Biomod/2011/Harvard/HarvarDNAnos:CurrentProgress

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Abstract

Previous work has showcased the use of scaffolded DNA origami to self-assemble nanoscale, three-dimensional shapes with enclosed, hollow interiors. We explore the possibility of using such structures to load, entrap, and release soluble nanoscale cargo, with potential future applications in drug delivery and molecular signal amplification. Specifically, we design a rectangular box structure that can encapsulate gold nanoparticles and subsequently release them by a two-step process: a) photocleavage, which solubilizes the nanoparticle within the box, and b) introduction of a DNA signal, which opens the box by strand-displacement. We further investigate the use of an existing spherical origami design for similar purposes, and demonstrate opening of the sphere.

Goals

Our goal is to create DNA origami containers that can load, hold, and release cargo. To do this, we must:

  1. Load various forms of cargo by attaching it to the inside of a container and then closing the container
  2. Solubilize this cargo without leakage to the exterior of the container
  3. Open our container, releasing our cargo


Designing our Containers

Sphere

  • Using caDNAno, we have replicated the sphere featured in the recent Science paper by Han et al. so that we can generate the exact same staple sequences published by Han et al.
  • Now we need to implement opening/closing and loading/solubilization mechanisms for the sphere
  • We also need to cap the holes (approximately 2-3 nm in diameter) at the poles of the sphere, or figure out how to exploit these holes
  • We are in the process of creating "SphereCAD," a computer program that synthesizes our caDNAno file and 3D models in order to streamline correlation between theta/phi, bp position, and whether that bp position points into or out of the sphere. This will help in the design process of O/C and L/S mechanisms.
3D model of our sphere, created in Autodesk Maya

Box with Lid

Schematic of Box with Lid with Parallel Locking Mechanism, to be locked by azobenzene-sequence strand
  • This design's strands have been ordered

  • We need to implement a L/S mechanism
  • We should also consider designing a box that has a lid with parallel helices so that the jagged interface between box and lid (due to crossover positions) will be complementary


Designing Our Cargo

Making chains of nanoparticles using "ultramer"

  • We are currently working on attaching the handles to the ultramer
    • We ran a reaction ladder where the first reaction had only the ultramer, the second had the ultramer with the first handle annealed, the second had the ultramer, first handle, and second handle, etc. Here is the PAGE result.
  • Next, we need to figure out how to attach thiolated DNA strands to gold nanoparticles

Synthesizing nanoparticles

  • We have made our own homemade gold nanoparticles, 5 nm (as per "Preparing Colloidal Gold for Electron Microscopy" by Polysciences, Inc.) and other sizes
  • We have used DLS to confirm their size and determine their hydrodynamic radius
  • Next, we will use TEM to image these nanoparticles, varying salt conditions to observe aggregation



Providing Functionality to our Designs

"One-pot" folding vs. Fold-then-load

Strand Displacement

Disadvantages

  • Single strands probably diffuse slowly into sphere


Photocleavable Spacers

From IDT


Azobenzene

Liang et al (2008)

  • First order of strands for Box with Lid has lock handles adapted to azobenzene sequence. Two options:
    • use actual azobenzene strand to lock, UV to unlock
    • use azobenzene-sequence strand with toehold to lock, strand displacement to unlock
  • We will first test both mechanisms on a "minimal box" which is just a strand of DNA with a central poly T representing the actual box



Testing the effectiveness of our containers

Sphere Design

  1. Normal sphere
  2. Sphere with disulfide on equator and cargo handle staples
  3. Sphere with strand displacement cargo handle staples, normal strands on equator --> tells us if single strands diffuse into sphere through top and bottom holes
  4. Sphere with strand displacement equator and cargo handle staples
  5. Sphere with strand displacement cargo handle staples, disulfide on equator (?)
  6. Sphere with strand displacement equator staples, disulfide handle staples (?)
  7. Sphere with restriction enzyme equator staples, disulfide cargo handle staples
  8. Sphere with azobenzene staples
  • NOTE 1: If from (3) we see that single strands do diffuse into the sphere, in the next round of design we can add capping mechanisms to seal off the top and bottom holes of the sphere. (Maybe even create a torus!)
  • NOTE 2: To test our program, we eventually need to attach cargo to outside.

Box Design

  • Bare box without cargo L/S mechanism
    • actual azobenzene: F_12X (folds open, lock, then UV release)
    • azobenzene sequence with toehold: F_n+ (folds open, lock, then strand displacement release)
    • intra-molecular locking (folds locked, then strand displacement release)
  • Box with cargo L/S mechanism


Staple Strands to order for Sphere Design

  1. Matching strands on equator
  2. Matching strands not on equator
  3. Non-matching strands on equator (from Han's paper)
  4. Matching on equator (from Han's paper)
  5. Non-matching strands on equator (from our caDNAno file)
  • Staples with disulfide
  • Staples with strand displacement mechanism
  • Staples with restriction enzyme sequences
  • Staples with azobenzene
  1. Matching on equator (from our caDNAno file)

Staple Strand to order for Box Design

  1. Core staples to not be manipulated
  2. Lock staples
  3. Staples that might be affected when direction of lock staples is changed
  4. Staples from which cargo-binding mechanism may be attached


Automation of our processes with CAD Software

SphereCADBasic

SphereCAD with standalone GUI


Additional Project Ideas (for another summer!)

See Brainstorming

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