Nanomechanical imaging of protein dynamics via programmable DNA interactions

项目摘要 拟议的研究将解决蛋白质和蛋白质动态结构成像的挑战 溶液中的络合物。荧光显微镜是目前用于化学特异性的主要技术。 在溶液中成像。尽管超分辨率技术已经极大地推动了 尽管荧光显微镜技术已经发展，但获得单分子和分子的图像仍然是一个重大挑战。 复合物的分辨率可与电子显微镜和X射线晶体学相媲美。我们提出的 研究将开发一种原子力显微镜（AFM）平台，利用短寿命DNA/DNA 相互作用以实现个体内化学基团的成像和三维重建 蛋白质，多酶复合物，蛋白质，RNA和DNA之间的复合物， 分辨率和生理相关条件。提高成像速度将能够观察到 结构转型。这些能力是通过一系列创新实现的：（1）使用短片段 DNA作为成像标记，其中DNA序列编码颜色信息，（2）使用短寿命， 远离提高分辨率和化学特异性的平衡相互作用，以及（3）使用特殊的 设计的T形原子力显微镜杠杆，允许检测短暂的相互作用。的 由此产生的显微镜平台将通过提供结构化的 通过现有方法难以研究的蛋白质和蛋白质复合物的信息。

PROJECT SUMMARY The proposed research will address challenges in imaging dynamic structures of proteins and protein complexes in solution. Fluorescence microscopy is currently the dominant technique for chemically specific imaging in solution. Although super-resolution techniques have dramatically pushed the resolution limits of fluorescence microscopy, it is still a major challenge to obtain images of single-molecules and molecular complexes with resolutions comparable to electron microscopy and X-ray crystallography. Our proposed research will develop an atomic force microscopy (AFM) platform that harnesses short-lived DNA/DNA interactions to achieve imaging and three-dimensional reconstruction of chemical groups within individual proteins, multi-enzyme complexes, and complexes between proteins, RNA and DNA with near atomic resolution and in physiologically relevant conditions. Enhancing the imaging speed will enable observation of structural transitions. These capabilities are enabled by a series of innovations: (1) the use of short fragments of DNA as imaging labels, wherein the sequence of DNA encodes color information, (2) the use of short-lived, far from equilibrium interactions that boost resolution and chemical specificity, and (3) the use of specially designed T-shaped atomic force microscopy cantilevers that allow detecting short-lived interactions. The resulting microscopy platform will have a broad impact across many biomedical fields by providing structural information about proteins and protein complexes that are difficult to study via the current methods.