Molecular Probes for Biomembrane Recognition

项目摘要/摘要 随着荧光成像的出现，荧光成像正在对普通医学产生革命性的影响 强大的临床前技术，如超分辨率显微镜和单分子跟踪，以及 新兴的临床程序，如荧光引导手术和快速病理学。每种方法都需要 一种特定类型的荧光分子探头，在许多情况下，探头性能不佳是一个主要原因 限制长期进展和广泛影响的因素。因此，这项研究的中心目标是 开发大大改进的荧光染料，并将其转化为分子探针，用于 临床前和临床影像。最大的一组项目是基于新的荧光菁七甲胺 (Cy7)染料，可发射波长为700 nm的近红外(NIR)光，并能对活体进行成像 在称为NIR I和NIR II的波长窗口中(后一窗口也称为短波长 红外线)。新的Cy7染料具有创新的分子设计特征，大大增强了图像对比度和 允许快速微调探针的药代动力学曲线。近红外染料将被转化为各种 靶向和生物响应性分子探针：(A)临床前显微镜，包括单分子 定位显微镜，(B)动物疾病模型的体内荧光成像，以及临床成像 接受手术的患者，(C)使用近红外荧光报告健康状况的长期植入式设备， 以及(D)生物医学诊断和测序方法。其中一些Cy7染料将非常稳定，以至于它们 可以首次并入自动合成多肽和寡核苷酸的方案中。这将是 对临床翻译有重大影响，因为它将允许研究人员可靠地将他们自己的Cy7标记为 使用标准的合成器机器进行大规模的多肽或寡核苷酸探针。第二套 项目将开发一种新的荧光分子，其不寻常的自丝线结构是 让人想起缠绕在一起的多肽。探头有坚硬的多肽环，可以促进卓越的稳定性和 靶向特性，以及非常适合高级细胞内的明亮的深红色荧光 显微镜。早期的焦点是自发渗透到活细胞中的分子探针，并允许 任何含有多组氨酸序列(组氨酸标记的)的细胞内蛋白的选择性荧光标记 蛋白质)。这项工作将创造第一个用于严格的纳米级成像的深红色荧光分子探针 在重要的生物医学动态过程中的亚细胞极性脂，如病毒感染和 内切体/外切体运输。我们将展示这些新型荧光探针的实用性和通用性。 通过与先进显微镜专家的合作。与商业供应商的密切协调将确保 这项研究开发的重要荧光探针很快就可以提供给所有感兴趣的人 调查人员。

Project Summary/Abstract Fluorescence imaging is having a transformative impact on general medical science with the advent of powerful preclinical techniques such as super-resolution microscopy and single-molecule-tracking, along with emerging clinical procedures such as fluorescence guided surgery and rapid pathology. Each method requires a specific type of fluorescent molecular probe, and in many cases non-optimal probe performance is a major factor that is limiting long term progress and broad impact. Thus, the central objective of this research is to develop greatly improved fluorescent dyes and convert them into molecular probes for important applications in preclinical and clinical imaging. The largest set of projects are based on new fluorescent cyanine heptamethine (Cy7) dyes that emit near-infrared (NIR) light with wavelengths >700 nm and enable imaging of living subjects in the wavelength windows known as NIR I and NIR II (the latter window is also called Short Wavelength Infrared). The new Cy7 dyes have innovative molecular design features that greatly enhance image contrast and permit rapid fine-tuning of probe pharmacokinetic profiles. The near-infrared dyes will be converted into various targeted and bioresponsive molecular probes for: (a) preclinical microscopy, including single-molecule localization microscopy, (b) in vivo fluorescence imaging of animal models of disease, and clinical imaging of patients undergoing surgery, (c) long-term implantable devices that use NIR fluorescence to report health status, and (d) biomedical diagnostics and sequencing methods. Some of these Cy7 dyes will be so stable that they can be incorporated, for the first time, into automated peptide and oligonucleotide synthesis protocols. This will have a major impact on clinical translation since it will allow researchers to reliably make their own Cy7 labeled peptide or oligonucleotide probes on a large scale using a standard synthesizer machine. A second set of projects will develop a new class of fluorescent molecules whose unusual self-threaded structures are reminiscent of entangled peptides. The probes have rigid peptide loops that promote outstanding stability and targeting properties, along with bright deep-red fluorescence that is perfectly suited for advanced intracellular microscopy. An early focus is on molecular probes that spontaneously permeate into living cells and permit selective fluorescence labeling of any intracellular protein containing a polyhistidine sequence (His-tagged protein). The work will create the first deep-red fluorescent molecular probes for rigorous nanoscale imaging of subcellular polar lipids during biomedically important dynamic processes such as viral infection and endosome/exosome trafficking. The utility and versatility of these new fluorescent probes will be demonstrated by collaborations with experts in advanced microscopy. Close coordination with a commercial vendor will ensure that the important fluorescent probes developed by this research quickly become available to all interested investigators.