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

长DNA的功能研究通常需要附加报告基团。这通常是通过非特异性标记方法实现的，其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，需要针对长DNA的序列特异性标记方法。最近，我们引入了一种新的DNA甲基转移酶辅因子，它与DNA底物定量、序列和碱基特异性偶联。该系统可以通过将报告基因或其他化学基团附加到新的辅因子上进行修饰，并用于DNA的序列特异性标记。已经获得了这种称为序列特异性甲基转移酶诱导DNA标记（smile DNA）的方法的原理证明。微笑DNA可以被视为一种使能技术，为DNA的功能研究和基于DNA的分子诊断或医学方法以及生物纳米技术提供了新的工具。在拟议的研究合作过程中，我们将进一步发展微笑DNA方法，并通过体内和体外DNA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，序列特异性地附着在质粒DNA上，并应用于双光子激光扫描显微镜对细胞中的质粒进行光学跟踪。与非特异性标记方法相比，使用smile 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，并且可以将标记定向到允许同时表达基因的非编码区域。在第二个应用中，smile DNA将扩展到一步序列特异性双荧光标记系统，并用于引入光激活氧化还原偶对，以研究天然DNA中的长距离电子转移过程。该系统将允许使用稳态和时间分辨荧光光谱探测长DNA中的电子转移，并可扩展到使用共聚焦或扫描隧道显微镜进行单分子测量。

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.