Fluorescence anisotropy and FRET studies of G-quadruplex formation in presence of different cations
Fluorescence anisotropy and FRET studies of G-quadruplex formation in presence of different cations
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DOI：
10.1016/j.saa.2005.08.012
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发表时间：
2006-07-01
影响因子：
4.4
通讯作者：
Takenaka, Shigeori
中科院分区：
文献类型：
--
作者：
Juskowiak, Bernard;Galezowska, Elzbieta;Takenaka, Shigeori
本文报道了在选定阳离子（Li+, Na+, K+， NEt4+和Mg2+）存在下g -四聚体形成的稳态荧光、各向异性和FRET测量结果。研究了荧光素（FAM）和四甲基罗丹明（TAMRA）标记的具有人类端粒序列的三种不同的荧光寡核苷酸：一种双标记的21聚体，称为PSO（钾感应寡核苷酸），两种5‘和3’单标记探针，分别为FAM-21和21- TAMRA。在所有体系中，FAM-21的荧光信号均显著增强，单价阳离子的荧光增强幅度相当，但Mg2+阳离子的荧光增强更为明显。这一现象是由于离子强度的变化影响了FAM的原解平衡。另一方面，Na(I)阳离子选择性地增强了TAMRA的荧光，这可以解释为由于Na(I)-四重体篮型结构的特殊性导致TAMRA发射脱淬。FAM-21的各向异性（而不是21-TAMRA）似乎对g -四聚体的形成很敏感，随着阳离子浓度的增加而显著增加，这表明FAM的旋转去极化有一定的限制。FRET实验显示，所有被测阳离子都引起PSO中FAM荧光猝灭，但只有Na+和K+离子产生TAMRA受体的敏化发射。在钠离子存在下观察到更高的FRET效率归因于篮型Na(I)-四联体中的特定光谱因子和空间相互作用。(c) 2005 Elsevier B.V.版权所有
Results of the steady-state fluorescence, anisotropy and FRET measurements of G-quadruplex formation in the presence of selected cations (Li+, Na+, K+, NEt4+ and Mg2+) are reported. Three different fluorescent oligonucleotides with human telomeric sequence labeled with fluorescein (FAM) and tetramethylrhodamine (TAMRA) were investigated: a dual-labeled 21-mer denoted as PSO (Potassium Sensing Oligonucleotide) and two 5'- and 3'- single-labeled probes, FAM-21 and 2 1-TAMRA, respectively. The fluorescence signal of FAM-21 increased significantly for all systems and the fluorescence enhancement was comparable in magnitude for monovalent cations but it was more pronounced for Mg2+ cation. This phenomenon was attributed to the protolytic equilibria of FAM affected by the variation in ionic strength. On the other hand, fluorescence of TAMRA was enhanced selectively by Na(I) cation that was explained by the dequenching of TAMRA emission originated from the peculiarity of the basket-type structure of Na(I)-quadruplex. Anisotropy of FAM-21 (but not 21-TAMRA) appeared to be sensitive to the G-quadruplex formation, showing significant increase with an increase in cation concentration and indicating some restrictions in rotational depolarization of FAM. FRET experiments revealed that all tested cations caused quenching of FAM fluorescence in PSO, but only Na+ and K+ ions produced sensitized emission of TAMRA acceptor. Higher FRET efficiency observed in the presence of sodium ion was attributed to the specific spectral factor and steric interactions in the basket-type Na(I)-quadruplex. (c) 2005 Elsevier B.V. All rights reserved.