Hairpin DNA在基片表面发卡状构型的高效形成、可控调控及其对弱结合刺激的响应研究

In view of their importance in fundamental research and in real application, surface-tethered hairpin DNA (i.e., HPD) probes are chosen as the research theme of this project. Particulary, this project is aimed to solve the difficulties existing in the assembly, conformation switching and sensing application of HPD probes and to finally develop high-performace biochips based on them. To reach such goals, we plan to develop in-situ observation, quantitative evaluation and controllable assembly techniques for actualizing the high yield formation and controllable modulation of the stem-loop conformations of the HPD probes tethered on substrates. We also plan to track the overall process and the related influence factors regarding conformation switching of the surface-tethered HPD probes, in order to uncover the underlying mechanism and to develop effective modulation technique for such process. We still plan to disclose the weak interactions between the HPD probes and non DNA targets and to design some efficient protocols for transducing and amplifying such interactions accordingly, so that we can finally achieve highly sensitive and specific detection of such targets. This project is therefore of particluar importance in developing high-performance sensing devices, well understanding the molecular behavior of the DNA probes on substrates, and better functionalizing DNA probes. The main novelty of this project lies in it not only affords a simple method for the high-yield formation and quantaitative evaluation of the stem-loop conformations of the HPD probes tethered on substrates but also provides an effective strategy for sceening the weak binding events.

鉴于在研究和应用中的重要价值，本项目选取固定在基片上的Hairpin DNA（HPD）探针为研究对象，着眼于解决其在组装、构型变换以及传感应用中面临的难题，构建高性能HPD基生物传感器。为此，本项目通过建立HPD在基片表面分子构型的原位观测、定量评价、可控组装技术，来实现其发卡构型在基片表面的高效形成；通过追踪HPD在基片表面发卡构型的演变过程及其关联因素的影响，来揭示其构型变换机制和发展可控调控技术；通过剖析HPD与非DNA类靶标物间的弱相互作用，来设计高效的构型转换、信号转导与放大策略，实现对弱结合刺激的高灵敏和特异性检测。因此，本项目无论是对发展高性能生物传感器，还是对理解DNA分子界面行为与构型，以至对DNA探针功能设计与应用，都有重要意义。本项目的特色之处在于开发了HPD探针构型的定量评价方法，建立了具有自修复和强制消除双功能的组装新工艺，提出了HPD探针对弱结合刺激的响应策略。

Hairpin DNA（以下简称hpDNA）是指一条能够形成茎环相连的“发卡状”构型的脱氧核苷酸单链，因其对靶标链的响应比直链DNA（即ssDNA）更特异、更灵敏、更显著，因而常作为分子探针固定在基片表面用于构造多种传感器件与分子器件。鉴于其重要的应用价值，本项目选取固定在基片上的hpDNA探针为研究对象，着眼于解决其在组装、构型变换以及传感应用中面临的难题，构造出基于hpDNA探针的高性能生物传感器。为此，本项目通过建立新颖的“Exo I水解辅助” hpDNA构型定量评测和可控组装技术，实现了发卡构型在基片表面的高效形成；通过建立“光电双通道”荧光观测平台原位追踪基片表面发卡构型的演变过程及其关联因素的影响，从微观尺度初步揭示了hpDNA的构型变换机制，开发了基于界面空间与环境效应的构型调控技术。此外，本项目还通过系统剖析hpDNA与非DNA类靶标物间相互作用强度及特征，设计了几种高效的构型转换、信号转导与放大策略，并辅以Exo I水解助力的背景信号消除技术，实现了hpDNA基生物传感器对弱结合刺激的高灵敏和特异性响应。本项目的特色之处在于，得益于对Exo I水解基片表面DNA行为的深入考察，我们以“Exo I的选择性水解能力”为破解问题的主要抓手，创新性解决了hpDNA在基片表面的构型评测、高效组装以及传感性能提升等一系列难题。因此，本项目无论是对发展高性能生物传感器，还是对理解DNA分子界面构型和Exo I的酶解行为，以至对DNA探针功能设计与应用，均具有重要意义。