Mechanical Signatures of Nucleic Acid Trefoil Knot Topology

ABSTRACT Molecular knots represent a fundamental form of polymer topology, yet their mechanical behavior in nucleic acids remains largely unexplored. Here, we engineer a single‐stranded DNA sequence that can fold into either a knot or a pseudoknot while maintaining identical base‐pairing interactions. Using single‐molecule force spectroscopy with optical tweezers, we show that molecular topology alone produces distinct mechanical behavior. Knotted ssDNA exhibits three characteristic signatures relative to the pseudoknot: higher unfolding forces, shorter unfolding extensions, and faster refolding kinetics. These features arise from the topological constraint imposed by strand threading and together provide a mechanical fingerprint that distinguishes knotted from unknotted nucleic acid structures. By analyzing the denatured state under tension, we further show that the knot tightens as force increases, entering a tight‐knot regime in which the molecule can be described as a compact knot core in series with a stretched single strand. The retained contour length reveals that the tight trefoil knot contains approximately 10 nucleotides at forces approaching 40 pN. These results establish mechanical signatures as a means of identifying nucleic acid topology and provide quantitative insight into the nanomechanics of molecular knots.