Here we investigated the stability of an aptamer, which is formed by two RNA strands and binds the antibiotic streptomycin. Molecular dynamics simulations in aqueous solution confirmed the geometry and the pattern of hydrogen bond interactions that was derived from the crystal structure (1NTB). The result of umbrella sampling simulations indicated a favored streptomycin binding with a free energy of Delta G(bind)(o) = -101.7 kJ mol(-1). Experimentally, the increase in oligonucleotide stability upon binding of streptomycin was probed by single-molecule force spectroscopy. Rate dependent force spectroscopy measurements revealed a decrease in the natural off-rate (k(off-COMPLEX) = 0.22 +/- 0.16 s(-1)) for the aptamer-streptomycin complex compared to the aptamer having an empty binding pocket (k(off-APTAMER) = 0.49 +/- 0.11 s(-1)). This decrease in the natural off-rate corresponds to a decrease in the Gibbs free energy of Delta Delta G(sheer) approximate to -3.4 kJ mol(-1). The simulated binding pattern and the experimental results led to the conclusion that hydrogen bonds between both RNA strands mainly contribute to the decrease in natural off-rate of the aptamer system studied.