技术先锋最新文章克服实验技术缺陷

　　

生物通报道：来自EurekAlert的消息，伊利诺大学基因组生物学研究所的研究人员突破了FRET技术的局限性，开发了一个共振传感器，来检测细胞内的氧化还原动力学的即时影像，这一研究成果公布在Experimental Biology and Medicine杂志上。

这项研究由Robert Clegg博士等人完成，Clegg博士是生物科学界发展光学显微镜新颖应用的先锋之一，他在荧光半衰期影像显微镜研究领域获得了许多重要的研究成果。其研究组在之前的研究中已建立了一系列的FTRET供体和受体荧光蛋白在氧化还原敏感开关。

在这篇文章中，研究人员在之前研究的基础上，将氧化还原敏感的绿色荧光蛋白（GFPs）的概念，转化为FRET的成像平台。以FRET为基础的感应器的原理主要是应用氧化还原反应引起氧化还原敏感开关的构象变化，改变连接在这个开关的两个荧光蛋白（供体和受体）间的距离，从而产生可被检测的共振效率之改变。在氧化状态下，感应器的荧光散射波长光谱红移（由于受体荧光增加），这和局部感应器浓度变化或是激发光的强度无关。

以FRET为基础的感应器克服了一般以荧光强度为基础的影像方法所衍生的障碍，因为只有能量转移的效率改变时，FRET受体分子的荧光才会增加。FRET的这种特异性和可区别性的特征，是推动发展以共振为基础，而不是只依赖单一组成的荧光强度变化作为分析方法的主要动机之一。

研究人员认为，这个研究最主要的突破在于改进光讯号的动态范围，也就是说加强了氧化态和还原态之间的讯号差。增加光讯号的动态范围使得探针更容易区别复杂的生物样品中的氧化还原状态。此外，新探针中较高的氧化中点电位是一个用来侦测哺乳类细胞中谷胱甘肽氧化还原电位的理想工具。

以FRET为基础的策略有以下的优点：（1）能够量化氧化还原状态的变化，（2）不依赖感应器的浓度，（3）模块化设计，可以通过改变开关或模块的荧光探针而精确地调整氧化还原敏感性和量测范围。研究人员认为新开发的氧化还原敏感性探针可能是至关重要的一个工具来协助我们了解化疗药物和氧化剂的药理和毒理反应。

最近，另外一个实验室也发展了一个以氧化还原敏感的萤光蛋白（roGFP）为基础的新颖位差量测探针。利用roGFP感应器来量测需要有两组被不同波长激发出来的萤光影像强度。利用两组巯基在双硫键形成/裂解的的氧化还原态之下，roGFP所产生的最大激发光波长来求其比值。

原文摘要：

Development of a high-dynamic range, GFP-based FRET probe sensitive to oxidative microenvironments

We report the optimization of a novel redox-sensitive probe with enhanced dynamic range and an exceptionally well-positioned oxidative midpoint redox potential. The present work characterizes factors that contribute to the improved Förster resonance energy transfer (FRET) performance of this green fluorescent protein (GFP)-based redox sensor. The α-helical linker, which separates the FRET donor and acceptor, has been extended in the new probe and leads to a decreased FRET efficiency in the linker's reduced, ‘FRET-off’ state. Unexpectedly, the FRET efficiency is increased in the new linker's oxidized, ‘FRET-on’ state compared with the parent probe, in spite of the longer linker sequence. The combination of a lowered baseline ‘FRET-off’ and an increased ‘FRET-on’ signal significantly improves the dynamic range of the probe for a more robust discrimination of its reduced and oxidized linker states. Mutagenesis of the cysteine residues within the α-helix linker reveals the importance of the fourth, C-terminal cysteine and the relative insignificance of the second cysteine in forming the disulfide bridge to clamp the linker into the high-FRET, oxidized state. To further optimize the performance of the redox probe, various cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) FRET pairs, placed at opposite ends of the improved redox linker (RL7), were quantitatively compared and exchanged. We found that the CyPet/YPet and ECFP/YPet FRET pairs when attached to RL7 do not function well as sensitive redox probes due to a strong tendency to form heterodimers, which disrupt the α-helix. However, monomeric versions of CyPet and YPet (mCyPet and mYPet) eliminate dimerization and restore redox sensitivity of the probe. The best performing probe, ECFP-RL7-EYFP, exhibits an approximately six-fold increase in FRET efficiency in vitro when passing from the oxidized to the reduced state. We determined the midpoint redox potential of the probe to be –143 ± 6 mV, which is ideal for measuring glutathione (GSH/GSSG) redox potentials in oxidative compartments of mammalian cells (e.g. the endoplasmic reticulum).