Inhibition of HSV-1 proliferation by decoy phosphodiester oligonucleotides containing ICP4 recognition sequences.

Transcriptional control in eukaryotes results from the interplay between DNA sequences in promoters, enhancers, or silencers and transcription factors. Selective control of gene expression can thus be achieved by inhibiting specific transcription factor/DNA interactions. Transcriptional activity of DNA binding transcription factors can be inhibited by competition with double-stranded oligonucleotides (decoys) that contain their specific recognition sequences. The immediate early protein ICP4 of herpes simplex virus type 1 (HSV-1) is a sequence-specific DNA binding protein that is essential for viral replication. We synthesized double-stranded hairpin phosphodiester oligonucleotides carrying ICP4 sites and demonstrated their ability to specifically titrate ICP4. Upon addition to Vero cells, ICP4 hairpin decoys significantly reduced HSV-1 titers (IC50 = 0.3 microM), whereas a control hairpin oligonucleotide had no activity. Antiviral activity of ICP4 hairpin decoys was correlated to their relative binding affinities. These results show that phosphodiester oligonucleotides can compete for binding of specific transcription factors within cells, thus providing a potential therapeutic tool to control disease-causing genes.

Retinoic acid receptor isoform RAR gamma 1: an antagonist of the transactivation of the RAR beta RARE in epithelial cell lines and normal human keratinocytes.

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR beta RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR beta RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR alpha 1 and RAR beta 2 isoforms, but not by RAR gamam 1. On the contrary, this latter isoform behaved towards the RAR beta RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR alpha 1 and RAR beta 2. RAR gamma 1 also behaved as an antagonist of the transactivation produced by cotransfected RXR alpha. The natural RAR beta gene promoter or RAR beta RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR gamma 1. It was, however, possible to activate to a certain extent RAR beta RARE-reporter constructs in NHK by co-transfecting RAR alpha 1, RAR beta 2 or RXR alpha. The antagonist behavior of RAR gamma 1 towards the RAR beta RARE may explain why in certain cell types such as keratinocytes, RAR beta is neither expressed nor induced by RA.

Ex vivo regulation of specific gene expression by nanomolar concentration of double-stranded dumbbell oligonucleotides.

Inhibition of specific transcriptional regulatory proteins is a new approach to control gene expression. Transcriptional activity of DNA-binding proteins can be inhibited by the use of double-stranded (ds) oligodeoxynucleotides that compete for the binding to their specific target sequences in promoters and enhancers. As a model, we used phosphodiester dumbbell oligonucleotides containing a binding site for the liver-enriched transcription factor HNF-1 (Hepatocyte Nuclear Factor 1). Binding affinity of HNF-1 to dumbbell oligonucleotides was the same as that to ds oligonucleotides, as determined by gel retardation assays. HNF-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 dumbbell oligonucleotide was added at nM concentration to transiently transfected C33 cells. On the contrary, HNF-1 ds oligonucleotides, which displayed the same activity as the dumbbell oligonucleotides in the in vitro 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 vitro with nucleolytic enzymes. Dumbbell oligonucleotides containing unmodified phosphodiester bonds may efficiently compete for binding of specific transcription factors within cells, then providing a potential therapeutic tool to control disease-causing genes.

Crosslinking of double-stranded oligonucleotides containing O-methyl-substituted pyrophosphate groups to the HNF1 transcription factor in nuclear cell extract.

Probing of the HNF1 (hepatocyte nuclear factor I) DNA-binding region using a set of DNA duplexes containing pyrophosphate or O-methyl-substituted pyrophosphate internucleotide groups at different positions of the HNF1 recognition sequence was performed. The histidine-tagged HNF1/1-281 DNA binding domain and nuclear extract from rat liver were used. We showed that HNF1 from these species specifically binds to modified DNA duplexes. A correlation in binding affinity of both types of duplexes was detected. Crosslinking of the HNF1 DNA-binding domain and HNF1 in nuclear liver extract to DNA duplexes carrying O-methyl-substituted pyrophosphate groups was observed. The crosslinking efficiency of HNF1 in liver extract to substituted pyrophosphate-modified DNA duplex, containing a reactive internucleotide group between nucleotides G and T of the GT dinucleotide immediately 5' to the TAAT recognition sequence, amounts to 40% of the efficiency of non-covalent association. Nonspecific crosslinking of the reactive DNA duplexes to other components of nuclear extract was not observed. These results indicate that DNA duplexes carrying substituted pyrophosphate internucleotide groups can specifically bind and crosslink with DNA-binding proteins, especially transcription factors in crude preparations and could constitute a potential tool to control the expression of disease-causing genes.