Formation of stable DNA triple helices within the human bcr promoter at a critical oligopurine target interrupted in the middle by two adjacent pyrimidines

Antigene strategies based on the use of tripler-forming oligonucleotides (T FO) as artificial repressors are constrained by the need for genomic target s with a polypurine polypyrimidine [poly (R.Y)] DNA moth. In this study, we demonstrate that both A/G and G/T moth oligonucleotides recognize and bind strongly to a critical polypurine sequence interrupted in the middle by tw o adjacent cytosines and located in the promoter of the human bcr gene at t he transcription initiation. The interaction between the designed TFO and t his irregular poly (R.Y) target has been studied using a number of techniqu es, including electrophoretic mobility shift assay (EMSA), circular dichroi sm (CD), DNase I, and dimethyl sulfate (DMS) footprinting. Although CD show s that the 24-mer TFO self-aggregate in solution, they bind to the bcr targ et at 37 degrees C, forming stable triplexes that do not dissociate during electrophoretic runs performed up to 50 degrees C in 50 mM Tris-acetate, pH 7.4, 10 mM MgCl2, 50 mM NaCl (buffer A). We used EMSA to determine the equ ilibrium dissociation constants (K-d) for the reaction T double left right arrow D + TFO at 37 degrees 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 mo novalent and divalent cation competition for binding to DNA. Footprinting e xperiments showed that the TFO interact with the irregular poly (R.Y) targe t in a highly sequence-specific way and that the A/G moth oligonucleotide, juxtaposing T to the double CG inversions of the target, formed the most st able tripler (e.g., 1 mu M TFO promoted strong footprints at 37 degrees C). These triplexes, except the one containing two A.C.G mismatched triads, ar e not destabilized under near physiologic conditions, that is, in 50 mM Tri s-acetate, pH 7.4, 80 mM KCl, 20 mM NaCl, 2 mM spermidine. Moreover, we fou nd 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 p romoter may be used as a potential binding site for TFO designed to repress artificially the transcription of the fused bcr/abl gene expressed in leuk emia cells.