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, interactions between antisense oligonucleotides (ASO) and cellular proteins are usually not considered. In this work, we have tested the ability of antisense phosphorothioate (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 factor that determines the success of RNA targeting. We have used a simple nuclease digestion assay to detect RNA/ASO hybrid formation in the presence of proteins. The results show the inability of a phosphorothioate oligonucleotide (SdT18) to reach the target RNA (rA18) in vitro in the presence of proteins. However, if proteins are absent, the RNA targeting was successful, as is usual in in vitro assays. Note that the target RNA concentration exceeded physiological values by several orders of magnitude while the crude protein extract was 20-fold diluted in the reaction tube. This finding is compatible with the notion that therapeutic properties of phosphorothioates could largely derive from a so-called "aptamer" effect.
Brukner
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Département de Biochimie, Université de Montréal, C.P. 6128, succursale Centre-ville, Montréal, Québec, Canada. ibrukner@hotmail.com
Tremblay
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GA
D000818:Animals; D002457:Cell Extracts / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism; D013873:Thionucleotides / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
D002457:Cell Extracts; D016376:Oligonucleotides, Antisense; D011506:Proteins; D013873:Thionucleotides; D012313:RNA; D016914:Ribonuclease H