Besch Robert, Giovannangeli Carine, Schuh Theda, Kammerbauer Claudia, Degitz Klaus
Department of Dermatology, Ludwig-Maximilians University, München, Germany.
J Mol Biol. 2004 Aug 20;341(4):979-89. doi: 10.1016/j.jmb.2004.05.079.
DNA-binding molecules that recognize specific sequences offer a high potential for the understanding of chromatin structure and associated biological processes in addition to their therapeutic potential, e.g. as positioning agents for validated anticancer drugs. A prerequisite for the development of DNA-binding molecules is the availability of appropriate methods to assess their binding properties quantitatively at the desired target sequence in the human genome. We have further developed a capture assay to assess triplex-forming oligonucleotide (TFO) binding efficiency quantitatively. This assay is based on bifunctional, psoralen and biotin-conjugated, TFOs and real-time PCR analysis. We have applied this novel quantification method to address two issues that are relevant for DNA-binding molecules. First, we have compared directly the extent of TFO-binding in three experimental settings with increasing similarity to the situation in vivo, i.e. naked genomic DNA, isolated cell nuclei, or whole cells. This comparison allows us to characterize factors that influence genomic triplex formation, e.g. chromosomal DNA organization or intracellular milieu. In isolated nuclei, the binding was threefold lower compared to naked DNA, consistent with a decreased target accessibility int he nucleosomal environment. Binding was detected in whole cells, indicating that the TFO enters the nucleus and binds to its target in intact cells in vivo, but the efficiency was decreased (tenfold) compared to nuclei. Secondly, we applied the method to characterize the binding properties of two different TFOs targeting the same sequence. We found that an antiparallel-binding GT-containing TFO bound more efficiently, but with less target sequence selectivity compared to a parallel-binding CU-containing TFO. Collectively, a sensitive method to characterize genomic triplex formation was described. This may be useful for the determination of factors driving TFO binding efficiency and, thus, may improve the usefulness of triplex-mediated gene targeting for studies of chromatin structure as well as for therapeutic antigene strategies.
识别特定序列的DNA结合分子，除了具有治疗潜力（例如作为经过验证的抗癌药物的定位剂）外，在理解染色质结构和相关生物学过程方面也具有很高的潜力。开发DNA结合分子的一个先决条件是要有合适的方法来定量评估它们在人类基因组中所需靶序列处的结合特性。我们进一步开发了一种捕获测定法，以定量评估三链体形成寡核苷酸（TFO）的结合效率。该测定法基于双功能的、补骨脂素和生物素共轭的TFO以及实时PCR分析。我们已应用这种新颖的定量方法来解决与DNA结合分子相关的两个问题。首先，我们在三种实验设置中直接比较了TFO结合的程度，这些设置与体内情况的相似性不断增加，即裸露的基因组DNA、分离的细胞核或完整细胞。这种比较使我们能够表征影响基因组三链体形成的因素，例如染色体DNA组织或细胞内环境。在分离的细胞核中，结合比裸露的DNA低三倍，这与核小体环境中靶标可及性降低一致。在完整细胞中检测到了结合，这表明TFO进入细胞核并在体内完整细胞中与其靶标结合，但与细胞核相比效率降低了（十倍）。其次，我们应用该方法来表征靶向相同序列的两种不同TFO的结合特性。我们发现，与平行结合的含CU的TFO相比，反平行结合的含GT的TFO结合更有效，但靶序列选择性较低。总的来说，描述了一种表征基因组三链体形成的灵敏方法。这可能有助于确定驱动TFO结合效率的因素，从而可能提高三链体介导的基因靶向在染色质结构研究以及治疗性反基因策略中的有用性。
