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) triplex with the upstream region of the c-myc gene was studied and found to bind to its target with Kd 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.
Geselowitz
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DA
Department of Nuclear Medicine, National Institutes of Health, Bethesda, Maryland 20892-1180, USA.
Neumann
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RD
D001386:Azides; D001483:Base Sequence; D001710:Biotin; D002874:Chromosome Mapping; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000528:radiation effects; D016259:Genes, myc; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D010777:Photochemistry; D010957:Plasmids; D013388:Succinimides
D001386:Azides; D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D013388:Succinimides; C096733:sulfosuccinimidyl 3-((2-(6-(biotinamido)-2-(4-azidobenzamido)hexanamido)ethyl)dithio)propionate; D001710:Biotin; D004247:DNA