Conformation‐Programmed Cyclization of Ultrashort ssDNA Through Electrostatic Nanoconfinement

ABSTRACT Enzymatic cyclization of ultrashort single‐stranded DNA (ssDNA) remains challenging. While conventional strategies have enabled the preparation of ultrashort ssDNA circles by passively suppressing intermolecular oligomerization, they lack the mechanical driving force to overcome the physical strain required for smaller circles. Herein, we report a conformation‐programmed cyclization (CPC) strategy that uses a V‐shaped DNA scaffold to guide DNA toward a cyclization‐favored conformation, with electrostatic nanoconfinement likely contributing to this conformational control. By utilizing a V‐shaped DNA scaffold with a flexible poly(thymine) hinge, we use the electrostatic interactions within the DNA scaffold under low ionic strength conditions to help pre‐organize the substrate into a pre‐bent, cyclization‐prone state. This strategy enables the near‐quantitative preparation of 15 nucleotide (nt) ssDNA circles (∼97% yield) and produces 14 nt circles with a yield of 71% through a stepwise loading protocol. The CPC platform is broadly applicable, achieving efficient cyclization across a wide size range from 14 to 70 nt. Importantly, the highly strained topology of these prepared circles enables strain‐promoted, toehold‐free strand displacement, significantly accelerating molecular switching kinetics compared to conventional linear duplexes. Our work moves beyond passive reaction‐pathway control toward active conformational engineering, providing a reliable framework for constructing and utilizing strained nucleic acid architectures in DNA nanotechnology.