Tying a molecular knot with optical tweezers

Filamentous structures are abundant in cells. Relatively rigid filaments, s uch as microtubules and actin, serve as intracellular scaffolds that suppor t movement and force, and their mechanical properties are crucial to their function in the cell. Some aspects of the behaviour of DNA, meanwhile, depe nd critically on ins flexibility-for example, DNA-binding proteins can indu ce sharp bends in the helix(1). The mechanical characterization of such fil aments has generally been conducted without controlling the filament shape, by the observation of thermal motions(2-5) or of the response to external forces(6-9) or flows(10-12). Controlled buckling of a microtubule has been reported(13), but the analysis of the buckled shape was complicated. Here w e report the continuous control of the radius of curvature of a molecular s trand by tying a knot in it, using optical tweezers to manipulate the stran d's ends. We find that actin filaments break at the knot when the knot diam eter falls below 0.4 mu m. The pulling force at breakage is around 1 pN, tw o orders of magnitude smaller than the tensile stress of a straight filamen t. The flexural rigidity of the filament remained unchanged down to this di ameter. We have also knotted a single DNA molecule, opening up the possibil ity of studying curvature-dependent interactions with associated proteins. We find that the knotted DNA is stronger than actin.