DNA G-quadruplex detection system employing a protein fibril ligand
DNA G-quadruplex detection system employing a protein fibril ligand
复制标题
DOI：
10.14800/tt.691
复制
发表时间：
2015-03
期刊：
影响因子：
--
通讯作者：
Ryuichi Maeda;Hidenobu Yaku;T. Nakabayashi;T. Murashima;N. Sugimoto;N. Ohta;D. Miyoshi
中科院分区：
文献类型：
--
作者：
Ryuichi Maeda;Hidenobu Yaku;T. Nakabayashi;T. Murashima;N. Sugimoto;N. Ohta;D. Miyoshi
ThioflavinT（ThT）是蛋白质纤维的典型探针，也对其他DNA结构具有高度特异性地结合人端粒G-四链体。结合后，ThT发出明亮的荧光，使其成为一种有前途的G-四链体探针。由于ThT最初用于检测蛋白质原纤维，因此在蛋白质存在的情况下，例如在活细胞中，DNA G-四链体的假阳性检测和成像是可能的。因此，我们开发了一个系统，通过测量从ThT的荧光共振能量转移（FRET）的DNA双链体探针的DNA G-四链体从蛋白质纤维的特异性歧视。我们设计了一种互补DNA（cDNA），它与DNA G-四链体形成区附近的单链区域杂交。碘化己啶（HI），一个典型的DNA双链荧光嵌入剂，被用作受体的FRET从ThT。仅当ThT结合G-四链体区域和HI结合cDNA的双链体区域时才发生FRET。荧光分析表明，该系统可以检测DNA G-四链体，即使在蛋白质纤维的存在。
Thioflavin T (ThT), a typical probe of protein fibrils, also binds human telomeric G-quadruplexes with high specificity for other DNA structures. Upon binding, ThT fluoresces brightly, making it a promising G-quadruplex probe. Because ThT was originally used for detecting protein fibrils, false-positive detection and imaging of DNA G-quadruplexes is possible in the presence of proteins, such as in living cells. We therefore developed a system for the specific discrimination of DNA G-quadruplexes from protein fibrils through measurement of fluorescence resonance energy transfer (FRET) from ThT to a DNA duplex probe. We designed a complementary DNA (cDNA) that hybridizes with the single-stranded region near the DNA G-quadruplex–forming region. Hexidium iodide (HI), a typical DNA duplex fluorescent intercalator, was utilized as an acceptor for FRET from ThT. FRET occurred only when ThT bound the G-quadruplex region and HI bound the duplex region of the cDNA. Fluorescence analysis showed that this system can detect DNA G-quadruplexes even in the presence of protein fibrils.