Modified Photoproteins as Labels and Molecular Switches in Bioanalysis

描述（由申请人提供）：生物工程的不断发现和更有效的分子生物学方法使科学家能够创造出具有独特和独特性质的新型设计生物分子。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高灵敏度技术的需求。生物发光光蛋白，如aequorin，具有巨大的潜力，为解决这一新挑战提供了一个解决方案，因为它们可以在低浓度下被检测到，并且根据所使用的蛋白质变体具有不同的发射波长，这一特性可以在多重分析中利用。我们现在建议制备新的aequorin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到的生物分子。具有光学特性的蛋白质分子开关是另一种设计生物分子，在靶配体存在的情况下，通过“开/关”信号表现出改变的反应。这些分子可用于各种应用，例如用于体外和体内检测的纳米传感器的开发。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aequorin变体提供的生物发光结合起来。为提出的工作制定的假设是基于我们在当前资助期间获得的知识，并研究了一系列计算和合成方法以及针对发色团电子环境改变的基因工程策略的使用，这些方法应该导致具有广泛光谱特性的新生物发光蛋白和分子开关。这些光蛋白将用于开发重要生物分子的检测方法。最后，我们将研究新制备的生物发光分子开关在生物分子的多重分析和单细胞分子的同时分析中的应用。我们预计，新的光蛋白将为体外和体内生物传感、成像和多重分析提供新的使能技术，这些技术比现有方法具有许多优势。

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.