Stable triple helices formed by oligonucleotide N3'-->P5' phosphoramidates inhibit transcription elongation.

Oligonucleotide analogs with N3'-->P5' phosphoramidate linkages bind to the major groove of double-helical DNA at specific oligopurine.oligopyrimidine sequences. These triple-helical complexes are much more stable than those formed by oligonucleotides with natural phosphodiester linkages. Oligonucleotide phosphoramidates containing thymine and cytosine or thymine, cytosine, and guanine bind strongly to the polypurine tract of human immunodeficiency virus proviral DNA under physiological conditions. Site-specific cleavage by the Dra I restriction enzyme at the 5' end of the polypurine sequence was inhibited by triplex formation. A eukaryotic transcription assay was used to investigate the effect of oligophosphoramidate binding to the polypurine tract sequence on transcription of the type 1 human immunodeficiency virus nef gene under the control of a cytomegalovirus promoter. An efficient arrest of RNA polymerase II was observed at the specific triplex site at submicromolar concentrations.

Oligonucleotide N3'-->P5' phosphoramidates.

Synthetic oligonucleotides and their analogs have attracted considerable interest recently. These compounds may lead to highly specific therapeutic agents, as well as to powerful diagnostic tools. Here, we present the synthesis of uniformly modified oligodeoxyribonucleotide N3'-->P5' phosphoramidates containing 3'-NHP(O)(O-)O-5' internucleoside linkages and the study of their hybridization properties. Thermal dissociation experiments show that these compounds form very stable duplexes with single-stranded DNA, RNA, and with themselves following Watson-Crick base pairing. The duplex thermal stability was enhanced by 2.2-2.6 degrees C per modified linkage compared with phosphodiesters. The structure of complexes formed by phosphoramidates closely resembles that of RNA oligomers and corresponds to an A form, as judged by CD spectroscopy. N3'-->P5' phosphoramidates also form stable triplexes with double-stranded DNA under near-physiological conditions when natural phosphodiesters fail to do so. Physicochemical characteristics of the amidates are similar to those of RNA oligomers, even though they are composed of 2'-deoxyfuranose-based nucleosides.