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 (FI) 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 FI 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 FI 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 FI fluorescence were used as sensitive tools to monitor the temperature-mediated and HIV nucleocapsid protein-mediated annealing of cTAR with its complementary sequence.
Bernacchi, S., Piémont, E., Potier, N., Van Dorsselaer, A., & Mély, Y. (2003). Excitonic heterodimer formation in an HIV-1 oligonucleotide labeled with a donor-acceptor pair used for fluorescence resonance energy transfer. Biophysical Journal, 84(1), 643–654. https://doi.org/10.1016/S0006-3495(03)74884-X