Understanding oligonucleotide-mediated inhibition of gene expression in Xenopus laevis oocytes

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Abstract
Triplex-forming oligonucleotides (TFOs) modified with N,N-diethylethylenediamine can inhibit the expression of a reporter plasmid in Xenopus oocytes if the triplex is preformed prior to injection while unmodified oligonucleotides cannot. Here we show that merely forming a triplex in a reporter plasmid does not disrupt transcription, but when TFOs are targeted to sites within the transcribed region of a reporter gene then gene activity is inhibited. TFO-based inhibition did not lead to large scale degradation or mutation of the reporter plasmid, but dramatically lowered mRNA levels. Finally, we investigated the accessibility of a triplex target site on a reporter plasmid after injection into nuclei. We found that the site used for our previous studies was inaccessible to restriction endonuclease after injection into nuclei. This observation may explain why inhibition was dependent on forming the triplex before injection into oocytes. Based on the assumption that oligonucleotide association, like restriction enzyme access, was excluded by nucleosome formation, additional target sites were inserted so that all sites could not simultaneously be associated with the octamer core of a nucleosome. With multiple target sites prior association of the plasmid with nuclear proteins does not prevent oligonucleotide-mediated inhibition of gene activity.