基于FRET机理的核酸适配体比率型荧光纳米探针的构建及其细胞内成像研究

Real-time monitoring of intracellular biomolecules would provide valuable information for early diagnosis of diseases, especially cancers. Due to the advantages of high affinity and specificity, good stability and low cost, aptamers have been widely used for biomolecules detection. However, most of the aptamer fluorescent probes are based on single signal mode, making it difficult for quantitative detection of biomolecules in living cells due to the environmental effects. Compared to intensity-based probes, the ratiometric probes using the built-in correction of two different emissions could reduce the environmental effects on detection. In this project, a series of ratiometric fluorescent nanoprobes with high sensitivity, good selectivity, high biocompatibility, good stability and high membrane penetration ability were constructed for ratiometric measurement of biomolecules in living cells by taking advantage of ratiometric self-calibration ability of the probe, the cell-specific recognition ability of aptamer, the good biocompatibility and cell membrane permeability of DNA nanostructures. This design strategy could solve problems of the traditional aptamer nanoprobes which suffer from poor stability, high cytotoxicity and difficult quantitation of intracellular biomolecules. Thus, the proposed design could achieve real-time imaging and quantifying of intracellular biomolecules, which are expected to provide valuable fundamental knowledge and technical support for studying the molecular mechanism underlying diseases.

实时监控生物体细胞内的生物分子对于疾病早期诊断具有重要的意义。核酸适配体由于亲和力高、特异性好、稳定性好、容易保存等优点广泛用于生物分子的传感检测。然而目前核酸适配体荧光探针多数是基于单个荧光发射峰的信号增强，易受环境因素干扰而难以实现活细胞内生物分子的定量检测。比率型荧光探针以双荧光发射峰的强度比值为信号输出，能够消除环境因素的干扰。本项目拟利用比率型荧光探针的比例自校准特征，结合核酸适配体特异性识别能力与DNA纳米结构生物相容性好和细胞膜穿透性强的优良性能，构建一系列灵敏高、选择性好、生物相容性好、稳定性好、膜穿透能力强的荧光能量共振转移（FRET）比率型荧光纳米探针，解决传统核酸适配体纳米探针存在的细胞毒性、稳定性差以及难以用于细胞内定量检测等问题，实现细胞内生物分子的定量检测及成像研究，为疾病的早期诊断及疾病发病机理研究提供有价值的理论依据和技术支持。

核酸适配体由于亲和力高、特异性好、稳定性好、容易保存等优点广泛用于生物分子的传感检测。然而目前核酸适配体探针多数是基于单个的信号峰，易受环境因素干扰，因此重现性差。本项目利用比率型探针的比例自校准特征，结合核酸适配体特异性识别能力与DNA纳米结构生物相容性好的优良性能，构建了一系列灵敏高、选择性好、生物相容性好和稳定性好的比率型纳米探针。比率型探针以强度比值为信号输出，能够消除环境因素的干扰，解决了传统核酸适配体纳米探针存在的细胞毒性、稳定性差以及难以用于细胞内定量检测等问题，为疾病的早期诊断及疾病发病机理研究提供有价值的理论依据和技术支持。