The First All-Nucleobase Analog FRET Pair

Abstract

The fluorescent nucleobase analogs of the tricyclic cytosine (tC) family are promising nucleic acid probes capable of being inserted into double-stranded DNA as a replacement for one of the natural bases without perturbing the overall double helical structure. The high fluorescence quantum yields in both single- and double stranded DNA, combined with a rigid and well-defined position inside the DNA double helix, make these molecules particularly well suited as fluorescence resonance energy transfer (FRET) probes in nucleic acid studies. Recently we reported the first all-nucleobase analog FRET-pair, consisting of tCO as the donor and the newly developed tCnitro as acceptor which will be the focus of this presentation.1-3 The FRET-pair successfully monitors distances covering up to more than one turn of the DNA duplex and, more importantly, the rigid stacking of the two base analogs, and consequently excellent control of the their exact positions, results in a very high control of the orientation factor in the FRET efficiency. A set of DNA strands containing the FRET-pair at wisely chosen locations will, thus, make it possible to accurately distinguish distance- from orientation-changes using FRET. We believe the development of this new tool opens up a wide range of possibilities in the structural investigation of nucleic acids, e.g. in characterizing DNA-protein complexes and in monitoring the inherent dynamics and the structural changes of nucleic acids in response to all kinds of stimuli.