Due to the discontinuous nature of HIV-1 plus-strand DNA synthesis, a 99-nt plus-strand overhang termed the central DNA flap is present near the center of the proviral DNA prior to integration. The flap appears to have stabilizing and/or protective effects on viral DNA, which has been hypothesized to be due to a specific conformation adopted by the three-stranded region. The 5' end of the flap sequence is very purine rich and has the potential to adopt different secondary structures (e.g., duplex, triplex or quadruplex). In the present work, circular dichroism spectroscopy and thermal unfolding techniques were used to characterize an 89-nt long DNA sequence designed to mimic the three-stranded region at the 5' end of HIV-1 proviral DNA. The effect of addition of the HIV-1 nucleocapsid protein (NC) on the nucleic acid structure was also examined. Although, guanine-rich short oligonucleotides derived from the DNA flap demonstrated CD spectra characteristic to parallel quadruplexes, this analysis reveals that the extended 89-nt construct folds into a canonical duplex with a flapping third strand both in the absence and presence of NC.