Effect of abasic linker substitution on triplex formation Sp1 binding and specificity in an oligonucleotide targeted to the human Ha-ras promoter.

摘要

A region of the human Ha-ras promoter (-8 to -28) which contains two of the three Sp1 binding sites essential for transcriptional activity forms a sequence specific oligonucleotide-directed pur*pur:pyr triple helix. The relative binding of oligonucleotides containing different substitutions, including an abasic propanediol linker, over three potentially destabilizing C:G interruptions in the otherwise poly G:poly C target was examined. DNase I footprint titrations reveal that substitution of the positively charged abasic propanediol linker results in approximately ten fold greater binding than cytosine substitution which in turn provides greater sequence specific binding than substitution of a guanine in the third strand oligonucleotide over the C:G interruptions. Protein binding assays demonstrate that triplex formation by the linker substituted oligomer (HR21Xap) is less effective in inhibiting Sp1 binding than the cytosine substituted oligomer (HR21ap) both to the target sequence as well as an upstream sequence. As an indication of the effect of linker substitution and targeting consensus Sp1 sites on triplex specificity, the relative ability of the Ha-ras promoter targeted oligonucleotides to interact with non-target Sp1 sequences within the Ha-ras promoter as well as in the DHFR promoter and HIV-1 LTR was also investigated. At concentrations which afford complete DNase I protection of the target sequence, HR21ap does not bind to the non-target sequences while HR21Xap interacts weakly only at a distal site in the DHFR promoter. Also, HR21ap as well as HR21Xap are specific in their inhibition of Sp1 binding. These results suggest that the propanediol linker is able to skip over interruptions in a target sequence thereby stabilizing triplex but, slightly compromises sequence specificity and the ability to inhibit Sp1 binding to the Ha-ras promoter.