Figure 3.

An oligonucleotide-linked etoposide core increases topoisomerase II-mediated DNA cleavage. (A) The central 30 base pairs of a double stranded 50-mer oligonucleotide sequence corresponding to bases 1471–1500 (top strand) of PML intron 6 is shown. The yellow box denotes the position of the tethered etoposide core and linker moieties on OTI28 or LIN28. Arrows indicate sites of DNA cleavage induced by free etoposide (blue) or OTI28 (yellow). (B) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML top strand hybridized to an unmodified PML bottom strand in the presence of free etoposide or hybridized to OTI28 (bottom strand). For each gel, lane 1 contains the unmodified PML oligonucleotide. Lanes 2–5 contain the unmodified PML duplex treated with 0–500 μM free etoposide. Lanes 7 and 8 contain the unmodified PML top strand hybridized with OTI28. Lanes 10 and 11 contain the unmodified top strand duplexed with LIN28 (bottom strand oligonucleotide that contains the linker at position 28 with no attached etoposide core). Lanes 6, 9, and 12 contain reference (R) oligonucleotides that were 24, 23 and 19 bases in length. Gels are representative of at least three independent experiments. (C) Quantification of the relative levels of DNA cleavage mediated by topoisomerase IIα (left) and topoisomerase IIβ (right). DNA cleavage at each site was normalized to the cleavage observed at site 24–25 in reactions containing unmodified duplex in the absence of etoposide (lane 2). Cleavage results of the unmodified duplex in the presence of 500 μM free etoposide are shown in blue (lane 5) and those with an unmodified top strand hybridized to OTI28 are shown in yellow (lane 8). Error bars represent the standard error of the mean of an average of two to five independent experiments. Significance was determined by paired t-tests. P-values are indicated by asterisks (*P < 0.05; **P < 0.005; ***P < 0.0005).