Fluorescent Probes for Multiplexed Intracellular Imaging

描述(申请人提供)：与可用于观察细胞内荧光的复杂仪器相比，用于细胞内成像的染料是不发达的。高级探测器将具有几个重要特征。它们应该是小的、无细胞毒性的、水溶性的实体，不干扰它们所结合的分子的功能。它们的光谱性质应该是，它们在与所用激发源对应的波长上吸收非常强烈，然后以高量子产额发射。如果要同时标记和观察几个生物分子(即多路复用)，则需要一组染料，这些染料在一个方便的激发波长上强烈吸收，但发出具有不同斯托克位移的尖锐强烈荧光信号，从而提供高分辨率。应进行修饰以促进在激发波长/功率下的吸收，以避免细胞生物分子的自发荧光和细胞损伤。发射波长应该出现在相对较长的波长(例如800 nm)，那里的电池基本上是透明的，从而更容易检测到。对于单分子检测方法，探针应抵抗涉及非荧光激发态的光解和闪烁。最后，还有其他特性可能会被改造成荧光探针。其中包括转换偶极子的各向同性，以消除基于FRET的距离测量中方位因素造成的不确定性，以及通过选通测量进行观测以增加信噪比的有利寿命特性。 在初步研究中，设计了一组用于DNA测序中的多路复用的新型探针。这些的概念基础也适用于细胞内成像。它们包含在一个激发波长强烈吸收的给体部分，扭曲但以其他方式共轭的连接体系统，促进快速的“通过键能量转移”，以及在远离激发源的波长发射的受体部分。因此，它们可以被单个激光以高截面激发，然后在不同的可分辨波长上发出强烈的荧光。这项提议是创造类似的“通过键的能量转移盒”，这些盒是针对细胞内成像进行优化的，即符合上述大多数或全部标准。它们将被制备(Burgess)，针对优异的光谱特性进行优化(Burgess/Hochstrasser)，并连接到一套四个模型蛋白质(Schroeder/Burgess)。标记的蛋白质将被输送到细胞内，并通过多组分、平衡的荧光测量来观察，以检测通过FRET供体(施罗德)的蛋白质-蛋白质相互作用。收集的数据将通过新的计算机算法(Liu)进行分析，然后与Hochstrasser等人在单分子水平上观察到的相同细胞内事件的数据进行比较。这项工作将为平衡和单分子检测方法提供更好的细胞内探针。

DESCRIPTION (provided by applicant): Dyes for intracellular imaging are underdeveloped relative to the sophisticated instrumentation available for observing fluorescence in cells. Superior probes would have several important characteristics. They should be small, noncytotoxic, water-soluble entities that do not interfere with the function of the molecules to which they are conjugated. Their spectroscopic properties should be such that they absorb very strongly at wavelengths that correspond to the excitation source used, then emit with high quantum yields. If several biomolecules are to be labeled and observed simultaneously (ie multiplexing), then sets of dyes are required that absorb strongly at one convenient excitation wavelength, but emit sharp intense fluorescence signals with different Stoke's shifts giving high resolutions. Modifications should be available to facilitate absorption at excitation wavelengths/powers that avoid autofluorescence from cellular biomolecules and cell damage. The emission wavelengths should occur at relatively long wavelengths (eg 800 nm) where cells are essentially transparent enabling easier detection. For single molecule detection methods, the probes should resist photodecomposition and "blinking" involving non-fluorescent excited states. Finally, there are other properties that might be engineered into fluorescent probes. These include isotropy of transition dipoles to remove uncertainties caused by orientation factors in FRET- based distance measurements, and favorable lifetime characteristics for observation via gated measurements to increase signal-to-noise. In preliminary studies, a set of novel probes was devised for multiplexing in DNA sequencing. The conceptual basis for these is also applicable to intracellular imaging. They contain donor parts that absorb strongly at one excitation wavelength, twisted, but otherwise conjugated, linker systems that facilitate rapid "through-bond energy transfer", and acceptor parts that emit at wavelengths far from the excitation source. Thus they can be excited with high cross-sections by a single laser, then fluoresce strongly at different, resolvable wavelengths. This proposal is to create similar sets of "through-bond energy transfer cassettes" that are optimized for intracellular imaging, ie that conform to most or all of the criteria described above. They will be prepared (Burgess), optimized for superior spectroscopic properties (Burgess/Hochstrasser), and attached to a set of four model proteins (Schroeder/Burgess). The labeled-proteins will be transported into cells and observed using multicomponent, equilibrium fluorescence measurements to detect protein-protein interactions via FRET donor (Schroeder). The data collected will be analyzed via novel computer algorithms (Liu), then compared with that obtained by Hochstrasser et al for the same intracellular events observed at the single molecule level. This work proposed will provide superior intracellular probes for equilibrium and single molecule detection methods.