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New Classes of Genetically-Encoded Fluorescence Anisotropy Probe

新型基因编码荧光各向异性探针

基本信息

批准号：
8919364
负责人：
GERARD MARRIOTT
金额：
$ 19.63万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8919364
关键词：
Achievement Actins Affinity Amino Acids Anisotropy Bacteria Binding Binding Proteins Biological Cells Complex Detection Development Diagnostic Doctor of Philosophy Drug Interactions Exhibits Flavoproteins Fluorescence Fluorescence Anisotropy Fluorescence Polarization Fluorescence Resonance Energy Transfer GTP Binding Goals Guanosine Triphosphate Health Image Image Analysis In Vitro Label Length Life Mammalian Cell Measures Medical Microfilaments Molecular Monomeric GTP-Binding Proteins Mutate Mutation Peptides Pharmaceutical Preparations Photobacterium Property Protein Analysis Proteins Proteome Quantitative Microscopy Research Riboflavin Sampling Sensitivity and Specificity Structure System Systems Biology Techniques Time Vibrio Vibrio fischeri Yeasts base chemical synthesis cofactor design drug discovery empowered fluorescence imaging fluorophore high throughput screening improved in vivo in vivo imaging innovation lumazine meetings mutant optical spectra protein complex quantum screening sensor targeted sequencing

项目摘要

DESCRIPTION (provided by applicant): Fluorescence techniques provide extraordinary high levels of sensitivity, specificity and selectivity and they are well-suited for high-throughput screening and imaging of specific proteins and drugs in biological samples. The most effective approach to quantify protein complexes within a sample is to use fluorescence anisotropy (FA) to quantify the formation of complexes between a FA-sensor and its target. FA has the distinct advantage over FRET in that it simply measures the increase in molecular volume of the bound FA sensor and using a single fluorophore. Currently, most FA-probes are prepared via laborious and specific chemical synthesis, reducing their appeal for high-throughput and in vivo screening of specific drugs or proteins. We will advance FA-based analyses of specific proteins for in vitro and in vivo systems through the introduction and optimization of a completely new class of genetically-encoded FA-sensor. The three sensors detailed in this proposal are truncated forms of: (i), a non-switchable mutant of Lov2 (fLov2), (ii), the yellow fluorescent protein from Vibrio fischeri (Y1); (iii), the lumazine binding protein (LUMP) from Photobacterium Leioghnati. The fluorescence properties of these flavoproteins proteins are similar to GFP, YFP and CFP respectively, although they only have 40%~67% of the mass, and exhibit far longer fluorescence lifetimes than GFP, a key property in the design of an FA-sensor for large proteins. The studies detailed are innovative on several counts and include the introduction of the smallest genetically-encoded fluorescent proteins for intracellular imaging of fused proteins, and the first encoded probes specifically designed for FA-based detection and imaging of specific protein targets in living cells. The research also identifies a promising approach for FA-based proteome-wide analysis of protein or drug interactions in vitro and in living bacteria and yeast.

说明(申请人提供)：荧光技术具有极高的灵敏度、特异度和选择性，非常适合对生物样品中的特定蛋白质和药物进行高通量筛选和成像。定量样品中蛋白质复合体的最有效方法是使用荧光各向异性(FA)来量化FA传感器与其目标之间的复合体的形成。与FRET相比，FA具有明显的优势，因为它只测量结合的FA传感器的分子体积的增加，并使用单个荧光团。目前，大多数FA探针是通过费力和特定的化学合成来制备的，这降低了它们对特定药物或蛋白质的高通量和体内筛选的吸引力。我们将通过引入和优化一类全新的遗传编码FA传感器来推进基于FA的体外和体内系统特定蛋白质的分析。本提案中详述的三个传感器是截断形式的：(I)Lov2的不可切换突变体(FLov2)，(Ii)来自鱼弧菌(Y1)的黄色荧光蛋白(Y1)，(Iii)来自Leioghnati的鲁马津结合蛋白(Lump)。这些黄素蛋白的荧光性质分别与GFP、YFP和CFP相似，尽管它们的质量仅为GFP的40%~67%，并且具有比GFP长得多的荧光寿命，这是设计大型蛋白质FA传感器的关键特性。详细的研究在几个方面都是创新的，包括引入最小的基因编码的荧光蛋白用于融合蛋白的细胞内成像，以及第一个专门设计用于基于FA的活细胞中特定蛋白质靶标的检测和成像的编码探针。这项研究还确定了一种基于FA的蛋白质组广泛分析体外以及活细菌和酵母中蛋白质或药物相互作用的有前途的方法。