Modified Photoproteins as Labels and Molecular Switches in Bioanalysis

修饰光蛋白作为生物分析中的标签和分子开关

• 批准号: 8291039
• 负责人: Sylvia Daunert
• 金额: $ 34.5万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8291039
• 关键词: Aequorin; Affect; Amber; Amino Acid Sequence; Amino Acids; Apoproteins; Binding; Binding Proteins; Biological; Biological Assay; Biological Markers; Bioluminescence; Biomedical Engineering; Biosensing Techniques; Biotechnology; Categories; Cells; Characteristics; Codon Nucleotides; Computer Analysis; Detection; Development; DsRed; Electronics; Engineering; Environment; Family; Funding; Genetic Code; Genetic Engineering; Goals; Hydrogen Bonding; Image; In Vitro; Knowledge; Label; Lead; Libraries; Life; Ligands; Liquid substance; Luciferases; Luminescent Proteins; Methods; Microfluidics; Molecular; Molecular Biology; Nanotechnology; Optics; Peptide Sequence Determination; Performance; Physiological; Play; Post-Translational Protein Processing; Preparation; Property; Protein Binding Domain; Proteins; Protocols documentation; Research; Resolution; Role; Sampling; Scaffolding Protein; Scheme; Scientist; Series; Signal Transduction; Site; Solutions; Source; Structure; System; Techniques; Technology; Time; Transfer RNA; Variant; Work; analog; assay development; base; chromophore; coelenterazine; computer studies; design; directed evolution; hybrid protein; in vivo; luminescence; molecular recognition; mutant; nanoscale; nanosensors; obelin; protein structure; public health relevance; response; scaffold; tool

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.

描述（由申请人提供）：生物工程和更有效的分子生物学方法中的连续发现使科学家可以创建具有独特和独特特性的新设计器生物分子。在这方面，生物纳米技术和纳米级分析变得越来越普遍，从而提高了对具有高灵敏度的技术的需求，可以检测生物样品中的生物分子。生物发光光蛋白（例如Aequorin）具有巨大的潜力，可以为新挑战提供解决方案，因为它们可以在低浓度下检测到它们，并且根据所使用的蛋白质变体具有不同的发射波长，该特性可以在多重分析中利用。现在，我们建议准备新的Aquorin变体，以扩大它们在生物分析中的使用范围，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类型的设计器生物分子，在靶配体的存在下，它表明了由“ ON/OFF”信号所表现出的改变的响应。这些分子在多种应用中很有用，例如用于体外和体内检测的纳米传感器的发展。为此，我们计划设计和开发生物发光的分子开关，这些开关结合了结合蛋白的识别特性与Aequorin变体提供的生物发光。为拟议工作制定的假设是基于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的使用，以及针对生物基团电子环境改变的基因工程策略，这些策略应导致新的生物发光蛋白质和具有广泛范围范围的生物发光蛋白质和分子开关。这些光蛋白将用于开发重要的生物分子测定法。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中以及单个细胞中分子的同时分析中的使用。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启示技术，这些技术比现有方法具有许多优势。 公共卫生相关性：叙事纳米级分析的越来越重要，提高了对可以检测生物样品中生物分子的高度敏感系统的需求。生物发光光蛋白（例如Aequorin）具有巨大的潜力，可以解决这一新挑战，因为可以在生理液的浓度很低的情况下检测到它们。在这方面，我们计划设计和制备基因修饰的光蛋白，构成了为检测相关生物分子和生物标记板的基础，用于体外和体内应用。