completely identical the listing below refers to the deoxy form used for testing in the finally used 2 o methyl ribonucleotides all t 39 s should be read as u 39 s the efficacy of the aons is determined by their binding affinity for the target sequence notwithstanding recent improvements in computer simulation programs for the prediction of rna folding it is difficult to speculate which of the designed aons would be capable of binding the target sequence with a relatively high affinity therefore we performed gel mobility shift assays according to protocols described by bruice et al 1997 the exon 46 target rna fragment was generated by in vitro t7 transcription from a pcr fragment amplified from either murine or human muscle mrna using a sense primer that contains the t7 promoter sequence in the presence of 32p ctp the binding affinity of the individual aons 0 5 pmol for the target transcript fragments was determined by hybridization at 37 u00b0 c for 30 minutes and subsequent polyacrylamide 8 gel electrophoresis we performed these assays for the screening of both the mouse and human specific aons fig3 at least 5 different mouse specific aons maon 4 6 8 9 and 11 and four corresponding human specific aons haon 4 6 8 and 9 generated a mobility shift demonstrating their binding affinity for the target rna the exon 46 specific aons which showed the highest target binding affinity in gel mobility shift assays were selected for analysis of their efficacy in inducing the skipping in muscle cells in vitro in all transfection experiments we included a non specific aon as a negative control for the specific skipping of exon 46 as mentioned the system was first set up.