SBIR Phase I: Time-Resolved Fluorescence (TRF) Microscopy of Live Cells with Cell-Penetrating Peptides and Other Targeting Agents

这个小型企业创新研究第一阶段项目将把Lumi 4 ®-Tb类似物与各种细胞穿透肽连接起来，作为选择性地将镧系元素探针分子递送到活细胞的手段。在空间和时间维度上，使用时间分辨显微镜在体内动态成像药物和生物相关蛋白质-蛋白质、蛋白质-DNA和核酸-核酸相互作用和动力学的能力对制药、诊断和药物发现行业具有重要价值。使用时间分辨显微镜（TRM），该项目将分析CPP探针结合物的细胞渗透和亚细胞分布。Lumi 4-Tb是新一代发光镧系络合剂，由于其高量子产率（60%）和摩尔吸光系数（26，000 M-1cm-1）而格外明亮。该提案还将利用独特的TRM和蛋白质标记技术，基于二氢叶酸还原酶对甲氧苄啶的亲和力。荧光镧系元素探针、FRET和CPP介导的细胞递送的组合将使以前所未有的灵敏度动态检测和化学计量定量活细胞中的蛋白质-蛋白质相互作用成为可能。将开发一种新的细胞成像试剂和技术，提高研究人员跟踪蛋白质的能力，蛋白质相互作用途径的定量准确性，以前没有。这不仅将影响基础和应用研究，还将通过发现新的药物靶点和机制来诊断和治疗疾病，从而影响初级卫生保健。设计和使用新的探针来研究活细胞中分子和亚细胞水平的结构和功能是一个非常重要的课题，越来越需要新的方法和工具来可视化细胞中分子种类的分布，但它们相互作用的方式。细胞内的蛋白质-蛋白质相互作用和其他动态事件在很大程度上是不可见的，但通过新的成像方式将越来越多地被观察到。特别是，镧系元素探针，通过时间门控实现的背景的显着降低，如果成功地与细胞渗透和分子靶向和识别相结合，可以实现新的显微成像。

This Small Business Innovative Research Phase I Project will link analogs of Lumi4®-Tb to various cell-penetrating peptides as a means of selectively delivering lanthanide probe molecules to living cells. The ability, in spatial and temporal dimensions, to dynamically image pharmaceutical and biologically relevant protein-protein, protein-DNA, and nucleic acid-nucleic acid interactions and kinetics in vivo using time-resolved microscopy is of significant value to pharmaceutical, diagnostic and drug-discovery industries. Using time-resolved microscopy (TRM), this project will analyze the CPP-probe conjugates for cell penetration and sub-cellular distribution. Lumi4-Tb is a new generation of luminescent lanthanide complexing agents that are exceptionally bright due to their high quantum yield (60%) and molar absorption coefficient (26,000 M-1cm-1). This proposal also will leverage unique TRM and protein labeling technology, based on the affinity of dihydrofolate reductase for trimethoprim. The combination of fluorescent lanthanide probes, FRET, and CPP-mediated cell delivery will make it possible to dynamically detect and stoichiometrically quantify protein-protein interactions in live cells with unprecedented sensitivity.The broader/commercial impact of this project, if successful, will be to develop a new class of cell imaging reagents and techniques that will improve the ability of researchers to follow protein-protein interaction pathways with quantitative accuracy that has not been available before. This will impact not only fundamental and applied research but also primary healthcare through the discovery of novel pharmaceutical targets and mechanisms to diagnose and treat disease. The design and use of novel probes to study structure and function at the molecular and subcellular level in living cells is a topic of great importance, with a growing need for new approaches and tools to visualize not only the distribution of molecular species in cells, but the manner in which they interact. Protein-protein interactions and other dynamic events within cells have been largely invisible, but will be increasingly observable with new imaging modalities. In particular, lanthanide probes, with the dramatic lowering of background achieved through time gating can enable new microscopic imaging if coupled successfully with cell penetration and molecular targeting and recognition.