QUANTITATION OF TRIPLE-HELIX FORMATION USING A PHOTO-CROSS-LINKABLE ARYL AZIDE BIOTIN OLIGONUCLEOTIDE CONJUGATE

DNA triple-helix formation has potential applications in gene mapping and as the basis of ''antigene'' pharmaceuticals; however, the methods for quantitation of triple-helix formation are limited, especially for purine(purine-pyrimidine)-based triplexes. We present a novel method for detection and quantitation of triple-helix formation by triple-helix-forming oligonucleotides. The oligonucleotide is conjugated to a photoactivatable cross-linker, sulfosuccinimidyl 3-[[2-[6-(biotinamido)-2-(p-azidobenzamido)hexanamido]ethyl]dithio]propionate. After incubation with the target DNA, exposure to light labels the target with biotin. The labeled target can be quantified by a chemiluminescent assay. A 26-mer oligonucleotide previously reported to form a purine(purine-pyrimidine) tripler with the upstream region of the c-myc gene was studied and found to bind to its target with K-d of approximately 100 nM at 37 degrees C, 10 mM MgCl2, pH 7.5, consistent with previous reports. This new technique can be used under a variety of conditions and in kinetic experiments and may be extendible to use in living cells.