单分子荧光应用级联反应催化性质研究

Organisms have evolved a special chemical conversion pathway which both timely and spatially regulate metabolism, reducing energy and substance losses caused by competitive reaction, avoiding cell injury caused by poisonous intermediates, therefore, guaranteeing the effectiveness of metabolic reactions. The orderly-arranged cascade reaction has attracted extensive attentions, researchers have discovered the obvious increase of enzyme activity when enzymes are in proximity from in vitro construction of cascade systems. Researchers have developed many models to explain the reaction mechanism, however, limited by assemble efficiency as well as determination and separation methods, traditional assays hardly distinguish real cascade enzymes from the free ones which hinder the detection of real reaction velocity of cascade enzymes. Therefore, this project proposes to establish a novel detection method to explore catalytic behaviors of cascade reaction on single molecule level. The cascade enzymes are orderly confined on DNA origami to achieve accurate space control. Through single molecule microscopy, catalytic dynamics of single cascade system are real-time imaged and monitored. Thus, we can make extensive study on increasement mechanism of reaction rate caused by close distance between cascade enzymes. This project is valuable to design novel single molecule fluorescence detection methods, as well as provide solid theoretical foundation and experimental support for single molecule enzymology.

生物体进化的化学转化方式可以从时间和空间上调控生化代谢,减少细胞内不同竞争反应所造成的能源及物质的浪费，避免有毒代谢中间物对细胞的损伤。这种有序的级联反应引起了强烈的关注，科研人员在体外建立级联反应体系时发现，当级联酶相互接近时，酶的活性发生显著提高。尽管已建立了很多方法试图解释这种活性提高的机理，然而受限于组装产率，及测定、分离方法，传统的测定很难真正将级联酶与未发生级联的酶区分开，很大程度影响了对级联反应速率的检测，故而所的数据在一定程度上并不能反应真实的级联催化反应。基于以上原因，本课题拟利用DNA折纸建立有序排列的级联酶体系，实现级联酶间距的精确调控。通过利用单分子荧光的方法，对级联反应进行实时成像分析，得到单个级联体系催化的动态信息，从而对级联增强催化速率的机理进行探索。本项目的开展有益于构建新型单分子荧光的检测方法，为单分子及酶学研究提供坚实的理论和实验支撑。

级联酶相互接近时，酶的活性发生显著提高。然而受限于组装产率，及测定、分离方法，传统的测定很难真正将级联酶与未发生级联的酶区分开，因此影响了对级联反应速率的检测，所的数据在一定程度上并不能反应真实的级联催化反应。本课题利用DNA折纸建立有序排列的级联酶体系，实现级联酶间距在10-70 nm的精确调控。建立了单分子条件下界面修饰、数据采集及数据分析的系统，采用TIRF显微镜对级联反应进行实时成像分析，得到单个级联体系催化的动态信息，研究了酶间距、拥挤试剂对级联酶催化速率的影响，并依此建立了肿瘤小分子标志物肌氨酸的检测方法。此外，我们进一步的设计了集肿瘤诊断和治疗一体化的纳米探针，并在体内外验证了纳米探针在肿瘤成像和肿瘤杀伤的效果。本项目的研究对构建新型单分子荧光的检测方法，单分子及酶学研究以及肿瘤诊疗提供了坚实的理论和实验支撑。