An approach to gene-specific transcription inhibition using oligonucleotides complementary to the template strand of the open complex

The single-stranded region of DNA within the open complex of transcriptiona lly active genes provides a unique target for the design of gene-specific t ranscription inhibitors. Using the Escherichia coil lac UV5 and trp EDCBA p romoters as in vitro models of open complex formation, we have identified t he sites inside these transcription bubbles that are accessible for hybridi zation by short, nuclease-resistant nonextendible oligoribonucleotides (ORN s), Binding of ORNs inside the open complex was determined by linking the c hemical nuclease bis(1,10-phenanthroline) cuprous chelate [(OP)(2)Cu+] to t he ORN and demonstrating template-specific DNA scission. In addition, these experiments were supported by in vitro transcription inhibition. We find t hat the most effective inhibitors are 5 nt long and have sequences that are complementary to the DNA template strand in the region near the transcript ion start site. The ORNs bind to the DNA template strand, forming an antipa rallel heteroduplex inside the open complex. In this system, RNA polymerase is essential not only to melt the duplex DNA but also to facilitate hybrid ization of the incoming ORN, This paradigm for gene-specific inactivation r elies on the base complementarity of the ORN and the catalytic activity and sequence specificity of RNA polymerase for the site- and sequence-specific recognition and inhibition of transcriptionally active DNA.