strands apparently are not substrates for RNase H in vitro. Despite that, these co mpounds exerted high sequence-specific antisense activity in various cell l ines and in SCID mice. The observed in vitro lack of RNase H recognition of the RNA:phosphoramidate duplexes may result in better specificity in biolo gical activity of these compounds relative to RNase Fl inducing oligonucleo tides. Experimental results also indicate that oligonucleotide phosphoramid ates can be used as efficient and specific modulators of gene expression by an antigene mechanism of action. Finally, the oligo-2'-deoxyphosphoramidat e double stranded complexes can structurally mimic native RNA complexes, wh ich could be efficiently and specifically recognized by the RNA binding pro teins, such as HIV-1 Rev and Tat. (C) 1999 Elsevier Science B.V. All rights reserved.