Base analogs as intrinsic labels for the study of DNA structure by fluorescence resonance energy transfer

Distance measurements using fluorescence resonance energy transfer (FRET) have been extensively applied to structural studies of proteins and protein:protein complexes. Only in a few instances FRET has been used for distance determination in oligonucleotides or oligonucleotide:protein complexes. In most of these studies donor acceptor systems with labels attached to the 5' ends of the individual oligonucleotide strands have been used. Since long flexible linkers are used to attach the dyes to the phosphate backbone of the oligonucleotide it is advantageous to analyze the experimental data in terms of a distribution of donor acceptor distances instead of a single distance. However, the length and the high flexibility of the linker arms result in broad distributions unsatisfactory for high precision distance determinations. In this study intrinsic labels like 2-aminopurine or dimethyllumazine are utilized instead. These fluorescent base analogs are incorporated into the DNA oligonucleotides and their viability for FRET measurements is tested. Time-domain measurements on oligonucleotides with one intrinsic label and one extrinsic label show that the recovered distance distributions are narrower. This improvement may even be enhanced by using intrinsic probes both as donor and as acceptor.