Figure 1.
Principle of the assay done with magnetic tweezers. (A) The assay involved three phases and three possible molecule states (A, B and C; described in detail in the text). In brief, the force value imposes the molecular state. At phase one, the force was close to 0 pN (Fzero), the hairpin was completely folded (state A), and its extension was defined as zero. At phase two, the force was increased (Fopen) to about ∼20 pN for a few seconds, and the hairpin was completely unwound and the DNA fully extended (state B). An oligonucleotide (RNA or DNA) complementary to the apical region of the DNA hairpin (shown in magenta) was able to hybridize to the DNA after a few seconds at Fopen. At phase three, the force was reduced to 6 pN (Fhold); however, the bound oligonucleotide prevented the DNA hairpin from reforming, and the DNA remained in a transiently extended (state C). During this phase, the oligonucleotide could dissociate from the DNA and return the molecule to state A, where the duration of state C was defined as Toff. Finally, the force was reduced to zero (Fzero), which caused any remaining molecules in state C to refold, and a new cycle was started. These phases were repeated numerous times with the same DNA molecule. (B) An example of the experimental assay. The top panel shows the force modulation acting on the hairpin while the lower panel shows the real time extension of three individual hairpins (red, blue, orange) with respect to the force applied. The red and blue traces were displaced slightly up and down, respectively, to facilitate viewing. The force modulation cycles were repeated ∼100–150 times with about 30 to 50 beads to obtain statistically significant values of the mean Toff, |Toff|.