Tetramolecular Fluorescence Complementation for Detection of Specific RNAs in Vitro
Tetramolecular Fluorescence Complementation for Detection of Specific RNAs in Vitro
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DOI：
10.1002/cbic.201200734
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发表时间：
2013-01-21
期刊：
影响因子：
3.2
通讯作者：
Rentmeister, Andrea
中科院分区：
文献类型：
--
作者：
Kellermann, Stefanie Julia;Rath, Anna Katharina;Rentmeister, Andrea
在许多细胞类型和生物体中发现了不对称定位的RNA，并参与了重要的发育过程，如酵母中的交配类型转换、果蝇的体轴形成[2]和哺乳动物大脑中轴突的定向引导。[3]不同的机制，包括定位转录、定向降解、扩散和锚定或主动运输，可以导致这种不对称定位。[4]为了更好地理解导致不对称定位的过程，需要能够在复杂细胞环境中进行RNA成像的探针。重要的是，这些探针在其结合和未结合的形式之间应该产生不同的信号，或者在与目标RNA结合时信号应该得到显著增强。到目前为止，已经制定了几种区分绑定和非绑定探针的策略。在最直接的方法中，通过将多个荧光团连接到RNA上来局部增强荧光。这一策略已经成功地应用于活细胞，但目标RNA必须延伸多达数百个核苷酸。[1，5]这个附件可能会损害原始RNA的结合或运输行为。在另一种方法中，利用FRET效应，当两个标记有合适发色团的核酸探针彼此相邻时，利用FRET效应提供明显的信号。[6]分子信标(由5о和3о处的荧光团和猝灭剂修饰的茎环结构，在与目标rna结合时发光)和强制插层探针(其中嵌入剂染料用作碱基替代物的多肽核酸探针)代表了其他广泛使用的方法。然而，标记的核酸不能在细胞内产生，因此将基于核酸的探针输送到细胞中仍然是一个挑战。在分子信标中实现的序列特异性和发光特性，以及对荧光进行基因编码的可能性，是一种有价值的替代方案。为此，识别特定RNA序列或结构的RNA结合蛋白被融合为分裂的荧光报告蛋白。几种蛋白质，如FragileX智力低下蛋白、真核启动因子4A和Pumilio，已经成功地用于RNA检测。[8，11]Pumilio是一种RNA结合蛋白，以序列特异性的方式与单链RNA的八个核苷酸(SsRNA)结合。[9]晶体结构分析揭示了RNA-蛋白质相互作用的分子细节。[10]Ozawa[11]和Tilsner[12]实现了Pumilio蛋白与分裂绿色荧光蛋白(GFP)变异体的结合，并将其应用于HeLa和表皮细胞。通过将两个融合成Split-GFP的RNA结合蛋白桥接起来，建立了一个三分子荧光互补系统。[8A]GFP或Venus的荧光互补已成功地用于探测蛋白质-蛋白质相互作用(双分子荧光互补)，以及较小程度的RNA-蛋白质相互作用(三分子荧光互补)，[11-13]，但仍存在一些局限性。首先，已经报道了荧光蛋白的两个部分的自组装，这导致了显著的背景信号。[14]这特别严重，因为它增加了背景荧光，从而损害了结合和未结合探针之间的区分。减少这种背景的突变提高了信噪比。[14]其次，Split-GFP和…
Asymmetrically localized RNA has been found in many cell types and organisms and contributes to important developmental processes, such as mating type switching in yeast,[1] body axis formation in Drosophila,[2] and directional steering in axons in the mammalian brain.[3] Different mechanisms, including localized transcription, directed degradation, diffusion and anchoring, or active transport, can lead to this asymmetric localization.[4] To better understand the processes leading to asymmetric localization, probes capable of RNA imaging in the complex cellular environment are required. Importantly, these probes should yield a different signal between their bound and unbound forms, or the signal should be substantially enhanced upon binding to the target RNA. Several strategies to discriminate between bound and unbound probe have been developed to date. In the most straightforward approach, the fluorescence is increased locally by attaching multiple fluorophores to the RNA. This strategy has been used successfully in living cells, but the RNA of interest had to be extended by up to hundreds of nucleotides.[1, 5] This appendage might impair binding or trafficking behavior of the original RNA. In another approach, the FRET effect has been harnessed to provide a distinct signal when two nucleic acid based probes labeled with suitable chromophores are bound adjacent to each other.[6] Molecular beacons (stem–loop structures modified by a fluorophore and a quencher at the 5оand 3о-ends that light up upon binding to target RNA) and forced intercalation probes (peptide nucleic acid based probes in which an intercalator dye serves as a base surrogate) represent other widely used approaches.[7] Labeled nucleic acids, however, cannot be produced inside cells, and delivery of nucleic acid-based probes into cells remains a challenge.[7b] Therefore, the sequence specificity and light-up properties realized in molecular beacons combined with the possibility to genetically encode fluorescence represents a valuable alternative. To this end, RNA binding proteins that recognize specific RNA sequences or structures have been fused to split fluorescent reporter proteins. Several proteins, such as fragileX mental retardation protein, the eukaryotic initiation factor 4A, and Pumilio, have been successfully implemented for RNA detection.[8, 11] Pumilio is an RNA binding protein that binds to a stretch of eight nucleotides of single-stranded RNA (ssRNA) in a sequence-specific manner.[9] Crystal structure analysis has revealed the molecular details of the RNA–protein interaction.[9] Therefore, it has become possible to rationally design Pumilio variants with altered specificity.[10] The combination of Pumilio proteins with split green fluorescent protein (GFP) variants was realized by Ozawa [11] and Tilsner [12] and applied to HeLa and epidermis cells. By bridging two RNA binding proteins fused to split-GFP, a trimolecular fluorescence complementation system was established.[8a] Fluorescence complementation of GFP or Venus has been successfully used to probe protein–protein interactions (bimolecular fluorescence complementation) and, to a lesser extent, RNA–protein interactions (trimolecular fluorescence complementation),[11–13] but still suffers from some limitations. Firstly, self-assembly of the two parts of the fluorescent protein has been reported, and this causes significant background signal.[14] This is particularly aggravating because it increases the background fluorescence and thus compromises discrimination between bound and unbound probe. Mutations that reduce this background improved the signal-to-noise ratio.[14] Secondly, split-GFP …