基于DNA仿真双向链置换组合回路的活细胞多水平高灵敏成像分析

Monitoring multilevel markers in living cell provides diverse molecular characteristics for biological research and clinical diagnostics. However, integrated probe design, intracellular signal amplification and single-nucleotide specificity remain challenging. In this project, we will construct DNA simulation-guided bidirectional strand displacement combinational circuits for highly sensitive and ultrahigh specific fluorescence imaging of telomerase and miRNA in living cell. All probes are delivered into cells by commercial transfection agents. Both bidirectional strand displacement probe and miRNA simulated probe are designed and generated as DNA duplexes by annealing bottom strands with top strands. The former is labeled with two fluorophores and one quencher, and contains two toeholds at the 3' end and 5' end of bottom strand for the recognition of telomerase and miRNA, respectively. The later one simulated the former, and is similar to its 5' part with single nucleotide variation for the binding of corresponding miRNA homologous sequence. The telomerase extending product and target miRNA recognize each toehold and initiate bidirectional strand displacement reactions, producing two new toeholds at the bottom strand. Subsequently, the helper probes for telomerase and target miRNA each induce second strand displacement reactions from new toehold sites, releasing the target sequences and fluorophore-labeled top strands. Two fluorescence signals are observed simultaneously. This copy of target sequence can bind another bidirectional probe molecule to initiate next strand displacement reactions. In this way, combinational circuits are formed with two increased detection signals. On the other hand, corresponding miRNA homologous sequence with single nucleotide variation is arrested by the simulated probe, and fails to induce fluorescence signal. Therefore, the highly sensitive imaging of multilevel markers with ultrahigh specificity in living cell is realized.

活细胞多水平成像提供多层次分子信息，对生物学和临床医学研究至关重要。本项目围绕胞内信号放大、单核苷酸分辨、多靶标探针设计等难题，拟构建DNA仿真双向链置换组合回路，建立活细胞多水平成像策略。通过序列设计及杂交组装，形成双向链置换组合探针和仿真链置换探针；前者可同时识别端粒酶和靶标miRNA，后者仿照前者设计，用于结合并消耗单核苷酸变异miRNA。探针经转染入胞后，端粒酶延伸序列和靶标miRNA分别从组合探针两端的toehold序列启动上游链置换反应，并形成两个新toehold位点。随后，相应的单链辅助探针可识别上述位点，分别引发下游链置换反应，释放靶标序列，产生对应荧光信号。两种靶标序列均可循环作用多分子双向探针，形成组合回路，放大端粒酶和miRNA检测信号；同时，单核苷酸变异miRNA快速结合仿真探针，而不作用组合探针，无法触发信号，从而实现活细胞多水平一体化、高灵敏、超特异成像分析。

活细胞多水平成像提供多层次分子信息，对生物学和临床医学研究至关重要。本项目围绕胞内高效信号放大、单核苷酸分辨、多靶标一体化探针设计等难题，构建了DNA仿真双向链置换组合回路，建立了活细胞多水平成像策略。通过一体化探针识别多水平靶标，显著放大检测信号、抑制同源序列干扰，实现了活细胞端粒酶和miRNA的高灵敏、超特异联合成像分析。并进一步设计了三价和四价DNA纳米回路，证明了已构建DNA回路系统的可扩展性，实现了活细胞端粒酶和多种miRNA的联合成像分析。本项目构建的活细胞熵驱动DNA纳米回路，可响应多种肿瘤标志分子，通过DNA计算生成多位的二进制编码，实现了活细胞中多种非编码调控RNA与端粒酶等肿瘤标志物的智能化分析。联合多种痕量细胞共培养微流控芯片，将肿瘤相关分子标志或复杂事件转化为数字信息的精确计算，实现了正常、致癌低转移性与高转移性乳腺细胞的分型及相关肿瘤进程区分。替换相应DNA组装与信号放大模块，即可实现多样化纳米机器或装置的定制，满足活细胞内多靶标的联合精准成像、多样化细胞过程监测和生物调控需求，适用范围广泛。本项目相关成果已发表SCI论文11篇，申请国家发明专利4项，其中已获授权2项，具有实际应用前景和推广开发价值，可制备相关生物分析试剂盒，应用于生物学基础研究与临床领域，如分析生物大分子结构与功能、细胞信号转导、肿瘤血管形成等。该方法或试剂盒整合微流控芯片、试纸条等成熟检测器件，有望制备小型或便携的分析检测设备，具有很高的经济和社会效益。

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