New Classes of Genetically-Encoded Fluorescence Anisotropy Probe

描述（由申请人提供）：荧光技术具有极高的灵敏度、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的最有效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。FA与FRET相比具有明显的优势，因为它简单地测量结合的FA传感器的分子体积的增加并且使用单个荧光团。目前，大多数FA探针是通过费力和特异性的化学合成制备的，降低了它们对高通量和体内筛选特异性药物或蛋白质的吸引力。我们将通过引入和优化一类全新的遗传编码FA传感器，推进体外和体内系统中特定蛋白质的基于FA的分析。本提案中详述的三种传感器是以下的截短形式：（i）Lov 2的不可切换突变体（fLov 2），（ii）来自费氏弧菌的黄色荧光蛋白（Y1）;（iii）来自雷氏发光杆菌的发光素结合蛋白（LUMP）。这些黄素蛋白质的荧光性质分别与GFP、YFP和CFP相似，尽管它们的质量仅为GFP的40%~67%，并且表现出比GFP长得多的荧光寿命，这是设计用于大蛋白质的FA传感器的关键特性。详细的研究在几个方面是创新的，包括引入最小的遗传编码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活细胞中特定蛋白质靶点的基于FA的检测和成像的编码探针。该研究还确定了一种有前途的方法，用于基于FA的蛋白质组范围内的蛋白质或药物在体外和活细菌和酵母中的相互作用分析。

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.