非蛋白酶核酸自催化扩增反应用于高灵敏度miRNA检测的研究

乳腺癌的早期诊断和疾病分型对小分子核酸（miRNA）的高效检测提出了更高的要求。针对现有miRNA检测方法灵敏度低、选择性低和易受干扰的难题，本项目拟构建具有幂指数信号扩增效率的非蛋白酶恒温核酸自催化扩增反应，用于miRNA的体外检测和活细胞分析；深入探究临床血清和复杂组织样本内的miRNA平行多重分析，考察该信号扩增技术对乳腺癌早期诊断和病理分型的适用性和准确性；构建恒温核酸自催化反应动力学模型，从分子水平上探究其反应机理，用于辅助核酸分子探针的精确设计和反应优化，同时采用实验结果修正动力学模型；最后将靶向特定信使RNA（mRNA）的核酸酶序列整合到核酸自催化扩增体系中，实现内源miRNA诱导激活扩增核酸酶，进而用于选择性剪切mRNA，最终实现高效基因沉默治疗目的。该核酸自催化扩增核酸酶集“传感-响应-处理”为一体，将会在癌症的智能诊疗领域得到广泛应用。

The earlier diagnosis and classification of breast cancer requires an efficient detection sensing strategy for microRNA (miRNA). The existing miRNA detection methods are however constrained by their low sensitivity and selectivity, as well as their susceptibility to external interference. Here we will develop two kinds of enzyme-free isothermal autocatalytic DNA amplifiers for sensitive in vitro miRNA detection and intracellular miRNA imaging. With powerful exponential signal amplification efficiency, these autocatalytic DNA amplifiers could thereby realize the sensitive miRNA assay in complex clinical serum and tissue samples. The applicability and accuracy of these two DNA amplifiers are furtherly investigated for early diagnosis and pathological classification of breast cancer. Then the autocatalytic reaction models are established for understating the underlying reaction mechanism at the molecular level, and are expected to assist in the optimization of DNA probes. On the contrary, these experimental results could also contribute to the appropriate adjustment of our autocatalytic reaction models. Finally, the messenger RNA (mRNA)-specific DNAzyme is integrated into the present autocatalytic DNA amplifiers to achieve the endogenous miRNA-amplified generation of therapeutic DNAzyme, which could selectively cleave mRNA target to achieve an efficient gene silencing operation. As a smart “sense-act-treat” theranostic tool, the enzyme-free isothermal autocatalytic DNAzyme amplifiers show great potential for earlier cancer diagnosis and therapy in the near future.