UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia , Universidade Nova de Lisboa , Campus da Caparica , 2829-516 Caparica , Portugal.
LAQV, Departamento de Química, Faculdade de Ciências e Tecnologia , Universidade Nova de Lisboa , Campus da Caparica , 2829-516 Caparica , Portugal.
ACS Chem Biol. 2018 May 18;13(5):1235-1242. doi: 10.1021/acschembio.8b00029. Epub 2018 Mar 23.
Selective base pairing is the foundation of DNA recognition. Here, we elucidate the molecular and structural details of a FRET-based two-component molecular beacon relying on steady-state fluorescence spectroscopy, small-angle X-ray scattering (SAXS), microscale thermophoresis (MST), and differential electrophoretic mobility. This molecular beacon was designed to detect the most common fusion sequences causing chronic myeloid leukemia, e14a2 and e13a2. The emission spectra indicate that the self-assembly of the different components of the biosensor occurs sequentially, triggered by the fully complementary target. We further assessed the structural alterations leading to the specific fluorescence FRET signature by SAXS, MST, and the differential electrophoretic mobility, where the size range observed is consistent with hybridization and formation of a 1:1:1 complex for the probe in the presence of the complementary target and revelator. These results highlight the importance of different techniques to explore conformational DNA changes in solution and its potential to design and characterize molecular biosensors for genetic disease diagnosis.
选择性碱基配对是 DNA 识别的基础。在这里，我们通过稳态荧光光谱、小角 X 射线散射（SAXS）、微尺度热泳（MST）和差示电泳迁移率阐明了基于荧光共振能量转移（FRET）的两部分分子信标的分子和结构细节。这种分子信标被设计用来检测导致慢性髓性白血病的最常见融合序列 e14a2 和 e13a2。发射光谱表明，生物传感器不同组件的自组装是顺序发生的，由完全互补的靶标触发。我们进一步通过 SAXS、MST 和差示电泳迁移率评估了导致特异性荧光 FRET 特征的结构变化，在存在互补靶标和揭示剂的情况下，观察到的大小范围与探针的杂交和 1:1:1 复合物的形成一致。这些结果强调了不同技术在探索溶液中构象 DNA 变化及其用于设计和表征遗传疾病诊断的分子生物传感器的重要性。
