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Biophysical Journal 84:643-654 (2003)
© 2003 The Biophysical Society

Excitonic Heterodimer Formation in an HIV-1 Oligonucleotide Labeled with a Donor-Acceptor Pair Used for Fluorescence Resonance Energy Transfer

Serena Bernacchi*, Etienne Piémont*, Noelle Potier{dagger}, Alain van Dorsselaer{dagger} and Yves Mély*

* Laboratoire de Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR 7034 CNRS, Faculté de Pharmacie, Université Louis Pasteur, 74, Route du Rhin 67401 Illkirch Cedex, France and {dagger} Laboratoire de Spectrométrie de Masse Bio-Organique, UMR 7509 CNRS, Ecole Européenne de Chimie Polymères et Matériaux, 25, Rue Becquerel, 67087 Strasbourg, France

Correspondence: Address reprint requests to Yves Mely, Tel.: +33 (0)3 90 24 42 63; Fax: +33 (0)3 90 24 43 12; E-mail: mely{at}pharma.u-strasbg.fr.

In this study, we investigated the absorbance and fluorescence properties of cTAR, the complementary DNA sequence of the transactivation response element of the HIV-1 genome, doubly end-labeled by different dyes, 5(and 6)-carboxyfluorescein (Fl) and 5(and 6)-carboxytetramethylrhodamine (TMR), frequently used in fluorescence resonance energy transfer (FRET) studies. This oligonucleotide forms a stable stem-loop structure. The absorption spectrum of this species clearly differed from that of a doubly labeled cTAR derivative in which the terminal part of the stem is melted and from an equimolecular mixture of singly labeled species. Moreover, no significant TMR fluorescence change accompanies the dramatic Fl intensity increase when the doubly labeled native cTAR was melted by temperature or annealed with its complementary sequence. Both elements suggest the formation of an H-type ground-state heterodimer between Fl and TMR that may be described by the molecular exciton model. Moreover, time-resolved fluorescence further suggests that the nonfluorescent heterodimer is in equilibrium with a small population of partially melted species showing FRET. Based on the spectral shifts associated with heterodimer formation, an interchromophore distance of 7.7 Å was calculated. Both the excitonic signal and the Fl fluorescence were used as sensitive tools to monitor the temperature-mediated and HIV nucleocapsid protein-mediated annealing of cTAR with its complementary sequence.




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