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Nick-and-Digest Strategy for Programmable Circular ssDNA Production and Scalable DNA Origami Assembly.

Jingyi Ye1,2, Ao Liu2, Hui Lv3

  • 1School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou International Campus, Guangzhou 511442, China.

JACS Au
|November 28, 2025
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Summary
This summary is machine-generated.

This study introduces a new method to create long DNA scaffolds for DNA origami, overcoming size limitations. The improved technique enables larger, more stable nanostructures for advanced applications.

Keywords:
Atomic force microscopy (AFM)Circular single-stranded DNA (cssDNA)DNA molecular dynamicsDNA origamiOne-pot assembly

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Area of Science:

  • Nanotechnology
  • Synthetic Biology
  • Biochemistry

Background:

  • DNA origami allows precise nanostructure assembly for diverse applications.
  • Scalability is limited by the length of available single-stranded DNA (ssDNA) scaffolds.

Purpose of the Study:

  • To develop a method for generating long circular ssDNA (cssDNA) scaffolds of customizable lengths.
  • To optimize large-scale DNA origami assembly for improved efficiency and stability.

Main Methods:

  • A "nick-and-digest" strategy using Cas9 nickase (Cas9n) and T7 exonuclease (T7 Exo) to produce cssDNA from plasmid DNA.
  • Optimization of denaturation temperature, annealing, and staple-to-scaffold ratios for one-pot folding.
  • Characterization using atomic force microscopy (AFM) and molecular dynamics simulations.

Main Results:

  • Successfully generated high-purity 7k-nt and 15k-nt cssDNA scaffolds with minimal sequence dependence.
  • Enabled one-pot folding of large-scale DNA origami structures (147 × 107 nm), doubling surface area compared to conventional designs.
  • Demonstrated higher yield, fewer structural defects, and enhanced mechanical stability in one-pot assembly versus a two-step method.

Conclusions:

  • The "nick-and-digest" strategy provides a robust method for generating long cssDNA scaffolds.
  • Optimized one-pot assembly overcomes key barriers in DNA origami scalability and programmability.
  • This approach facilitates applications in large-scale nanoelectronics, data storage, and therapeutics.