Mapping of the accessibility of the 5′-terminal region of ΔNp53utr-Luc transcript to oligonucleotide hybridization.

<p>Semi-random libraries of DNA 6-mers and RNase H hydrolysis of RNA-DNA hybrids were used to search for sites accessible to hybridization in the 5′-terminal region of ΔNp53utr-Luc transcript. (<b>A</b>) The cleavage sites were identified by reverse transcription with the 5′-end-[³²P]-labelled DNA primer that was bound to nucleotides 232–257 of the ΔNp53utr-Luc sequence. The cDNA products were analyzed on 8% polyacrylamide gels. Selected cytosine and guanosine residues are marked on the left and the short and long run of the gel is shown. Lane (−) denotes the control reaction in the absence of semi-random oligonucleotide libraries. A, T, C, G – sequencing reaction with adenosine, thymidine, cytosine and guanosine dideoxy terminating nucleotides, respectively. (<b>B</b>) The cleavage sites occurring in the presence of libraries <i>a</i>, <i>c</i>, <i>g</i>, <i>t</i> displayed on the RNA secondary structure model. Continuous grey lines along the RNA sequence show the most probable positions of oligonucleotide hybridization on the ΔNp53utr-Luc transcript. The designed specific antisense oligonucleotides are marked with black lines and numbered respectively.