DNA逻辑电路调控的新型智能分析器件的设计与应用

Recognizing the surroundings is a main challenge for in vivo applications of DNA aptasensors to in situ analysis. Towards this goal, we design environment-responding intelligent DNA devices controlled by logic circuits for analysis, based on the fact that the intracellular ATP concentration is several orders of magnitude higher than the extracellular. With a DNA tetrahedron as the scaffold, we integrate a DNA computing network consists of three AND logic gates with ATP, thrombin, lysozyme aptasensors. The difference between intracellular and extracellular ATP concentrations can induce a DNA structural change of ATP aptasensor, by which the environmental information is delivered to DNA logic circuit that performs the information processing and then logic control of the secondary structures of thrombin and lysozyme aptasensors. This makes the two sensors able to distinguish the intracellular and extracellular surroundings, and so detect target proteins only inside cells. The above DNA structural changes can be monitored by gel electrophoresis, fluorescence resonance energy transfer, atomic force microscopy and circular dichroism. Further, we apply the designed DNA intelligent device to in situ cell analysis, to demonstrate its application potential in bioanalysis.

环境识别判断是DNA适配体传感器应用于体内原位分析时所面临的一个重要挑战。针对这一问题，我们在细胞体内外ATP浓度相差几个数量级的事实基础上，设计了一种对细胞环境响应的DNA逻辑电路调控的智能分析器件。我们以DNA四面体为脚手架，将三个AND逻辑门串联和并联组成的DNA计算电路与ATP、凝血酶、溶菌酶等适配体传感器有机整合在一起。细胞体内外ATP浓度变化可诱导ATP适配体传感器发生DNA结构变化，以此方式将外部环境信息传递给DNA逻辑电路，由其做出判断，并逻辑控制凝血酶、溶菌酶适配体传感器的二级结构，使其能够区分细胞体内与体外，且只在细胞体内执行目标蛋白的分析检测任务。以上过程中的DNA结构变化可用凝胶电泳、荧光共振能量转移、原子力显微镜及圆二色谱等手段进行监控。我们进一步将设计的DNA智能分析器件应用到细胞活体原位分析方面，以显示其在生物传感分析方面的应用潜力。

本项目主要围绕细胞环境识别的智能DNA逻辑分析器件的构建与体内外应用开展工作。首先，我们构建了一系列ATP适配体纳米传感器，探讨ATP诱导DNA纳米器件发生结构变化的能力；基于此，我们把ATP适配体与G-四链体、i-motif等功能核酸作为逻辑控制单元，用于构建对ATP、K+、H+等细胞内源物质响应的DNA计算电路，能够识别细胞溶解液、溶酶体等体内外细胞环境。在此基础上，我们发展了一系列DNA逻辑电路调控的新型智能纳米器件，在体外或细胞活体内工作良好。在其应用方面，我们发现原方案中凝血酶和溶菌酶不适合细胞活体原位检测，因此，我们把该设计的智能分析器件的应用对象调整为疾病相关的核酸标志物如DNA基因和mRNA等，取得了良好效果。进一步地，我们把DNA逻辑电路控制的智能纳米器件用于细胞体内靶向传输分子载荷，探讨其在细胞基因表达的逻辑调控方面的应用前景。