Novel Fluorophores for Molecular and Cellular Imaging

描述（由申请人提供）：生物学功能的核心是蛋白质相互作用和相互缔合的能力，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，并且对于通过各种信号传导机制研究具有高特异性和灵敏度的分子过程至关重要。每年都有数十种荧光探针被开发出来，用于蛋白质/DNA/RNA标记，研究分子通路和相互作用以及组织成像。然而，经过多年的重大努力，我们仍然缺少“完美”的探测器。对于可用于研究细胞和组织中的生物过程的标记物，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的共同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要需要具有与相互作用分子伴侣的流动性相当的荧光寿命的探针。许多膜和细胞蛋白质是大的，分子量从20 kDa到远远超过100 kDa，翻滚时间和构象变化在几十和几百纳秒内。在今天可用的大量染料中，我们有许多荧光寿命为几纳秒或更短的明亮荧光团，以及一些发光体，如激发态寿命为微秒的镧系元素。目前，我们缺乏在红色光谱范围内的荧光寿命超过10纳秒的荧光团。 在本申请中，我们提出利用和进一步开发新的小有机化合物组[1，2]。这组新的氮杂-triangulenium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和小三角形框架具有非常有利的光谱特性，包括高的光稳定性和最重要的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，用于在单分子水平、细胞水平和组织成像上研究分子过程和相互作用。 在染料开发和调整其光谱特性的同时，我们将开发基于时间门控检测的新方法，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。这将使：（1）精确的时间分辨成像，其带来关于在大分子复合物中观察到的过程的动态信息，该动态信息不能从稳态测量中获得;（2）使用时间门控检测，其将显著降低背景并将成像灵敏度提高100倍以上;（3）基于FRET、长寿命供体和时间门控检测的新型分子信标型探针，其将具有105的巨大信号增益。 此外，10-30 ns的荧光寿命比典型背景信号的寿命长得多，同时易于门控。重要的是，可以用重复率为1-5 MHz的脉冲激光源轻松地进行这种寿命的时间分辨测量，相反，在重复率仅为kHz的镧系元素的情况下，将需要显著更长的时间来收集足够的光子。

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.