DIFFERENTIAL REGULATION OF GENE-EXPRESSION IN-VIVO BY TRIPLE HELIX-FORMING OLIGONUCLEOTIDES AS DETECTED BY A REPORTER ENZYME

In an attempt to assay the capability of various oligonucleotides to i nhibit gene transcription in vivo through triplex formation, we develo ped a cellular system employing transfection of a reporter plasmid and putative triplex-forming oligonucleotides targeted to Sp1-binding sit es contained within the SV40 early promoter. Using this approach, we d emonstrated that the activity of the reporter enzyme, alkaline phospha tase, was highly dependent on the sequence of the oligonucleotides: ol igonucleotides utilizing G:GC triplets, but not C:GC triplets, promote d a dose-dependent decrease in reporter enzyme activity. Evidence of p hysical interaction between Sp1-binding sites within the SV40 promoter and sequence-specific G-rich oligonucleotides has been demonstrated, suggesting triple-helix formation as the most probable explanation for the inhibitory effect on alkaline phosphatase activity observed for t hese oligonucleotides. Surprisingly, Southern analysis of isolated nuc lear DNA indicates that the differences in alkaline phosphatase activi ty associated with transfection of the different oligonucleotides appe ar to correlate with internalized plasmid DNA copy number rather than inhibition of transcription. It is intriguing to postulate the existen ce of a nuclease that is able to recognize and cleave triple-helical D NA structures. This hypothesis implies the existence of a novel mechan ism of gene regulation specific for triplex structures and, presumably , independent of transcription.