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1.
Introduction
In the range of important techniques used for studying nucleic acid-containing systems (including e.g. nuclear magnetic resonance (NMR), X-ray crystallography, electrophoresis, footprinting, calorimetry, enzymatic methods and UV-Vis absorption), fluorescence is one of the most sensitive, versatile, easily accessible, fast and straightforward ones. For this reason, it has found use in single-molecule real-time dynamics of nucleic acids as well as proteins, cell microscopy, nucleic acid detection and nucleic acid-protein interaction measurements. Thus, fluorescence studies at both the ensemble and single-molecule level are at the fore of biochemical as well as biophysical research on nucleic acids, and will increase in importance in the expanding field of DNA nanotechnology as these constructs keep decreasing in dimensions (Aldaye & Sleiman, 2006; Seeman, 2003; Tumpane et al. 2007). However, to investigate nucleic acids using fluorescence techniques, an emissive reporter group, a fluorophore, first has to be covalently or non-covalently introduced to the system since the nucleobases themselves are virtually non-fluorescent under normal conditions. This fluorophore labelling will be the focus of this review and most attention will be given to the covalent internal modifications and more specifically to fluorescent nucleobase analogues. Since most of the fluorescent modifications in nucleic acid research have been performed on DNA-containing systems, the review will primarily discuss these; however, for most of the modifications, one can equally well imagine using them for RNA systems.
2.
Fluorescent labelling of nucleic acids
The lack of adequate intrinsic emission in DNA and RNA has resulted in the development of a variety of classes of molecules for fluorescent labelling of nucleic acids. One of those classes has been developed to interact non-covalently with the nucleic acid and has been used, for example, to visualize DNA in gel electrophoresis or cell microscopy. Examples of such molecules are DNA intercalators like ethidium bromide, YO (oxazole yellow) and its homodimer YOYO, as well as DNA groove binders such as 4[variant prime],6-diamidino-2-phenylindole (DAPI) and Hoechst derivatives. More importantly, molecules that enable external and internal covalent modifications of nucleic acids have also been developed and will be described in more detail below.
2.1
External modifications
Modifying nucleic acids by covalently attaching fluorophores to the backbone at the end of or within an oligonucleotide sequence, but outside...