Novel Fluorophores for Molecular and Cellular Imaging

用于分子和细胞成像的新型荧光团

• 批准号: 8097201
• 负责人: ZYGMUNT GRYCZYNSKI
• 金额: $ 46.7万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8097201
• 关键词: 2-cyclopentyl-5-(5-isoquinolylsulfonyl)-6-nitro-1H-benzo(D)imidazole; Anisotropy; Binding; Biological Process; Cancer Detection; Cell Culture Techniques; Cells; Cellular Membrane; Chemistry; Communication; Complex; DNA; Detection; Development; Disease; Drops; Dyes; Energy Transfer; Event; Extinction (Psychology); Fluorescence; Fluorescence Anisotropy; Fluorescence Polarization; Goals; Image; Interest Group; Kinetics; Label; Lanthanoid Series Elements; Lasers; Life; Living Donors; Malignant Neoplasms; Matrix Metalloproteinases; Measurement; Measures; Medical; Methodology; Methods; Molecular; Monitor; Organ; Pathway interactions; Peptides; Photons; Physiologic pulse; Physiological; Process; Property; Proteins; RNA; Reporting; Resolution; Sampling; Sensitivity and Specificity; Signal Transduction; Source; System; Technology; Testing; Time; Tissues; Universities; absorption; base; bioimaging; biological systems; cellular imaging; chemical property; drug testing; fluorophore; imaging modality; improved; in vivo; molecular dynamics; molecular imaging; molecular mass; molecular/cellular imaging; nanoscience; nanosecond; novel; outcome forecast; protein protein interaction; quantum; response; single molecule; time use; two-photon; whole body imaging

DESCRIPTION (provided by applicant): At the core of biological function lays the ability of proteins to interact and associate with each other and the imaging methods capable of reporting on molecular events and interactions with high sensitivity and high resolution become indispensible. Fluorescence methods have a potential for highly sensitive detection and become essential for studying molecular processes with high specificity and sensitivity through a variety of signaling mechanisms. Tens of fluorescence probes are developed every year to be used for proteins/DNA/RNA labeling and to study molecular pathways and interactions as well as tissue imaging. However after many years of significant effort we are still missing "perfect" probes. There are many obstacles for markers that can be used for studying biological processes in cells and tissue. Two fundamental common problems for biomedical imaging, from single molecule studies and cellular imaging to whole body imaging are the background signal and availability of highly bright probes with suitable fluorescence lifetimes. The background signal (sample autofluorescence, scattering, and non- specific probe binding) always compromise sensitivity and specificity. The need for imaging kinetics and dynamics of molecular interactions/processes (like protein-protein interactions) requires probes with fluorescence lifetimes comparable to the mobility of interacting molecular partners. Many membrane and cellular proteins are large with molecular masses from 20 kDa to much over 100 kDa for which tumbling time and conformational changes are within tens and hundreds of nanoseconds. Within the large arsenal of dyes available today we have many bright fluorophores with fluorescence lifetimes of few nanoseconds or less and some luminophores like lanthanides with exited state lifetimes in microseconds. At present we lack fluorophores in red spectral range with fluorescence lifetimes over 10 ns. In this application we propose to utilize and further develop new group of small organic compounds [1,2]. The new group of azaoxa-triangulenium dyes offers excellent physico-chemical properties that will have unprecedented impact on molecular imaging. The rigid and small triangular frame of this organic compound has very favorable spectral properties including high photostability and most importantly unprecedented long single exponential fluorescence lifetime (~20 ns). We now propose to develop active and enhanced forms of these compounds to be used for studying molecular processes and interactions on a single molecule level, cellular level, and tissue imaging. In parallel to dyes development and tuning their spectral properties we will develop novel methodologies based on time gated detection to eliminate background signal and study dynamics of molecular processes and interactions by fluorescence polarization and FRET. This will enable: (1) precise time-resolved imaging that brings dynamic information about observed processes in large molecular complexes not available from steady-state measurements; (2) use of time-gated detection that will dramatically decrease background and improve imaging sensitivity over 100 folds; (3) new molecular beacon-type probes based on FRET, long lived donor, and time-gated detection that will have enormous signal gain of 105. In addition, 10-30 ns fluorescence lifetimes are much longer from the lifetime of typical background signal and in the same time easy for gating. Importantly time-resolved measurements for such lifetimes can be comfortably made with a pulsed laser source with a repetition rate of 1-5 MHz, in contrast it will require significantly longer time to collect enough photons in case of the lanthanides where the repetition rates are only in kHz. PUBLIC HEALTH RELEVANCE: In order to understand many complex biological processes, it is important to characterize complex interactions that occur in living subject by developing strategies, technologies, and probes that can monitor intracellular communication pathways, including protein-protein interactions. Our immediate goal is to develop probes and technologies that will allow for noninvasive imaging of molecular events openings new ways for studying complex interactions, as well as testing of drugs in cell cultures and in living subjects. The long term goal is to develop methods to image arrays of diverse molecular processes simultaneously to provide adequate in vivo characterization of diseases and allow accurate prognosis and rational disease treatment.

