Modified Photoproteins as Labels and Molecular Switches in Bioanalysis

描述（由申请人提供）：生物工程和更有效的分子生物学方法的不断发现使科学家能够创造具有独特和独特性质的新设计生物分子。在这方面，生物纳米技术和纳米级分析正变得越来越普遍，提高了对能够检测生物样品中的生物分子的具有高灵敏度的技术的需求。生物发光发光蛋白，如水母发光蛋白，具有很大的潜力，提供了一个解决这个新的挑战，因为它们可以在低浓度下检测，并具有不同的发射波长，这取决于所使用的蛋白质变体，一个属性，可以利用多重分析。我们现在建议制备新的水母发光蛋白变体，以扩大其在生物分析中的应用范围，从而允许检测其他技术无法检测的生物分子。具有光学性质的蛋白质分子开关是另一种类型的设计生物分子，其在靶配体的存在下，表现出由“开/关”信号表现的改变的响应。这些分子可用于多种应用，例如用于开发用于体外和体内检测的纳米传感器。为此，我们计划设计和开发生物发光分子开关，将结合蛋白的识别特性与水母发光蛋白变体提供的生物发光结合起来。为拟议的工作制定的假设是基于在我们目前的资助期间获得的知识，并调查使用一系列的计算和合成方法沿着基因工程策略，针对改变的发色团的电子环境，应该导致新的生物发光蛋白质和分子开关具有广泛的光谱特性。这些发光蛋白将被用于重要生物分子的分析开发。最后，我们将探讨新制备的生物发光分子开关在生物分子多重分析和单细胞分子同时分析中的应用。我们预计，新的发光蛋白将提供新的使能技术，用于体外和体内生物传感，成像和多重分析，这些技术与现有方法相比具有许多优势。 公共卫生相关性：叙述纳米级分析的重要性日益增加，提高了对能够检测生物样品中生物分子的高灵敏度系统的需求。生物发光蛋白，如水母发光蛋白，具有很大的潜力，提供了一个解决这一新的挑战，因为它们可以在非常低的浓度在生理液体中检测。在这方面，我们计划设计和制备转基因发光蛋白，为检测相关生物分子和生物标志物的体外和体内应用奠定基础。

DESCRIPTION (provided by applicant): Continuous discoveries in bioengineering and more efficient molecular biology methods have allowed scientists to create new designer biomolecules with unique and distinct properties. In that regard, bio- nanotechnology and nanoscale analysis are becoming increasingly prevalent, raising the demand for techniques with high sensitivity that can detect biomolecules in biological samples. Bioluminescent photoproteins, such as aequorin, possess great potential to provide with a solution to this new challenge because they can be detected at low concentrations, and have different emission wavelengths depending on the protein variant used, a property that can be exploited in multiplex analysis. We now propose to prepare new aequorin variants to broaden the scope of their use in bioanalysis, thus allowing for detection of biomolecules that are not detectable by other technologies. Protein molecular switches with optical properties are another type of designer biomolecules that, in the presence of a target ligand, demonstrate an altered response manifested by an "on/off" signal. These molecules can be useful in a variety of applications, such as in the development of nanosensors for in vitro and in vivo detection. To that end, we plan to design and develop bioluminescent molecular switches that incorporate the recognition properties of binding proteins with the bioluminescence afforded by the aequorin variants. The hypotheses formulated for the proposed work are based on knowledge gained during our current funding period, and investigate the use of a series of computational and synthetic approaches along with genetic engineering strategies targeting the alteration of the electronic environment of the chromophore that should lead to new bioluminescent proteins and molecular switches with a wide range of spectral properties. These photoproteins will be employed in the development of assays for important biomolecules. Finally, we will investigate the use of the newly prepared bioluminescent molecular switches in the multiplex analysis of biomolecules and in the simultaneous analysis of molecules in single cells. We anticipate that the new photoproteins will provide with new enabling technologies for in vitro and in vivo biosensing, imaging, and multiplex analysis that have a number of advantages over existing methods. PUBLIC HEALTH RELEVANCE: NARRATIVE The increasing importance of nanoscale analysis has raised the demand for highly sensitive systems that can detect biomolecules in biological samples. Bioluminescent photoproteins, such as aequorin, possess great potential to provide a solution to this new challenge because they can be detected at very low concentrations in physiological fluids. In that regard, we plan to design and prepare genetically modified photoproteins that form the basis of enabling technologies for the detection of relevant biomolecules and panels of biomarkers for in vitro and in vivo applications.