THE HIGH-STABILITY OF THE TRIPLE HELICES FORMED BETWEEN SHORT PURINE OLIGONUCLEOTIDES AND SIV HIV-2 VPX GENES IS DETERMINED BY THE TARGETEDDNA-STRUCTURE/

In our previous works we have shown that the oligonucleotides 5'-GGGGA GGGGGAGG-3' and 5'-GGAGGGGGAGGGG-3', give very stable and specific tri plexes with their target double stranded DNAs [Svinarchuk, F., Bertran d, J.-R. and Malvy, C. (1994) Nucleic Acids Res., 22, 3742-3747; Svina rchuk, F., Paoletti, J. and Malvy, C. (1995) J. Biol. Chem., 270, 14 0 68-14 071]. The target for the invariable part of these oligonucleotid es, 5'-GGAGGGGGAGG-3', is found in a highly conserved 20 bp long purin e/pyrimidine tract of the vpx gene of the SIV and HIV-2 viruses and co uld be a target for oligonucleotide directed antivirus therapy. Here w e report on the ability of four purine oligonucleotides with different lengths (11-, 14-, 17- and 20-mer) to form triplexes with the purine/ pyrimidine stretch of the vpx gene. Tripler formation was tested by jo int dimethyl sulfate (DMS) footprint, gel-retardation assay, circular dichroism (CD) and UV-melting studies. Dimethyl sulfate footprint stud ies revealed the antiparallel orientation of the third strand to the p urine strand of the Watson-Crick duplex. However, the protection of th e guanines at the ends of the target sequence decreased as the length of the third strand oligonucleotide increased. Melting temperature stu dies provided profiles with only one transition for all of the triplex es. The melting temperatures of the triplexes were found to be the sam e as for the targeted duplex in the case of the 11- and 14-mer third s trands while for the 17- and 20-mer third strands the melting temperat ure of the triplexes were correspondingly 4 and 8 degrees C higher tha n for the duplex. Heating and cooling melting curves were reversible f or all of the tested triplexes except one with the 20-mer third strand oligonucleotide. Circular-dichroism spectra showed the ability of the target DNA to adopt an A-like DNA conformation.