Nucleic Acids Research, 1995, Vol. 23, No. 19 3831-3836
© 1995
MOLECULAR BIOLOGY |
The high stability of the triple helices formed between short purine oligonucleotides and SIV/HIV-2 vpx genes is determined by the targeted DNA structure
1Laboatoire de Biochimie-Enzymologie CNRS URA 147, Institut Gustave Roussy, rue Camille Desmoulins, 94805 Villejuif Cedex, France 3Deparment of Biochemistry, Novosibirsk Institute of Bioorganic Chemistry 8 Prospect Lavrenteva, Novosibirsk, 630090, Russia 2Department de Biologie et Pharmacologie Structurales CNRS URA 147, institute Gustave Roussy, rue Camille Desmoulins, 94805 Villejuif Cedex, France
*To whom correspondence should be addressed
Received July 20, 1995. Revised September 1, 1995. Accepted September 1, 1995.
In our previous works we have shown that the oligonucleotides 5'-GGGGAGGGGGAGG-3' and 5'-GGAGGGGGAGGGG-3' give very stable and specific triplexes with their target double stranded DNAs [Svinarchuk, F., Bertrand, J.-R. and Malvy, C. (1994) Nucleic Acids Res., 22, 3742-3747; Svinarchuk, F., Paoletti, J. and Malvy, C. (1995) J. Biol Chem., 270, 14 068-14 071]. The target for the invariable part of these oligonucleotldes, 5'-GGAGGGGGAGG-3', is found in a highly conserved 20 bp long purine/pyrimidine tract of the vpx gene of the SIV and HIV-2 viruses and could be a target for oligonucleotide directed antivirus therapy. Here we report on the ability of four purine oligonucieotides with different lengths (11-, 14-, 17- and 20-mer) to form triplexes with the purine/pyrimidine stretch of the vpx gene. Triplex formation was tested by Joint dimethyl sulfate (DMS) footprint, gel-retardation assay, circular dichrolsm (CD) and UV-melting studies. Dimethyl sulfate footprint studies revealed the antiparallel orientation of the third strand to the purine strand of the Watson-Crick duplex. However, the protection of the guanines at the ends of the target sequence decreased as the length of the third strand oligonucleotide increased. Melting temperature studies provided profiles with only one transition for all of the tripiexes. The melting tenpe t werei as for the targeted duplex in the case of the 11- and 14-mer third strands while for the 17- and 20-mer third strands the melting temperature of the triplexes were correspondingly 4 and 8°C higher than for the duplex. Heating and cooling melting curves were reversible for all of the tested triplexes except one with the 20-mer third strand oligonucleo tide. Circular dichroism spectra showed the ability of the target DNA to adopt an A-like DNA conformation.Upon triplex formation the A-DNA form becomes even more pronounced. This effect depends on the length of the thlrd strand oligonucleotide: the CD spectrum shows a ‘classical’ A-DNA shape wtth the 20-mer. This is not observed wtth the purin/pyrimidine stretch of the HN-1 DNA which keeps an-like spectrum even after triplex formation. We suggest, that an A-like duplex DNA is required for the formation of a stable DNA purlne(purine-pyrimidine) triplex.
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