We directly measured at the single-molecule level the forces and lifetimes of DNA base-pair stacking interactions for all stack sequence combinations. Our experimental approach combined dual-beam optical tweezers with DNA origami components to allow positioning of blunt-end DNA helices so that the weak stacking force could be isolated. Base-pair stack arrays that lacked a covalent backbone connection spontaneously dissociated at average rates ranging from 0.02 to 500 per second, depending on the sequence combination and stack array size. Forces in the range from 2 to 8 piconewtons that act along the helical direction only mildly accelerated the stochastic unstacking process. The free-energy increments per stack that we estimate from the measured forward and backward kinetic rates ranged from -0.8 to -3.4 kilocalories per mole, depending on the sequence combination. Our data contributes to understanding the mechanics of DNA processing in biology, and it is helpful for designing the kinetics of DNA-based nanoscale devices according to user specifications.
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