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.

中文翻译：

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慢性粒细胞白血病DNA生物传感器的光学和结构表征。

选择性碱基配对是DNA识别的基础。在这里，我们阐明了基于稳态荧光光谱，小角度X射线散射（SAXS），微型热电泳（MST）和差分电泳迁移率的基于FRET的两组分分子信标的分子和结构细节。该分子信标旨在检测引起慢性髓细胞性白血病的最常见融合序列e14a2和e13a2。发射光谱表明，由完全互补的靶标触发，生物传感器的不同组件的自组装顺序发生。我们进一步评估了SAXS，MST和差分电泳迁移率导致结构荧光FRET签名的结构改变，其中观察到的大小范围与在互补靶标和揭示试剂存在下探针的杂交和形成1：1：1复合物相一致。这些结果凸显了探索溶液中构象DNA变化的不同技术的重要性以及其设计和表征用于遗传疾病诊断的分子生物传感器的潜力。