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Improving the production of highly custom single-stranded DNA sequences for applications in DNA nanotechnology through the development of novel bacteriophage-based DNA-production methods

  • Author(s): Nafisi, Parsa
  • Advisor(s): Douglas, Shawn M
  • et al.
Abstract

DNA origami is a powerful tool for the production of self-assembled nanostructures and has shown promising applications in a wide range of fields. These structures are typically assembled through a reaction between one long single-stranded DNA (ssDNA) scaffold and many short oligonucleotide staples. In our efforts towards developing applications of DNA origami, we determined that two important properties for scaffold production include the ability to customize its size and sequence, and the ability to produce scaffolds cost effectively at scale. Several in vitro methods exist for producing custom scaffolds. However, because they rely on enzymatic processes, these methods are unreliable at large scale. On the other hand, scalable production of scaffolds based on the M13 bacteriophage genome has been demonstrated. However, the M13 genome will only accommodate insertions up to 2.5 kilobases (kb) reliably, limiting customizability. Likewise, phagemid vectors allow for the production of phage-based scaffolds with larger custom regions, but they still require 2.9 kb of fixed sequence. To overcome these issues, we developed a modified expression system using a novel phagemid, which we call pScaf. pScaf includes a standard M13 origin of replication (M13 ori) as well as a modified M13 ori which behaves as a terminator of ssDNA synthesis, allowing for packaging and export of the intervening sequence as a custom phage particle, while reducing the fixed-sequence region to only 381 bases.

Here, I discuss the steps taken to develop this novel phagemid vector. I also describe the resulting custom scaffolds which we produced using this vector and demonstrate the usefulness of these scaffolds for applications in DNA origami.

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