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We present the first nucleobase analog fluorescence resonance energy transfer (FRET)-pair. The pair consists of tC(O), 1,3-diaza-2-oxophenoxazine, as an energy donor and the newly developed tC(nitro), 7-nitro-1,3-diaza-2-oxophenothiazine, as an energy acceptor. The FRET-pair successfully monitors distances covering up to more than one turn of the DNA duplex. Importantly, we show that the rigid stacking of the two base analogs, and consequently excellent control of their exact positions and orientations, results in a high control of the orientation factor and hence very distinct FRET changes as the number of bases separating tC(O) and tC(nitro) is varied. 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. In combination with the good nucleobase analog properties, this points toward detailed studies of the inherent dynamics of nucleic acid structures. Moreover, the placement of FRET-pair chromophores inside the base stack will be a great advantage in studies where other (biomacro)molecules interact with the nucleic acid. Lastly, our study gives possibly the first truly solid experimental support to the dependence of energy transfer efficiency on orientation of involved transition dipoles as predicted by the Forster theory.

Original publication

DOI

10.1021/ja806944w

Type

Journal article

Journal

J Am Chem Soc

Publication Date

01/04/2009

Volume

131

Pages

4288 - 4293

Keywords

Cytosine, DNA, Fluorescence Resonance Energy Transfer, Models, Chemical, Models, Theoretical, Nitro Compounds, Nucleic Acid Conformation, Nucleic Acids, Oligonucleotides, Oxazines, Phenothiazines, Photochemistry, Spectrophotometry, Ultraviolet, Temperature