ANTISENSE OLIGONUCLEOTIDE STRATEGIES IN PHYSIOLOGY

Antisense oligonucleotides can inhibit gene expression in living cells by binding to complementary sequences of DNA, RNA or mRNA. The mechan isms include inhibition of RNA synthesis, RNA splicing, mRNA export, b inding of initiation factors, assembly of ribosome subunits and of sli ding of the ribosome along the mRNA coding sequence. The most efficien t antisense oligonucleotides also activate RNAse H, an ubiquitous enzy me that cleaves the mRNA at sites of mRNA/oligonucleotide duplex forma tion. A staggering number of oligonucleotide modifications have been p roposed to retard degradation by nucleases, enhance cellular uptake, i ncrease binding to the target sequence, and minimize non-specific bind ing to related nucleic acid sequences. Phosphorothioates are the most popular oligonucleotides used in cell culture and in vivo, although se quence non-specificity remains an underreported problem. Recently deve loped chimeras between methylphosphonates and phosphodiester oligonucl eotides appear to combine the advantages of water solubility, nuclease resistance, enhanced cellular uptake, activation of RNAse H, and high sequence selectivity. Antigene oligonucleotides are also promising, b ecause they can inhibit gene expression by triple helix formation with DNA or by binding to one of the DNA strands. They have so far been li ttle used in physiological studies. Cost is still a prohibitive factor , especially for suppressing the expression of a hormone or hormone re ceptor gene in rats, for example. However, patch-clamp dialysis of sin gle cells or nuclear microinjections in culture, exposure of cultures to extracellular oligonucleotides, and intra-cerebral microinjections of oligonucleotides are feasible and highly rewarding approaches in ph ysiology.