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

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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.