INHIBITION OF T7 AND T3 RNA-POLYMERASE DIRECTED TRANSCRIPTION ELONGATION IN-VITRO

A class of oligonucleotides which binds to naturally-occurring duplex DNA sites at physiologic pH to form triple helical structures was used as transcription attenuators in an in vitro transcription assay. Olig onucleotides were designed to form triple helices with a purine-rich, double-stranded target by binding in the major groove in an orientatio n anti-parallel to the most purine-rich strand of the target. A 45 bas e-pair purine-rich region located within the gag gene of Friend Murine Leukemia Virus (FMLV) was used as the duplex target. The target DNA w as inserted by molecular cloning downstream of either the bacterial T7 - or T3 promoter. The sequence-specific interaction of the triple heli x-forming oligonucleotide (TFO) with the FMLV target was confirmed by DNAse I footprint analysis. The affinity of the TFO, as measured by th e equilibrium dissociation constant of the TFO for the duplex, was det ermined by band shift analysis. When a TFO was allowed to form a tripl e helix with the target duplex in well-defined buffer conditions befor e the transcription reaction, truncated transcripts of a predicted siz e were observed. Attenuation of transcription was observed only when b uffer conditions favorable to triple helix formation were used. In add ition, oligonucleotides containing a high percentage of guanosine resi dues were able to inhibit mRNA production of the bacterial T7 polymera se by a mechanism independent of transcription attenuation. The abilit y of an oligonucleotide-directed triple helical structure to slow down , or even completely stop, RNA chain elongation may expand the utility of triple helix technology in the area of gene regulation.