Since toeprinting experiments suggested reduced structural stability of the lower stem, we investigated the mechanical stability of the HIV-1 hairpins. Individual hairpins, sandwiched between two differentially endlabeled hybrid DNA-RNA handles were unfolded using optical tweezers (42) . Molecules were tethered between a streptavidin-coated glass cover slip and anti-digoxygenin coated polystyrene beads (diameter 1 mm), and stretched by moving the piezo-actuated microscope stage while holding the bead with optical tweezers. Force versus extension curves were computed taking into account this experimental geometry (58) . Stage velocities were 67 nm/s, so that the system remained at or close to thermodynamic equilibrium as confirmed by the overlap of the stretching and relaxation force versus extension curves ( Figure 5B ). In addition, repeated unfolding and folding can be observed within single force versus extension curves ( Figure 5B) , a further indication that the loading rate is sufficiently low as to assure thermodynamic equilibrium. The increase in contour length (expressed in number of nucleotides) upon unfolding was subsequently computed using the worm-like chain model for polymer elasticity assuming a stretching modulus of 1000 pN, a persistence length of 1 nm (43,59) and a inter-phosphate distance of 0.59 nm (see 'Materials and Methods' section). Results are summarized in Figure 5C , yielding a mean increase of 25.3 AE 3.4 nm (mean AE SD) nucleotides, consistent with the contour length of the upper stem of the HIV-1 hairpin. The mean unfolding force is 12.8 AE 1.0 pN (mean AE SD), whereas the standard free-energy change at zero force was found as ÁG = 10 AE 2 kcal/mol (mean AE SD). Refolding statistics are summarized in the Figures S4 and S5 and yield a decrease of contour length of 26.0 AE 4.7 nm (mean AE SD) at an average refolding force of 13.1 AE 1.1 pN (mean AE SD), and ÁG = 11 AE 3 kcal/mol (mean AE SD), essentially unchanged from the unfolding statistics, as one would expect when at thermal equilibrium.