SPECIFIC-INHIBITION OF IN-VITRO TRANSCRIPTION ELONGATION BY TRIPLEX-FORMING OLIGONUCLEOTIDE-INTERCALATOR CONJUGATES TARGETED TO HIV PROVIRAL DNA

A 16-base pair oligo(purine). oligo(pyrimidine) sequence present in th e coding region of two HIV 1 proviral genes (pol and nef) was chosen a s a target for tripler-forming oligonucleotides in in vitro transcript ion assays. Inhibition of transcription elongation was observed with t ripler-forming oligonucleotide-acridine conjugates (Acr-15-TCG:5'-Acr- T(4)CT(4)G(6)-3' and Acr-9-TC:5'-Acr-T4CT4-3' where C is 5-methylcytos ine) under conditions where the unsubstituted oligomers did not exhibi t any inhibitory effect. Both SP6 bacteriophage RNA polymerase and euk aryotic RNA polymerase II were physically blocked by such a tripler ba rrier. The polymerase arrest is caused by the triple-helical complex i nvolving the hydrogen-bonded oligonucleotide stabilized by the interca lated moiety and not solely by the acridine molecule specifically inte rcalated at the duplex-tripler junction. The stability of the triple-h elical complex formed by the 15-mer containing thymines, cytosine, and guanines (15-TCG) and involving the formation of six contiguous C . G xG base triplets was strongly enhanced in the presence of a benzopyrid oindole derivative (BePI), which intercalates in tripler structures. T his improvement of the binding affinity led to an increased inhibition of transcription elongation. The present results demonstrate the nece ssity to use tripler-forming oligonucleotides with high binding affini ty and a long residence time on their double-stranded target to effici ently inhibit transcription elongation. These data provide a rational basis for the optimization and the development of triplex-forming olig onucleotides as transcriptional blockers, even when they are targeted to the transcribed portion of a gene, downstream of the transcription initiation site.