Geselowitz D A, Neumann R D
Department of Nuclear Medicine, National Institutes of Health, Bethesda, Maryland 20892-1180, USA.
Bioconjug Chem. 1995 Jul-Aug;6(4):502-6. doi: 10.1021/bc00034a021.
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.
DNA三链螺旋的形成在基因图谱绘制以及作为“反基因”药物的基础方面具有潜在应用；然而，三链螺旋形成的定量方法有限，特别是对于基于嘌呤（嘌呤 - 嘧啶）的三链体。我们提出了一种通过三链螺旋形成寡核苷酸检测和定量三链螺旋形成的新方法。该寡核苷酸与一种可光活化的交联剂——磺基琥珀酰亚胺3 - [[2 - [6 - （生物素酰胺基） - 2 - （对叠氮苯甲酰胺基）己酰胺基]乙基]二硫代]丙酸酯相连。与靶DNA孵育后，光照用生物素标记靶标。标记的靶标可通过化学发光测定法进行定量。研究了一种先前报道的与c - myc基因上游区域形成嘌呤（嘌呤 - 嘧啶）三链体的26聚体寡核苷酸，发现在37℃、10 mM MgCl₂、pH 7.5条件下，其与靶标的结合解离常数（Kd）约为100 nM，与先前报道一致。这种新技术可在多种条件下以及动力学实验中使用，并且可能扩展到活细胞中的应用。
