Biomod/2011/Harvard/HarvarDNAnos:Design Sphere

From OpenWetWare

Jump to: navigation, search


Home              Mission              Process              Designs              Results              Resources              Team


Contents

Spherical Container

...Return to Designs     Go to Methods...    Go to Results...

Introduction

Figure 1. A three-dimensional model of the sphere (Han et al. 2011).
Figure 1. A three-dimensional model of the sphere (Han et al. 2011).

In our search for a robust and elegant design, we were inspired by the origami sphere that Dongran Han demonstrated in their 2011 Science paper "DNA Origami with Complex Curvatures in Three-Dimensional Space".

  • The spherical design appealed to us because of its efficient use of DNA and lack of weak points--that is, instead of having edges, it only has two holes at each pole, minimizing spots where cargo can leak out. We imagined that we would be able to change the design of the Han sphere to make it an openable and closable container.

The design principles for an origami sphere (and other 3D origami with complex curvatures) employed by Han are the following (Figure 1):

  • Multi-planar arrangement of parallel double helices with in-plane curvature of helices into rings, and
  • Curvature across planes caused by different ring sizes and greater distance between crossovers in larger rings than in smaller rings.

Ultimately, the Han sphere is comprised of 24 parallel rings of helices (12 rings in each hemisphere) and is 6626 bases long. Its largest ring is 42 nm in diameter and the hole at each pole is 4 nm.

To adapt the Han sphere to our purposes, we generated a caDNAno file from diagrams provided in Han's supplementary materials. We succeeded in reconstructing the Han sphere down to the base and matched all staple strands generated by caDNAno to staple strands used by Han et al.


Loading Cargo

Figure 2. A cross-sectional view of the sphere depicting our strategy for attachining gold nanoparticles. The ssDNA conjugate and extension strands are complementary.
Figure 2. A cross-sectional view of the sphere depicting our strategy for attachining gold nanoparticles. The ssDNA conjugate and extension strands are complementary.

The cargo that we chose to place inside our containers was gold nanoparticles of 5 nm in diameter. As gold nanoparticles are highly visible under the transmission electron microscope (TEM), we can easily tell if cargo was placed inside our containers or released from our containers.

To place gold nanoparticles inside the sphere:

  • We conjugated ssDNA to the nanoparticles that are complementary to staple strand extensions on the inside of the sphere (Figure 3). That is, the ssDNA on the nanoparticles would bind to the staple extensions on the inside of the sphere, tethering the nanoparticle to the sphere.
  • The ssDNA conjugated to gold contains a photocleavable (PC) spacer from IDT; once the cargo is inside the sphere, we can solubilize it by exposing the solution to UV light, which cleaves the PC spacer.
  • To maximize the chance that the staple extension on the sphere points inside rather than outside, we chose to extend the staple strand at a base where it is oriented inside the sphere in its helical turn. This was possible because we mapped out the orientation (inside the sphere, upward crossover, downward crossover, or outside the sphere) of every base in the sphere in SphereCAD.
Figure 3. We created the open state of the sphere by removing all staples holding together the two hemispheres.
Figure 3. We created the open state of the sphere by removing all staples holding together the two hemispheres.

Since the Han sphere in its unaltered form is closed, next came the question of how to incorporate cargo. Our options were:

  • Fold the sphere in the presence of gold nanoparticles
  • Fold the sphere in an "open state," add gold nanoparticles, and then close the sphere.

Since gold nanoparticles aggregate under high temperatures, we opted for the latter strategy.

To create an open state of the sphere, we removed all staple strands holding the twelfth and thirteenth helices (the helices at the "equator" of the sphere) together (Figure 3). This results in two hemispheres held together by a scaffold strand crossover.


Locking Mechanism

Figure 4. Azobenzene tweezers (Liang et al. 2008).
Figure 4. Azobenzene tweezers (Liang et al. 2008).

To close the sphere after loading of cargo and reopen it to release cargo, we designed a series of "locks" based on azobenzene tweezers detailed by Liang et al. in this paper (Figure 4). Recall that we had cut all staple strand crossovers holding the two hemispheres together.

To create the locks, we extended staples on helices 12 and 13 where they are oriented outward; again, these locations were found with SphereCAD.

  • We were able to find nine positions around the equator of the sphere where symmetric staples on the Northern and Southern hemisphere both face outward. Thus, we designed the closable sphere with a maximum of nine locks.
  • We extended the 3' ends of staple strands on the Northern Hemisphere and the 5' ends of staple strands on the Southern Hemisphere. Onto all Northern staples were appended the sequence 5'-CTGGTAACAATCACG-3', onto all Southern staples 5'-CTGTCTGAACTAACG-3'. These sequences are from the Liang tweezer design.

To close the sphere, we used Liang's "lock strand" that is complementary to both the Northern and Southern staple extensions.


Re-opening Mechanism

In order to have a re-openable sphere, we designed a strand displacement mechanism and a photocleavage mechanism.

  • In the strand displacement mechanism, a "key strand" with greater complementarity to the lock strand than the staple extensions is added to solution and displaces the lock strands from the staples, opening the sphere (Figure 5). Thus we added a toehold to the lock strand and designed a complementary key strand. To make sure these strands do not have problematic secondary structures and that lock:key interactions are stronger than staple:lock:staple interactions, we consulted NUPACK.
  • Lock strand sequence, with toehold bolded: 5'-CGTGATTGTTACCAGTTCGTTAGTTCAGACAG-CAGACAGACAG-3'
  • Key strand sequence: 5'-CTGTCTGTCTG-CTGTCTGAACTAACGAACTGGTAACAATCACG-3'
Figure 5. A diagram explaining our strand displacement mechanism for opening the sphere.
Figure 5. A diagram explaining our strand displacement mechanism for opening the sphere.
  • In the photocleavage mechanism, rather than use a key strand to displace the lock strand, we simply insert a photocleavable (PC) spacer (ordered from IDT) between the halves complementary to the Northern and Southern staple extensions (Figure 6).
  • When exposed to UV light, the spacer cleaves, splitting the lock strand in half and opening the sphere (FIGURE). The lock strand used in this mechanism is the same as that used for the strand displacement mechanism except for the presence of a photocleavable spacer.
  • Lock strand sequence, with PC spacer and toehold bolded: 5'-CGTGATTGTTACCAG-/iSpPC/-TTCGTTAGTTCAGACAG-CAGACAGACAG-3' (Note: the toehold is not of use in the photocleavable mechanism.)
Figure 6. A diagram explaining our photocleavage mechanism for opening the sphere.
Figure 6. A diagram explaining our photocleavage mechanism for opening the sphere.


Source Files

The final caDNAno file can be downloaded here (Figure 7).

An Excel file containing all staple sequences for the sphere can be downloaded here. The file includes staples needed to recreate the original, closed Han sphere, as well as the staples we modified to create our locks.

  • To create the open state of the sphere, add all staples to solution except for those labeled orange in the Excel file.
  • We used M13mp18 scaffold from New England Biolabs and ordered our staple strands from Integrated DNA Technologies.
Figure 7. A strand diagram corresponding to the caDNAno representation of the Han sphere. Click to expand!
Figure 7. A strand diagram corresponding to the caDNAno representation of the Han sphere. Click to expand!

Personal tools