Cellular proteins prevent antisense phosphorothioate oligonucleotide (SdT18) to target sense RNA (rA18): Development of a new in vitro assay

There are numerous indications that the "antisense" mechanism alone cannot account for the observed effects in living cells. Despite that, interaction s between antisense oligonucleotides (ASO) and cellular proteins are usuall y not considered. In this work, we have tested the ability of antisense pho sphorothioate (SdT) oligonucleotides and natural deoxyoligonucleotides (dT) for their ability to interact with target RNA in the presence of cellular proteins. We show that the affinity for cellular proteins is an essential f actor that determines the success of RNA targeting. We have used a simple n uclease digestion assay to detect RNA/ASO hybrid formation in the presence of proteins. The results show the inability of a phosphorothioate oligonucl eotide (SdT18) to reach the target RNA (rA18) in vitro in the presence of p roteins. However, if proteins are absent, the RNA targeting was successful, as is usual in in vitro assays. Note that the target RNA concentration exc eeded physiological values by several orders of magnitude while the crude p rotein extract was 20-fold diluted in the reaction tube. This finding is co mpatible with the notion that therapeutic properties of phosphorothioates c ould largely derive from a so-called "aptamer" effect.