Mergny J L, Lacroix L, Teulade-Fichou M P, Hounsou C, Guittat L, Hoarau M, Arimondo P B, Vigneron J P, Lehn J M, Riou J F, Garestier T, Hélène C
Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, Institut National de la Santé et de la Recherche Médicale Unité 201, Centre National de la Recherche Scientifique Unité Mixte 8646, 43 Rue Cuvier, 75005 Paris, France.
Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3062-7. doi: 10.1073/pnas.051620698.
The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5' and 3' ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2-20 degrees C at 1 microM dye concentration. This increase in T(m) value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC(50) value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.
大多数癌细胞中端粒酶活性的重新激活支持了端粒酶是肿瘤学中一个相关靶点的概念，并且端粒酶抑制剂已被提议作为新的潜在抗癌药物。富含鸟嘌呤的端粒单链DNA在体外可形成分子内四链体结构，该结构已被证明能抑制端粒酶活性。我们使用荧光测定法来鉴定能稳定G-四链体的分子。具有四个人类端粒序列重复片段的寡核苷酸分子内折叠成G-四链体结构会导致供体（荧光素）和分别共价连接到寡核苷酸5'和3'末端的受体（四甲基罗丹明）之间发生荧光激发能量转移。通过测量供体的荧光发射来监测在假定的G-四链体结合分子存在下G-四链体的解链情况。在1 microM染料浓度下，一系列化合物（五环新月形二苯并菲衍生物）显示能使G-四链体的解链温度升高2 - 20摄氏度。这种熔解温度（T(m)）值的升高与体外端粒酶抑制效率的提高密切相关。在标准的端粒酶重复序列扩增协议测定中，最佳的端粒酶抑制剂显示IC(50)值为28 nM。因此，荧光能量转移可用于揭示四链DNA结构的形成及其被四链体结合剂的稳定化情况，以努力发现新的强效端粒酶抑制剂。
