The aims of this project were to build switchable DNA origami nanostructures, or ‘nanorobots’, and develop methods to control them using external signals, such as electromagnetic radiation. The long- term application is to develop these nanostructures as multifunctional drug delivery vehicles, that can be modified to carry many different cargos, and respond to a complex combination of cell surface and external signals

To this end, we have made important advances in developing both switchable nanostructures and testing methods for controlling their activity:

1. Switchable DNA origami structures

• -  we designed a new DNA origami nanostructure capsule with an open and closed state

• -  the active part of the nanostructures consists of a hydrophobic ‘pore’, that can penetrate

  cell membranes to improve drug delivery. We confirmed that the pore forms correctly, and

  demonstrated its ability to transport molecules across lipid membranes

• -  we designed a protective DNA-origami barrel ‘shell’ to enclose the active pore. We

  confirmed the pore and barrel form together as a single structure by transmission electron

  microscopy

• -  a tether strand of DNA links the pore inside the DNA barrel. We confirmed that by altering

  the length of the tether we could control whether the pore is either contained inside the barrel, in the ‘closed’ state, or able to diffuse out of the barrel and interact with cell membranes, in the ‘open’ or active state.

• -  We are in the process of validating switching control between the two states with toe-hold mediated DNA strand-displacement.

  2. Triggered change in activity

• -  We modified DNA strands with gold (AuNPs, 2-5 nm) or superparamagnetic iron oxide

  nanoparticles (SPION, 25 nm). This included optimising nanoparticle surface chemistry for

  covalent DNA attachment

• -  We developed a DNA-based temperature sensor to detect local short-term melting of DNA

  strands, and validated it with known external temperature gradients

• -  we are in the process of testing the use of externally applied radio and microwaves to trigger

  local melting of the gold functionalised DNA strands that hold our nanostructure in the

  closed state

• -  We have also designed and successfully tested DNA motifs for alternative switching triggers,

  including pH and salt concentration. These have been confirmed on simple DNA structures, and will now be applied to our larger pore-shell DNA nanostructure

  3. In vivo stability

- Our ultimate goal is to use these nanostructures for drug delivery, which requires they be stable in cell culture and in vivo systems

• -  We have successfully used chemical functionalisation to protect our nanostructure barrels, and confirmed that they do not undergo denaturation and enzymatic degradation in cell media conditions

• -  We are now confirming the compatibility of these stabilisation methods with the switchable design described in (1) above

  With the funding, I have been able to:

• -  purchase commercially-available DNA strands to assemble the switchable nanostructures

• -  purchase reagents necessary to prepare gold and iron oxide nanoparticle DNA conjugates

• -  paid for myself and PhD student Jasleen Kaur Daljit Singh to travel to the 25th International DNA

  Computing conference (Seattle, USA) to present this work in August 2019
  Dissemination of work
  The work that has been carried out in this project has been presented at a number of meetings:

  • -  25th International Conference in DNA Computing (DNA25) (Seattle,USA), poster presentation by Jasleen Kaur Daljit Singh, August 2019

  • -  The Poladian Project Conference (Sydney, Australia), oral presentation by Shelley Wickham, February 2019

  • -  Organic18, RACI Organic Division National Conference (Perth, Australia), oral presentation by Shelley Wickham, December 2018

  • -  ComBio 2019 Conference (Sydney, Australia), oral presentation by Shelley Wickham, September 2018

  • -  Seminar visits in 2018-19 to the University of Melbourne, University of Tasmania, UNSW, and public outreach talk for Sydney Design Week at Children's Medical Research Institute, Westmead

We have published the following review paper as a part of this work:

Daljit Singh, Jasleen Kaur, Minh Tri Luu, Ali Abbas, and Shelley F. J. Wickham. “Switchable DNA- Origami Nanostructures That Respond to Their Environment and Their Applications.” Biophysical Reviews, October 2, 2018.

The following publications are now being prepared:

• -  Switchable DNA-origami nanopore for drug delivery

• -  a temperature sensor for local melting of DNA