Figure 1

Activity of bifunctional oligonucleotides carrying a 5' tail. In vitro splicing assays using a model 964-nt long Bcl-x pre-mRNA incubated in HeLa nuclear extracts in the presence of increasing concentrations of oligonucleotides lacking a tail (M4) or carrying a 5' tail (M4A1, M4A1M, M4JV2, M4BC2 and M4B5). (A) Structure of the human Bcl-x pre-mRNA. Boxes and lines represent exons and introns, respectively. The dashed lines indicate plasmid sequences. The position of the 5' splice sites is shown as well as the position of the primers used in the RT-PCR assay. The portion of the pre-mRNA targeted by hybridization with various oligomers corresponds to nucleotide position -4 to -23, upstream of the Bcl-xL 5' splice junction. (B) Schematic structure of the oligonucleotides. The horizontal portion is complementary to position -4 to -23, upstream of the Bcl-xL 5' splice site. The presence of A1/A2 binding sites (A1BS), mutated A1/A2 binding sites (mutA1BS) or a branch site (BS) is indicated. (C) The RT-PCR assay was carried out on total RNA isolated from splicing reactions performed in triplicate. The RT-PCR products were fractionated by electrophoresis on an Agilent 2100 bioanalyser and the ratio of Bcl-xL/Bcl-xS products was calculated with standard deviations. The results were plotted in a graph that indicates the xL/xS ratio at various concentrations of oligonucleotides. (D) A computer generated image of fluorescent Bcl-x RT-PCR products is shown for the reactions performed in the absence of oligonucleotide (control, lane 2) and in the presence of oligonucleotides M4, M4A1 and M4A1M at concentrations of 1.5, 3.0 and 6.0 nM. The position and size of the Bcl-xL and Bcl-xS products and molecular weight markers are indicated.