Sequence-specific Fluorescence Labelling of long DNA for Optical Tracking in Cells and Electron Transfer Studies

用于细胞光学跟踪和电子转移研究的长 DNA 序列特异性荧光标记

• 批准号: 5408197
• 负责人: Professor Dr. Elmar Weinhold
• 金额: --
• 依托单位: Reconnaissance moléculaire des acides nucléiques : sondes de structures et sondes fluorescentes (UMR 176)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2003
• 资助国家: 德国
• 起止时间: 2002-12-31 至 2007-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5408197?language=en
• 关键词: Sequence; specific; Fluorescence; Labelling; long

Functional studies of long DNA often require the attachment of reporter groups. This is commonly achieved by non-specific labelling methods which have the draw-back that an ensemble of different molecules will be analysed and that the label might interfere with the function of DNA. In order to avoid these problems, sequence-specific labelling methods for long DNA are needed. Recently, we introduced a new cofactor for DNA methyltransferases which is quantitatively, sequence- and base-specifically coupled with the DNA substrate. This system can be modified by attaching reporter or other chemical groups to the new cofactor and used for sequence-specific labelling of DNA. Proof of principle for this method called Sequence-specific Methyltransferase-Induced Labelling of DNA (SMILing DNA) was already obtained. SMILing DNA can be viewed as an enabling technology to provide new tools for functional studies of DNA and for DNA-based approaches in molecular diagnostics or medicine as well as in bionanotechnology. In the course of the proposed research cooperation we will further develop the SMILing DNA method and demonstrate its potential by two important applications for in vivo and in vitro studies of DNA. In the first application triphenylamine two-photon absorption fluorophores will be optimised, sequence-specifically attached to plasmid DNA and applied for optical tracking of plasmids in the cell by two-photon laser-scanning microscopy. The advantages of using SMILing DNA compared to non-specific labelling methods are that the degree of labelling can be fully controlled, the integrity of the plasmid DNA is maintained and the label can be directed to non-coding regions allowing simultaneous gene expression. In the second application SMILing DNA will be extended to a one-step sequence-specific two-fluorophore labelling system and used to introduce a photo-activated redox couple for studying the electron transfer process in native DNA over long distances. This system will allow to probe electron transfer in long DNA using steady-state and time-resolved fluorescence spectroscopy and can be extended to single molecule measurements using confocal or scanning tunnelling microscopy.

长DNA的功能研究通常需要连接报告基团。这通常通过非特异性标记方法来实现，该方法的缺点是将分析不同分子的集合，并且标记可能干扰DNA的功能。为了避免这些问题，需要长DNA的序列特异性标记方法。最近，我们介绍了一种新的辅助因子的DNA甲基转移酶，这是定量，序列和碱基特异性耦合的DNA基板。该系统可以通过将报告基因或其他化学基团连接到新的辅因子上来修饰，并用于DNA的序列特异性标记。这种称为序列特异性甲基转移酶诱导的DNA标记（SMILing DNA）的方法的原理已经得到了证明。SMILing DNA可以被视为一种使能技术，为DNA的功能研究和分子诊断或医学以及生物纳米技术中基于DNA的方法提供新的工具。在拟议的研究合作过程中，我们将进一步开发SMILing DNA方法，并通过DNA体内和体外研究的两个重要应用来展示其潜力。在第一个应用中，三苯胺双光子吸收荧光团将被优化，序列特异性地连接到质粒DNA上，并通过双光子激光扫描显微镜应用于细胞中质粒的光学跟踪。与非特异性标记方法相比，使用SMILing DNA的优点是可以完全控制标记的程度，保持质粒DNA的完整性，并且可以将标记引导至允许同时基因表达的非编码区。在第二个应用SMILing DNA将扩展到一个一步序列特异性的两个荧光团标记系统，并用于引入光活化的氧化还原对，用于研究天然DNA中的电子转移过程在长距离。该系统将允许探测电子转移长DNA使用稳态和时间分辨荧光光谱，并可以扩展到单分子测量使用共聚焦或扫描隧道显微镜。