EX-VIVO REGULATION OF SPECIFIC GENE-EXPRESSION BY NANOMOLAR CONCENTRATION OF DOUBLE-STRANDED DUMBBELL OLIGONUCLEOTIDES

Inhibition of specific transcriptional regulatory proteins is a new ap proach to control gene expression. Transcriptional activity of DNA-bin ding proteins can be inhibited by the use of double-stranded (ds) olig odeoxynucleotides that compete for the binding to their specific targe t sequences in promoters and enhancers. As a model, we used phosphodie ster dumbbell oligonucleotides containing a binding site for the liver -enriched transcription factor HNF-1 (Hepatocyte Nuclear Factor 1). Bi nding affinity of HNF-1 to dumbbell oligonucleotides was the same as t hat to ds oligonucleotides, as determined by gel retardation assays. H NF-1 dumbbells specifically inhibited in vitro transcription driven by the albumin promoter by more than 90%. HNF-1-dependent activation of a CAT reporter plasmid was specifically inhibited when the HNF-1 dumbb ell oligonucleotide was added at nM concentration to transiently trans fected C33 cells. On the contrary, HNF-1 ds oligonucleotides, which di splayed the same activity as the dumbbell oligonucleotides in the in v itro assays, were no more effective in the ex vivo experiments. These results might reflect the increased stability of the circular dumbbell oligonucleotides towards cellular nuclease degradation, as shown in v itro with nucleolytic enzymes. Dumbbell oligonucleotides containing un modified phosphodiester bonds may efficiently compete for binding of s pecific transcription factors within cells, then providing a potential therapeutic tool to control disease-causing genes.