In HIV-1, translational frameshifting leads to synthesis of the Gag-Pol fusion protein which gives rise to the viral protease, reverse transcriptase (RT) and integrase. This HIV-1 RNA frameshifting signal is a potential target for antiviral therapy (23) (24) (25) (26) . The exact structure of this RNA frameshifting signal has been the subject of debate. Jacks and collaborators initially proposed that it is a stem-loop structure downstream of the slippery sequence that is essential for efficient frameshifting (4) . Alternative structures have subsequently been proposed-reviewed by Brierley and Dos Ramos (27)in which the stimulatory RNA folds as a pseudoknot (28, 29) ; a pseudoknot with an RNA triple helix motif (30) and two-stem helix containing a three-purine bulge (8) . Recently however, two independent nuclear magnetic resonance (NMR) studies have shown that, in the absence of the ribosome, the fold is a long hairpin ( Figure 1 ) with an internal three-nucleotide bulge (9, 10) . A more recent structure-function analysis of the ribosomal -1 FS signal of two human HIV-1 isolates (31) favors the two-stem helix model of Dulude et al. (8) . The internal loop of the long stem-loop introduces a distinct bend between the lower and upper helical regions, a structural feature which, remarkably is also often seen with frameshifting pseudoknots. It has been proposed that the lower stem and the bend serve to initiate contacts between the upper stem-loop and the ribosome. Subsequently the lower stem melts allowing the slippery sequence to bind at the decoding site (10, 32) . Based on the identification of position +11 as the limit of accessibility of an RNA double helix approaching the ribosome (15), we previously proposed that the upper segment of the lower stem and the bulged region could be structured and/or contact the ribosomal surface (9) . NMR studies also pointed out that the upper stem, rich in conserved G-C Watson-Crick base pairs, is highly stable whereas the bulge region and the lower stem are much less so, and may readily unfold/melt. We therefore decided to unfold individual HIV-1 hairpins using optical tweezers, which complement existing methods, such as thermal denaturing, chemical probing or NMR spectroscopy, that address local features within the context of the entire global RNA structure. In principle, optical tweezers aid in applying forces locally to a folded RNA molecule in a way that may be more similar to in vivo conditions than, for example, thermal or chemical denaturing. In the case of a hairpin, the mechanical force will act locally on the 5 0and 3 0 -ends of the RNA unzipping the stem base-pair by base-pair toward the loop. A similar situation may be found in the ribosome where the translocation movement will generate a force pulling the 5 0 -end of the mRNA inside the ribosome.