SWITCHING OF FLUORESCENCE LIFETIME UPON FUOROBODY BINDING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要 生物科学部的安德鲁·布拉德伯里博士正在开发一种新的荧光配体，被称为‘氟抗体’，这是一种绿色荧光蛋白分子，在其一端添加了一系列配体识别环，产生了一个准抗体结合部位(1)。如果结合时荧光信号发生一些变化，这种荧光体将更加有用。我们将研究荧光体与其靶配体的结合是否会导致GFP的荧光寿命发生可测量的变化。如果结合和未结合的分子有两个不同的荧光寿命，这基本上是一个检测到结合的开关。我们将测量与暴露在不同浓度配体的微球上的荧光体的荧光寿命，并确定我们是否可以检测到结合时寿命的变化。布拉德伯里博士的团队已经设计了几种不同的GFP分子，它们折叠的稳定性不同，我们或许能够通过使用蛋白质折叠不太稳定的GFP来选择更大的配体结合寿命效应。 背景 我们正在开发一种新的荧光配体，称为‘氟抗体’。这些都是GFP分子，抗体结合环已经插入其中。在其目前的实施中，对应于重链可变区的第三高变区的单环被插入到GFP中的特定位置。用来自淋巴细胞的随机HCDR3‘S(见附录)和通过噬菌体展示选择的特定结合剂创建了这种荧光体的文库。尽管GFP中只显示了一个环路，但他已经能够选择识别高纳米分子范围(400-1000 nm)内具有亲和力的许多不同目标的结合剂。这比传统的肽噬菌体展示获得的亲和力要高得多，而且所选择的结合剂保持了它们的荧光。事实上，亲和力的测定是使用流式细胞术进行的，将抗原偶联到聚苯乙烯微珠上，并检测到荧光体结合产生的荧光。 方法 如果结合时荧光信号发生一些变化，荧光体将更加有用。虽然我们试图开发基于结合后改变其荧光的荧光蛋白的结合剂，但另一种方法是确定其他荧光性质是否在结合时改变。我们将研究荧光体与其靶配体的结合是否会导致GFP的荧光寿命发生可测量的变化。如果结合和未结合的分子有两个不同的荧光寿命，这基本上是一个检测到结合的开关。 我们将采取一种不断发展的方法来实施和应用这项技术。首先，我们将测量与微球结合并暴露在不同浓度配体下的荧光体的荧光寿命，并确定是否可以检测到结合时寿命的变化。这项工作可以使用升级版的分离式PS细胞仪来完成。其次，我们将确定我们是否可以设计出结合后寿命发生变化的荧光体。我们已经设计了几种不同的荧光蛋白，它们的折叠稳定性不同，我们或许能够通过使用不太稳定的蛋白质折叠的GFP来选择更大的配体结合寿命效应。这一应用将很好地利用同时测量多个寿命的能力，将在相敏和集成相谱仪器上实现。第三，我们将确定是否可以开发具有不同光谱发射光谱的荧光体，如YFP、BFP。这将打开使用具有不同发射光谱的荧光体组合来同时定量细胞中几种配体的结合形式和未结合形式以及基于微珠的分析的可能性。如果这项技术成功，它将为集成的相位光谱仪提供一个潜在的非常强大的生物学应用，它将提供区分许多荧光体混合物的光谱发射和寿命的能力。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Abstract Dr. Andrew Bradbury of the Bioscience Division is developing a new class of fluorescent ligand termed 'fluorobodies', which are GFP molecules to which a series of ligand-recognition loops have been added at one end, producing a quasi-antibody binding site (1). Such fluorobodies would be much more useful if there was some change in the fluorescence signal upon binding. We will investigate whether binding of fluorbodies to their target ligand induces a measurable change in the fluorescence lifetime of the GFP. If the bound and unbound molecules had two different fluorescence lifetimes, this would essentially be a switch that detected binding. We will measure the fluorescence lifetime of fluorobodies bound to microspheres exposed to differing concentrations of ligand, and determine if we can detect a difference in shift in lifetime upon binding. Dr. Bradbury's group has engineered several different GFP molecules that vary in the robustness of their folding, and we may be able to select larger lifetime effects of ligand binding by using GFPs having less stable protein folding. Background We are developing a new class of fluorescent ligand termed 'fluorobodies'. These are GFP molecules into which antibody binding loops have been inserted. In its present incarnation, single loops corresponding to the third hypervariable region of the heavy chain variable region have been inserted into a specific site in GFP. A library of such fluorobodies has been created using random HCDR3's derived from lymphocytes (see attached publication in appendix) and specific binders selected by phage display. Even though only single loops are displayed within the GFP, he has been able to select binders recognizing a number of different targets with affinities in the high nanomolar range (400-1000nM). This is significantly higher than the affinities obtained by traditional peptide phage display, and the selected binders retain their fluorescence. In fact, the determination of the affinity was carried out using flow cytometry with antigen coupled to polystyrene beads and the detected fluorescence arising from fluorobody binding. Approach Fluorobodies would be much more useful if there was some change in the fluorescence signal upon binding. Although we are attempting to develop binders based on fluorescent proteins that change their fluorescence upon binding, an alternative approach is to determine whether other fluorescence properties change upon binding. We will investigate whether binding of fluorobodies to their target ligands induces a measurable change in the fluorescence lifetime of the GFP. If the bound and unbound molecules had two different fluorescence lifetimes, this would essentially be a switch that detected binding. We will take an evolving approach to the implementation and application of this technology. First, we will measure the fluorescence lifetimes of fluorobodies bound to microspheres and exposed to differing concentrations of ligand, and determine if we can detect a difference in shift in lifetime upon binding. This work can be done with the upgraded version of the separated PS cytometer. Second, we will determine if we can engineer fluorobodies with enhance lifetime changes upon binding. We have already engineered several different fluorescent proteins that vary in the robustness of their folding, and we may be able to select larger lifetime effects of ligand binding by using GFPs having less stable protein folding. This application will take good advantage of the capability of measuring multiple lifetimes simultaneously, to be implemented on both the phase sensitive and integrated phase-spectral instruments. Third, we will determine whether we can develop fluorobodies with different spectral emission spectra, e.g. YFP, BFP. This would then open the potential to use combinations of fluorobodies with differing emission spectra to simultaneously to quantitate bound and unbound forms of several ligands in cells and bead-based assays. If this technology is successful, it will provide a potentially very powerful biological application for the integrated phase-spectral instrument, which would provide the ability to distinguish spectral emission and lifetime on a mixture of many fluorobodies.