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Nucleic Acids Research, 1992, Vol. 20, No. 16 4275-4281
© 1992

MOLECULAR BIOLOGY

Oligodeoxynucleotide-directed photo-induced cross-linking of HIV proviral DNA via triple-helix formation

Carine Giovannangéli, Nguyen T. Thuong1 and Claude Héléne*

Laboratoire de Biophysique, Muséum Naitonal d'Histoire Naturelle INSEERM U201, CNRS UA 481, 43 Rue Cuvier, 75231 Paris Cedex 05 1Centre de Biophysique Moléculaire CNRS, 45071 Orléans Cedex 02, France

*To whom correspondence should be addressed

Received April 29, 1992. Revised June 22, 1992. Accepted June 22, 1992.

The HIV proviral genome contains two copies of a 16 bp homopurine.homopyrlmidlne sequence which overlaps the recognition and cleavage site of the Dra I restriction enzyme. Psoralen was attached to the 16–mer homopyrimldlne oligonucleotide, d5'(TTTTCTTTTCCCCCCT)3' which forms a triple helix with this HIV proviral sequence. Two plasmids, containing part of the HIV proviral DNA, with either one (pLTR) or two (pBT1) copies of the 16–bp homopurine.homopyrimidine sequence and either 4 or 14 Dra I cleavage sites, respectively, were used as substrates for the psoralerv oligonucleotide conjugate. Following UV Irradiation the two strands of the DNA targeted sequence were crosslinked at the triplex-duplex junction. The psoralenollgonucleotide conjugate selectively inhibited Dra I enzymatic cleavage at sites overlapping the two triple helix-forming sequences. A secondary triplex-forming site of 8 contiguous base pairs was observed on the pBT1 plasmid when binding of the 16 base-long oligonucleotide was allowed to take place at high oligonucleotide concentrations. Replacement of a stretch of six cytoslnes in the 16–mer oligomer by a stretch of six guanlnes increased binding to the primary sites and abolished binding to the secondary site under physiological conditions. These results demonstrate that oligonucleotides can be designed to selectively recognize and modify specific sequences in HIV proviral DNA.


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