SINGLE CELL FLUORESCENT PROBES

单细胞荧光探针

• 批准号: 7598437
• 负责人: KEVIN BURGESS
• 金额: $ 2.38万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598437
• 关键词: Active Biological Transport; Biochemical; Cell membrane; Cells; Chariot peptide; Chemicals; Complex; Computer Retrieval of Information on Scientific Projects Database; Coupling; Cytosol; Data; Degradation Pathway; Diffusion; Dyes; Electroporation; Encapsulated; Energy Transfer; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Funding; Grant; Harvest; Institution; Label; Lasers; Life; Light; Liposomes; Methods; Microinjections; Nature; Object Attachment; Peptides; Photons; Protein Import; Proteins; Research; Research Personnel; Resolution; Resources; Scheme; Solutions; Source; System; Time; United States National Institutes of Health; Vesicle; Viral Vector; Work; absorption; base; cytotoxic; design; receptor; single molecule

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. It is often desirable to simultaneously observe several fluorescently tagged components in a biochemical mixture, i.e. using multiplexing. The apparatus for this is simplest when all the tags are excited using the same laser source, and this arrangement also has the advantage that data are more easily interpreted because all the labels receive the same input power. Multiplexing with one excitation source is, however, difficult. This is because dyes that emit close to the excitation source tend to absorb the light most efficiently since they have the greatest absorption at that wavelength, while those dyes that emit further into the red have red-shifted absorption spectra and harvest less photons at the excitation wavelength. Combinations of dyes arranged to maximize FRET have been used to alleviate this problem. Unfortunately, this is only a partial solution because the efficiency of the energy transfer in FRET systems is governed by the overlap integral. If the overlap of the emission of the donor dye with the absorption of the acceptor dye is small, then the energy transfer will be small. The central hypothesis of this work is that when a donor and acceptor systems are connected via a conjugated linker that does not allow them to become planar then rapid energy transfer from the donor to the acceptor may occur through bonds. Through-bond energy transfer is mechanistically different to the F¿rster basis for FRET, and there is no known requirement for overlap of the emission of the donor fragment with the absorption of the acceptor part. Thus, appropriately designed through-bond energy transfer cassettes could absorb photons via a donor part, or parts, at a convenient wavelength (eg 488 nm: excitation from an Ar-laser), transfer the energy rapidly through the conjugated linker to the acceptor fragment that emits at a far longer wavelength. There is no constraint on the difference between the donor absorption and the acceptor emission wavelengths in this scheme. It therefore is possible to design dyes that absorb strongly at a short wavelength and emit brightly with very similar intensities at several wavelengths (governed by the chemical nature of the acceptor) that are many wavenumbers apart, ie with excellent resolution. Coupling more than one donor in a conjugated system with an acceptor facilitates absorption of more light thereby increasing the intensity of the emission. In summary, through bond energy transfer cassettes have the potential to increase both the resolution and fluorescence intensities obtained from several probes excited by a laser source operating at a single wavelength. Proteins generally cannot enter cells by passive diffusion, but require active transport. While some proteins can also be transported into cells by microinjection, entrapped in liposomes, viral vectors, and electroporation, such methods are laborious, time consuming, and often have low efficiencies. A recently developed method involving a peptide called Chariot (Active Motif, Carlsbad, CA) overcomes these problems. Chariot non-covalently complexes with proteins to peptides and facilitates their transport into cells. The Chariot peptide is non-cytotoxic, and crosses plasma membranes independent of transporters or specific receptors, thus avoiding the lysosomal degradative pathway. The Chariot peptide has high transport efficiency (65-95%) and has already been shown to rapidly co-transport large fluorescent proteins. Once internalized, the fluorescent protein-Chariot peptide complex rapidly dissociates, thereby allowing the fluorescent-tagged protein to proceed to its intracellular target while concomitantly the Chariot peptide is rapidly degraded. Use of the Pep1 peptide (or similar carrier systems) to transfer protein/through-bond cassette conjugates into living cells opens new vistas of research. However, it is not evident that proteins imported into cells using the Chariot system are free in the cytosol; they could be encapsulated in intracellular vesicles. One of the objectives of our research is to elucidate this with single molecule studies performed at the center.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 通常需要同时观察生化混合物中的多个荧光标记成分，即使用多路复用，当使用相同的激光源激发所有标记时，这种装置是最简单的，并且这种布置还具有数据更容易的优点。然而，用一个激发源进行多路复用是很困难的，因为靠近激发源发射的染料往往会最有效地吸收光，因为它们在该波长下具有最大的吸收。而那些进一步发射红光的染料具有红移吸收光谱，并在激发波长处收获较少的光子。不幸的是，这只是部分解决方案，因为效率较高。 FRET 系统中的能量转移由重叠积分控制。如果供体染料的发射与受体染料的吸收的重叠较小，则能量转移也会较小。 这项工作的中心假设是，当供体和受体系统通过不允许它们变成平面的共轭连接体连接时，可能会通过键发生从供体到受体的快速能量转移。与 F 不同??供体片段的发射与受体部分的吸收重叠没有已知的要求，因此，适当设计的通过键合能量转移盒可以通过一个或多个供体部分吸收光子。方便的波长（例如 488 nm：来自 Ar 激光的激发），通过共轭连接体将能量快速转移到以更长波长发射的受体片段。对供体之间的差异没有限制。因此，可以设计出在短波长下吸收并在相隔许多波数的几个波长（由受体的化学性质决定）下以非常相似的强度发射的染料。具有出色的分辨率，在共轭系统中将多个供体与受体结合有利于吸收更多的光，从而增加发射强度。 总之，通过键合能量转移盒有可能增加两者。由在单一波长下工作的激光源激发的多个探针获得的分辨率和荧光强度。 蛋白质一般不能通过被动扩散进入细胞，而需要主动转运，虽然有些蛋白质也可以通过显微注射、脂质体包埋、病毒载体和电穿孔转运到细胞内，但此类方法费力、耗时，且效率往往较低。最近开发的一种涉及称为 Chariot（Active Motif，卡尔斯巴德，加利福尼亚州）的肽的方法克服了这些问题，Chariot 与蛋白质非共价复合成肽并促进其运输。 Chariot 肽不具有细胞毒性，并且不依赖于转运蛋白或特定受体而穿过质膜，因此避免了溶酶体降解途径。Chariot 肽具有高转运效率 (65-95%)，并且已被证明可以快速降解。 -运输大的荧光蛋白。一旦内化，荧光蛋白-Chariot肽复合物迅速解离，从而允许荧光标记的蛋白同时进入其细胞内靶标。 Chariot 肽迅速降解。 使用 Pep1 肽（或类似的载体系统）将蛋白质/直通键盒缀合物转移到活细胞中开辟了新的研究前景。它们可以被封装在细胞内囊泡中，我们研究的目标之一是通过在该中心进行的单分子研究来阐明这一点。