Novel Fluorophores for Molecular and Cellular Imaging

描述（申请人提供）：生物功能的核心是蛋白质相互作用和相互作用的能力，能够报告分子事件和相互作用的高灵敏度和高分辨率的成像方法是必不可少的。荧光方法具有高灵敏度检测的潜力，并成为通过多种信号机制研究具有高特异性和高灵敏度的分子过程的必要手段。每年都会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，经过多年的重大努力，我们仍然缺少“完美”的探测器。利用标记物研究细胞和组织中的生物过程存在许多障碍。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（样品自身荧光、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求要求探针具有与相互作用分子伙伴的迁移率相当的荧光寿命。许多膜和细胞蛋白的分子质量从20 kDa到100 kDa以上，翻滚时间和构象变化在几十到几百纳秒内。在今天可用的大量染料中，我们有许多荧光寿命只有几纳秒或更短的明亮荧光团，还有一些荧光团，如镧系元素，其激发态寿命在微秒内。目前我们还没有在红色光谱范围内荧光寿命超过10ns的荧光团。在这一应用中，我们提出利用和进一步开发新的一类小有机化合物[1,2]。新的氮杂-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和小三角形框架具有非常良好的光谱特性，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。这将实现：(1)精确的时间分辨成像，提供稳态测量无法获得的大分子复合物中观察过程的动态信息；(2)使用时间门控检测，这将大大降低背景，提高成像灵敏度超过100倍；(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，将具有105的巨大信号增益。此外，10 ~ 30ns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，这种寿命的时间分辨测量可以用重复频率为1-5 MHz的脉冲激光源轻松完成，相比之下，在重复频率仅为kHz的镧系元素的情况下，收集足够的光子需要更长的时间。

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.