Designing, Building, and Using DNA Nanoboxes: - Inside-Outside Specific Protection of Sites-

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Designing, Building, and Using DNA Nanoboxes
-Inside-Outside Specific Protection of Sites-

• Tiffany Chan
• -
• Harvard International Genetically Engineered
  Machines (iGEM) Team

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1. Intro to DNA Nanoboxes

• WHAT Building DNA nanostructures that act as
  boxes
• -
• WHY Boxes as drug-delivery vehicles
• Cargo or Sequestration
• -
• HOW
• DNA origami!
• -
• Honeycomb lattice system

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HOW DNA Nanostructure Origami
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HOW Honeycomb Lattice and Program
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2. The Process Designing and Building a DNA
Nanobox

1. Coming up with ideas
2. lllustrator diagrams
3. ASCII diagrams for program inputs
4. Running the program with the ASCII and scaffold
   sequence input
5. Schematics of the oligo-scaffold hybridizations,
   for organization and addition of any
   modifications
6. Ordering oligos, with any necessary modifications
7. Organizing and making up oligo solutions
8. Folding the oligos and scaffold to make the
   structure
9. Imaging through gel shifts and electron
   micrography

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A C Ideas and Pictures

• A. Coming up with ideas B. lllustrator
  diagrams C. ASCII diagrams for program
  inputs

Double-ply hexagonal barrel, smaller so no lids
necessary?
Single-ply rectangular barrel, lids extending
from barrel?
Double-ply hexagonal barrel, two lids on a
separate scaffold?
Single-ply hexagonal barrel, two lids on the
same scaffold?
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D Running the Program and Getting the Oligos
D. Running the program with the ASCII and
scaffold sequence input to get list of oligos and
their positions


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E-G Schematic-making, Ordering, and Organizing
E. Schematics of the oligo locations on the
scaffold F. Ordering oligos, with any necessary
modifications G. Organizing and making up oligo
solutions
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H Folding
H. Mixing and folding the oligos and scaffold to
make the structure
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I. Imaging
I. Imaging through gel shifts and electron
micrography
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I. Imaging (cont.)
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I. Imaging (cont.)
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3. Using DNA Nanoboxes

• GOAL Make sure theyre actually boxes!
• A box defines a separate inside and outside
• -
• SOLUTION Protection assays
• Protect what on the inside?
• Thrombin (stuck to thrombin DNA-aptamers built
  into the oligos
• Streptavidin (stuck to the biotin molecules on
  the oligos ordered that way)
• Oligo-ligand (a double-stranded DNA segment made
  from a ssDNA staple-end and a ssDNA added to the
  box mixture after folding)
• From what on the outside?
• Proteases (trypsin and proteinase K)
• Restriction enzyme (AscIII)

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4. Future Plans

• More designs
• More protection assays
• More electron microscopy and imaging

snakes on a grid!

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Acknowledgments

• Harvard iGEM Team
• Nanostructure Team
• Katie Fifer
• Matt Meisel
• Val Lau
• Cyanobacteria Team
• Hetmann Hsieh
• Jeff Lau
• Dave Ramos
• Zhipeng Sun
• Cell-Targeters
• Lewis Han
• Perry Tsai

• TFs
• Shawn Douglas
• Nick Stroustrup
• Chris Doucette
• Faculty
• William Shih
• George Church
• Pam Silver
• Radhika Nagpal
• Jagesh Shah

PRISE and Greg Llacer!
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THE END!