Designing, Building, and Using DNA Nanoboxes:

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Inside-Outside Specific Protection of Sites-

Tiffany Chan-

Harvard International Genetically Engineered Machines (iGEM) Team

1. Intro to DNA Nanoboxes

• WHAT: Building DNA nanostructures that act as boxes-

• WHY: Boxes as drug-delivery vehicles-

• HOW: – Staple-oligonucleotides + single-stranded scaffold DNA =

DNA origami!-

– Honeycomb lattice system

HOW: DNA Nanostructures

2. The Process: Designing and Building a DNA Nanobox

A. Coming up with ideasB. lllustrator diagramsC. ASCII diagrams for program inputsD. Schematics of the oligo-scaffold hybridizationsE. Running the program to get a list of oligosF. Organizing oligosG. Ordering oligos (with any necessary modifications)H. Making up oligo solutionsI. Folding the oligos and scaffold to make the structureJ. Imaging through gel shifts and electron micrography

A – C: Ideas and PicturesA. Coming up with ideas B. lllustrator diagrams C. ASCII diagrams for program inputs

Single-ply hexagonal barrel, two lids on the same scaffold?

Double-ply hexagonal barrel,two lids on a separate scaffold?

Double-ply hexagonal barrel,smaller so no lids necessary?

Single-ply rectangular barrel,lids extending from barrel?

3. Using DNA Nanoboxes: • GOAL: Make sure they’re 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)

4. Future Plans• More designs

• More protection assays

• More electron microscopy

snakes on a grid

THE END!