框架核酸组合探针界面精确调控及其在长片段ctDNA检测中的应用

Circulating tumor DNA (ctDNA) carries the key genetic information of the tumor. Achieving the sensitive analysis of ctDNA plays an important role for the early diagnosis and prognosis of tumor. However, how to avoid the interference of short ctDNA fragments for achieving long fragment ctDNA detection with high sensitivity and specificity is the major bottleneck that restrict long fragment ctDNA to use for liquid biopsy. In this project, based on the framework nucleic acid interface regulation technique, framework nucleic acid combinatorial probe with precisely controlled probe spacing is constructed. Combined with the enzyme catalyzed signal amplification system, long fragment ctDNA detection strategy with high sensitivity and high specificity is developed. Using framework nucleic acid as the building blocks for combinatorial probe can avoid the non-specifically interaction between probes and improve the interface recognition efficiency by precisely regulating the nano-space between probes. Using combinatorial probe to recognize and capture long fragment ctDNA can improve the long fragment ctDNA detection specificity by avoiding the interference of ctDNA fragments. The success implementation of the project will be helpful to provide new means of diagnosis for tumor early diagnosis, treatment effect evaluation and prognosis of tumor.

循环肿瘤DNA（ctDNA）携带着肿瘤的关键基因信息，实现对ctDNA的灵敏检测对肿瘤早期诊断及预后判断有重要意义。然而，如何排除ctDNA碎片的干扰，实现对长片段ctDNA的高灵敏、高特异的检测是制约ctDNA应用于实际液体活检的主要瓶颈问题之一。本项目拟利用框架核酸界面调控技术，构建探针间距精确可控的框架核酸组合探针，结合酶催化信号放大系统，发展高灵敏度、高特异性的长片段ctDNA检测方法。以框架核酸作为组合探针构建基元，可以保证探针之间纳米间距精确可调，避免探针之间发生非特异性的相互作用，提高探针的界面识别效率；用组合探针对长片段ctDNA进行识别和捕获，能够有效避免ctDNA碎片对长片段ctDNA检测的干扰，提高对长片段ctDNA检测的特异性。项目的成功实施将为肿瘤的早期诊断、治疗效果评估及预后复查等提供重要检测手段。

利用框架核酸精准定位能力，我们构建了一系列框架核酸探针，用于生物传感及细胞成像。首先借助DNA精确自组装及调控能力，构建了基于框架核酸的拓扑核酸探针，实现了核酸探针价态和空间分布的精确调控，将探针的识别性能增强了19倍；利用该探针实现了金纳米颗粒在流动界面的几何重排，此外，利用该探针研究了不同价态探针与细胞膜受体之间的相互作用，并实现了探针与受体之间结合强度的调控；进一步地，我们探究了框架核酸探针在复杂体系中的稳定性，发现在全血中孵育1小时探针的完整性可以保持~80%；同时，我们利用该框架核酸探针实现了病人血样中循环肿瘤细胞的无损捕获与释放；为了实现核酸的高通量检测，我们发展了基于框架核酸的微阵列检测芯片，为实现病人样本的高通量检测提供了新策略。