Formation of Stable DNA Triple Helices Within the HumanbcrPromoter at a Critical Oligopurine Target Interrupted in the Middle by Two Adjacent Pyrimidines

Abstract

Antigene strategies based on the use of triplex-forming oligonucleotides (TFO) as artificial repressors are constrained by the need for genomic targets with a polypurine polypyrimidine [poly (R·Y)] DNA motif. In this study, we demonstrate that both A/G and G/T motif oligonucleotides recognize and bind strongly to a critical polypurine sequence interrupted in the middle by two adjacent cytosines and located in the promoter of the human bcr gene at the transcription initiation. The interaction between the designed TFO and this irregular poly (R·Y) target has been studied using a number of techniques, including electrophoretic mobility shift assay (EMSA), circular dichroism (CD), DNase I, and dimethyl sulfate (DMS) footprinting. Although CD shows that the 24-mer TFO self-aggregate in solution, they bind to the bcr target at 37°C, forming stable triplexes that do not dissociate during electrophoretic runs performed up to 50°C in 50 mM Tris-acetate, pH 7.4,10 mM MgCl₂, 50 mM NaCl (buffer A). We used EMSA to determine the equilibrium dissociation constants (K_d) for the reaction T ⇔ D + TFO at 37°C, either in buffer A or in 50 mM Tris-acetate, pH 7.4,10 mM MgCl₂, 5 mM NaCl (buffer B). The triplexes were found to be more stable in buffer B, a behavior that can be rationalized in terms of monovalent and divalent cation competition for binding to DNA. Footprinting experiments showed that the TFO interact with the irregular poly (R·Y) target in a highly sequence-specific way and that the A/G motif oligonucleotide, juxtaposing T to the double CG inversions of the target, formed the most stable triplex (e.g., 1 μM TFO promoted strong footprints at 37°C). These triplexes, except the one containing two A·C·G mismatched triads, are not destabilized under near physiologic conditions, that is, in 50 mM Tris-acetate, pH 7.4, 80 mM KCl, 20 mM NaCl, 2 mM spermidine. Moreover, we found that guanine N7 in T·C·G and guanine N7 in A·C·G are both accessible to DMS and that the first is less reactive than the second. In conclusion, the results of this study indicate that a critical sequence in the human bcr promoter may be used as a potential binding site for TFO designed to repress artificially the transcription of the fused bcr/abl gene expressed in leukemia cells.