SWITCHING OF FLUORESCENCE LIFETIME UPON FUOROBODY BINDING

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 摘要 博士生物科学部的Andrew Bradbury正在开发一类新的荧光配体，称为“荧光体”，这是一种GFP分子，其一端添加了一系列配体识别环，产生准抗体结合位点（1）。 如果在结合时荧光信号有一些变化，则这样的荧光体将更加有用。 我们将研究荧光体与其靶配体的结合是否会诱导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，这将基本上是一个检测结合的开关。 我们将测量与暴露于不同浓度配体的微球结合的荧光体的荧光寿命，并确定我们是否可以检测到结合后寿命偏移的差异。 Bradbury博士的研究小组已经设计了几种不同的GFP分子，这些分子在折叠的稳健性上各不相同，我们可以通过使用具有较不稳定蛋白质折叠的GFP来选择更大的配体结合寿命效应。 背景 我们正在开发一类新的荧光配体，称为“fluorobodies”。这些是GFP分子，其中插入了抗体结合环。在其目前的形式中，对应于重链可变区的第三高变区的单环已被插入GFP中的特定位点。已经使用来自淋巴细胞的随机HCDR 3（参见附录中的所附出版物）和通过噬菌体展示选择的特异性结合剂创建了这样的荧光体文库。尽管在GFP中只显示了单环，但他已经能够选择识别许多不同靶点的结合剂，其亲和力在高纳摩尔范围（400- 1000 nM）。这显著高于通过传统肽噬菌体展示获得的亲和力，并且所选择的结合剂保留其荧光。事实上，亲和力的测定是使用流式细胞术进行的，其中抗原偶联到聚苯乙烯珠，并且检测到荧光体结合产生的荧光。 方法 如果在结合时荧光信号有一些变化，则荧光体将更加有用。 虽然我们试图开发基于荧光蛋白的结合剂，其在结合时改变其荧光，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诱导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，这将基本上是一个检测结合的开关。 我们将采取不断发展的方法来实施和应用这项技术。 首先，我们将测量结合到微球并暴露于不同浓度的配体的荧光体的荧光寿命，并确定我们是否可以检测到结合后寿命变化的差异。 这项工作可以用升级版的分离式PS细胞仪完成。 第二，我们将确定我们是否可以设计结合后具有增强寿命变化的荧光体。 我们已经设计了几种不同的荧光蛋白，其折叠的鲁棒性不同，并且我们可能能够通过使用具有较不稳定的蛋白质折叠的GFP来选择更大的配体结合寿命效应。该应用将充分利用同时测量多个寿命的能力，在相敏和集成相位谱仪器上实现。第三，我们将确定我们是否可以开发具有不同光谱发射光谱的荧光体，例如YFP，BFP。 这将开启使用具有不同发射光谱的荧光体的组合以同时定量细胞和基于珠的测定中的几种配体的结合和未结合形式的可能性。 如果这项技术是成功的，它将提供一个潜在的非常强大的生物应用的集成相位光谱仪器，这将提供区分光谱发射和寿命的混合物的许多荧光体的能力。

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.