1. Designing, Building, and Using DNA Nanoboxes: - Inside-Outside Specific Protection of Sites - Tiffany Chan - Harvard International Genetically Engineered Machines (iGEM) Team

2. 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

3. HOW: DNA Nanostructure Origami

4. HOW: Honeycomb Lattice and Program

5. 2. The Process: Designing and Building a DNA Nanobox A.Coming up with ideas B.lllustrator diagrams C.ASCII diagrams for program inputs D.Running the program with the ASCII and scaffold sequence input E.Schematics of the oligo-scaffold hybridizations, for organization and addition of any modifications F.Ordering oligos, with any necessary modifications G.Organizing and making up oligo solutions H.Folding the oligos and scaffold to make the structure I.Imaging through gel shifts and electron micrography

6. A – C: Ideas and Pictures A. Coming up with ideas B. lllustrator diagrams C. ASCII diagrams for program inputs Double-ply hexagonal barrel, two lids on a separate scaffold? Single-ply hexagonal barrel, two lids on the same scaffold? Double-ply hexagonal barrel, smaller so no lids necessary? Single-ply rectangular barrel, lids extending from barrel?

7. 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 & +

8. 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

9. H: Folding H. Mixing and folding the oligos and scaffold to make the structure

10. I. Imaging I. Imaging through gel shifts and electron micrography

11. I. Imaging (cont.)

13. 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)

14. 4. Future Plans More designs More protection assays More electron microscopy and imaging snakes on a grid! =

15. 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!

16. THE END!