描述（由申请人提供）：生物功能的核心在于蛋白质相互作用和相互关联的能力，并且能够以高灵敏度和高分辨率报告分子事件和相互作用的成像方法变得不可或缺。荧光方法具有高灵敏度检测的潜力，对于通过各种信号机制研究具有高特异性和灵敏度的分子过程至关重要。每年开发出数十种荧光探针，用于蛋白质/DNA/RNA 标记、研究分子途径和相互作用以及组织成像。然而，经过多年的努力，我们仍然缺少“完美”的探测器。可用于研究细胞和组织生物过程的标记物存在许多障碍。从单分子研究和细胞成像到全身成像，生物医学成像的两个基本常见问题是背景信号和具有合适荧光寿命的高亮度探针的可用性。背景信号（样品自发荧光、散射和非特异性探针结合）总是会损害灵敏度和特异性。对分子相互作用/过程（如蛋白质-蛋白质相互作用）的成像动力学和动力学的需求需要荧光寿命与相互作用的分子伙伴的迁移率相当的探针。许多膜和细胞蛋白都很大，分子量从 20 kDa 到远超过 100 kDa，其翻滚时间和构象变化在数十和数百纳秒之内。在当今可用的大量染料中，我们有许多明亮的荧光团，其荧光寿命为几纳秒或更短，以及一些发光团，如激发态寿命为微秒的镧系元素。目前我们缺乏红色光谱范围内荧光寿命超过 10 ns 的荧光团。 在此应用中，我们建议利用并进一步开发一组新的小有机化合物[1,2]。这组新的氮杂氧三角染料具有优异的物理化学性质，将对分子成像产生前所未有的影响。这种有机化合物的刚性小三角形框架具有非常有利的光谱特性，包括高光稳定性和最重要的是前所未有的长单指数荧光寿命（~20 ns）。我们现在建议开发这些化合物的活性和增强形式，用于研究单分子水平、细胞水平和组织成像上的分子过程和相互作用。 在开发染料和调整其光谱特性的同时，我们将开发基于时间选通检测的新颖方法，以消除背景信号，并通过荧光偏振和 FRET 研究分子过程和相互作用的动力学。这将实现：（1）精确的时间分辨成像，提供有关大分子复合物中观察到的过程的动态信息，这是稳态测量无法获得的； (2) 采用时间选通检测，可显着降低背景并将成像灵敏度提高 100 倍以上； (3) 基于 FRET、长寿命供体和时间选通检测的新型分子信标型探针，其信号增益高达 105。此外，10-30 ns 荧光寿命比典型背景信号的寿命长得多同时易于浇口。重要的是，可以使用重复率为 1-5 MHz 的脉冲激光源轻松进行此类寿命的时间分辨测量，相比之下，对于重复率仅为以千赫兹为单位。 公共卫生相关性：为了了解许多复杂的生物过程，重要的是通过开发可以监测细胞内通讯途径（包括蛋白质-蛋白质相互作用）的策略、技术和探针来表征活体中发生的复杂相互作用。我们的近期目标是开发能够对分子事件进行无创成像的探针和技术，为研究复杂的相互作用以及在细胞培养物和活体受试者中测试药物开辟新的途径。 长期目标是开发同时对不同分子过程阵列进行成像的方法，以提供疾病的充分体内特征，并实现准确的预后和合理的疾病治疗。