New Classes of Genetically-Encoded Fluorescence Anisotropy Probe

描述（由申请人提供）：荧光技术提供了非常高的灵敏度、特异性和选择性，非常适合生物样品中特定蛋白质和药物的高通量筛选和成像。定量样品中蛋白质复合物最有效的方法是使用荧光各向异性（FA）来定量FA传感器与其靶标之间复合物的形成。与FRET相比，FA具有明显的优势，因为它只是测量结合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探针都是通过费力和特定的化学合成来制备的，这降低了它们在高通量和体内筛选特定药物或蛋白质的吸引力。我们将通过引入和优化一种全新的遗传编码fa传感器，推进基于fa的体外和体内系统特定蛋白质的分析。本提案中详细介绍的三个传感器是截断形式：(i) Lov2的不可切换突变体（fLov2）， （ii）来自费氏弧菌的黄色荧光蛋白（Y1）；（iii）来自光杆菌的lumazine binding protein （LUMP）。这些黄蛋白蛋白的荧光特性分别与GFP、YFP和CFP相似，尽管它们的质量仅为GFP的40%~67%，但它们的荧光寿命比GFP长得多，这是设计用于大蛋白的fa传感器的关键特性。详细的研究在几个方面都是创新的，包括引入用于融合蛋白细胞内成像的最小遗传编码荧光蛋白，以及专门设计用于基于fa的活细胞中特定蛋白靶点检测和成像的第一个编码探针。该研究还确定了一种有前途的方法，用于基于fa的蛋白质组分析蛋白质或药物在体外、活细菌和酵母中的相互作用。

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.