核酸纳米条纹带“一键式”组装及恶性肿瘤基因诊断新方法研究

Lung cancer has the highest incidence rate and highest mortality rate among malignant tumors, becoming the leading cause of cancer deaths and the biggest threat to human health and life safety. The mutation of p53 gene is the most common phenomenon occurring early in carcinogenesis and tumor progression, being the biomarker for diagnosis of lung cancer. It is well known that DNA nanostructures are of great importance in life science, bio-nanomaterials, disease diagnosis and target therapy. However, the assemblies of DNA nanostructures often require considerable number of nucleotide components; for example, tens or hundreds of DNA species. Moreover, tedious assembling procedures and stringent assembling conditions are involved. To detect selectively and sensitively the mutant p53 gene, the present project will focus on the following aspects: an efficient technology would be developed to prepare the DNA nanoribbon via one-button-operation, and only several or less (e.g., two) types of nucleotide molecules are used, addressing the technical bottleneck encountered in the construction of DNA nanostructures; then, we would attempt to attach DNA and/or Fc-modified gold nanoparticles (GNPs) or conjugated macromolecules with high biological activity onto DNA nanoribbon via the sticky end-based hybridization. Moreover, the preparation of this complicated DNA nanoribbon could be accomplished via one-button-operation even on the substrate surface. Along this line, the electrochemical sensing system is expected to be capable of being developed for the genetic diagnosis of diseases. Via utilizing the developed systems, the mutant p53 genes are expected to be selectively identified, providing the methods for reference and preliminary scientific data for the research of early discovery, early diagnosis and early treatment of malignant tumors.

肺癌发病率与死亡率居全球恶性肿瘤第—位，是人类健康与生命安全的最大威胁。p53抑癌基因变化是肺癌发生过程中最常见的早期分子事件之一。核酸纳米器件在生命科学、生物纳米材料、医疗诊断与药物靶向治疗等学科领域中具有重要的作用。可是，传统组装技术往往需要几十到几百种核酸元件，而且组装步骤繁杂、条件苛刻。本项目以p53抑癌基因的特异性高灵敏检测为出发点与终极目标，发展核酸纳米条纹带“一键式”组装技术，并将参与组装的核酸元件减少到几种，甚至两种，解决传统的核酸纳米器件组装技术的瓶颈问题；借助粘性末端杂交作用，将金纳米颗粒衍生物或者生物活性分子有序排列在核酸纳米条纹带上；然后发展金纳米颗粒衍生物或生物活性分子-核酸纳米条纹带界面修饰一键式组装技术，构建基因诊断电化学检测新方法，用于p53 抑癌基因的识别与分析，为肺癌的早发现、早诊断与早治疗的研究提供方法借鉴与数据支持。

根据2019年《中国恶性肿瘤流行情况分析》，全国恶性肿瘤新发病例约有392.9万例，死亡病例约233.8万例，居全球首位，每年所需医疗费用超过2200亿，癌症防控迫在眉睫。p53基因编码p53蛋白，是与癌症发生、发展与预后直接相关的突变率最高的基因之一。.通过设计分子内有规律杂交的RCA产物，本项目发展了核酸纳米条纹带，不但用侧面的双寡核苷酸对其进行规整、安装核酸适体，而且在金纳米颗粒表面进行原位组装；通过普通碱基互补片段纵向交替杂交和回文粘性末端交叉横向交联粘合作用，只用两个DNA发夹探针就可“一键式”组装成三维核酸纳米片。由于组装简便、三维空间能高容量容纳电化学信号分子，三维核酸纳米片被用来构建电化学传感界面，用于对p53基因点突变的特异性检测，并评价其在恶性肿瘤基因诊断中的应用潜力。.本项目还研发了聚合链取代回文分子信标、细胞内自动运行的DNA分子机器、四面体保护的荧光成像探针、在球形基底表面运行的具有抗降解保护盾的DNAzyme步行器和能沿着Y型、X型钢化多边形轨道运行的DNAzyme步行器等, 用于KRAS基因、STAT3基因和miRNAs等肿瘤相关基因的检测或细胞内成像。.此外，利用回文序列简化组装过程，本项目用几条甚至一条寡核苷酸序列，还组装了核酸纳米棒、核酸纳米线、核酸纳米珠串、分枝状核酸纳米结节、单层核酸纳米阵列和抗降解核酸纳米球等，发展细胞内分析方法、活体成像系统和癌症靶向治疗药物载体等。.总之，本项目完成了恶性肿瘤基因检测、荧光成像和靶向治疗等多项课题研究，发表SCI论文34篇，其中顶级期刊学术论文5篇，JCR一区期刊论文26篇；获国家发明专利3件，培养硕士、博士和博士后等科技人才38名，参与全国性大型学术会议12人次；项目负责人获批国家自然科学基金面上项目和福建省自然科学基金重点项目，并入选福建省高校领军人才计划；项目组积极开展国际研究合作，取得4项标志性学术成果，并同生物医药公司就成果转化达成初步协议，打开了科技成果转化的契机。