Single-molecule magnetic tweezers reveal distinct dynamics and enhanced mechanical stability of ssDNA 31 and 52 knots.

Nucleic acid knots play essential roles in diverse systems, ranging from RNA pseudoknot in Zika virus to DNA knot in viral DNA ejection. To investigate the mechanical properties of DNA knots, we designed single-stranded DNA (ssDNA) sequences with the capability to form $3_1$ and $5_2$ knots through base pairing interactions and quantitatively characterized their tightening and relaxation dynamic processes using single-molecule magnetic tweezers. In typical force-increasing and force-decreasing processes, the tightening and relaxation transitions show distinct signals for different types of knots. The ssDNA sequence designed for $3_1$ knot successfully forms $3_1$ knot in an annealing process, while two ssDNA sequences designed for $5_2$ knot can form three types of distinct conformations, including DNA hairpin, $3_1$ knot, and $5_2$ knot. Our results show that knot topology can significantly enhance the mechanical stability of ssDNA structures from ${\sim} 10$ pN to >30 pN at typical 1 pN/s loading rate. The step sizes of transitions reveal that the tightened $3_1$ knot and $5_2$ knot absorb 16-18 nt and 25-30 nt, respectively. This study improves our understanding of the dynamic processes and mechanical stability of ssDNA knots and provides a foundational framework to explore the functions and potential applications of nucleic acid knots.