1. Triggered Sequestration with DNA Nanoboxes: A New Drug Delivery Method July 24, 2006 iGEM Week 7: Progress Report Tiffany Chan, Katherine Fifer, Valerie Lau, Matthew Meisel, Lewis Hahn

2. Mission: Visualization (not Impossible, though at times difficult) 1. Visualizing boxes c3.2 & 4.0 2. Visualizing design c5.0: double-ply 3. Visualizing protein protection by the containers

3. 1. Container 3.2 Scale Bar: 100nm for left, 20nm for right; 0.23% uranyl formate stain; +latch2

4. 1. Container 4.0 Lane 4 (-L –A), 5 (L1 –A), 6 (L2 –A), 7 (6hb), 8 (scaffold), 9 (oligos) Scale Bar: 100nm, 0.5% UF, -latches Gel shift for folded boxes (4-6) from scaffold alone (8)

5. 1. Container 4.0 (cont.) Scale Bar 100nm, 0.1% UF, -latchesScale Bar 20nm, 0.5% UF, -latches

6. 1. Container 4.0 (cont.) Scale Bar 100nm, 0.2% UF, latch1 -aptamers

7. 2. Container 5.0: Why? Greater rigidity Hard to visualize by EM Stain seems to crush single-ply boxes Single-ply lids not very visible Closer packing approximately 2.1nm per barrel, as opposed to single-ply 3nm Images courtesy Dr. Shih and Shawn Douglas Double-Ply Sheets

8. 2. Container 5.0 Process 1. Designing barrel and lids for efficacy 2. Plotting oligos 3. Pinpointing oligo and scaffold entrance and exit points for latches 4. Splitting up the scaffold sequences 5. Designing latches 6. Organizing and ordering oligos

9. 2. Container 5.0: Design

10. 2. Container 5.0: Plotting Oligos and Entrance & Exit Points Red Up, Green Down Points LID BARREL By Hand By Program Red Entrance, Green Exit Points

11. 3. Visualizing protein protection by the containers Goals: develop a way of differentiating whether or not biotinylated nanostructures have been decorated with streptavidin Prove that we’re not seeing streptavidin interactions with scaffold or free oligos Challenges: Imaging streptavidin on a PA gel Visualizing a gel shift on a PA gel

12. 3. Visualizing protein protection by the containers Streptavidin-+-+-+-++-++ Scaffold--++-------- Biotinylated oligos----++------ Nanostructures------outoutoutininin (w/ excess unused oligos) Latches--------+--+ 123456789101112 Scaffold/nanostructures Biotinylated oligos Unused oligos Latch oligos Thank you, uncooperative ladder and loading dye.

13. 3. Visualizing protein protection by the containers Free streptavidin Traces of oligo-bound streptavidin (lanes 8, 9, 11, 12)? Streptavidin-+-+-+-++-++ Scaffold--++-------- Biotinylated oligos----++------ Nanostructures------outoutoutininin (w/ excess unused oligos) Latches--------+--+ 123456789101112

14. 3. Visualizing protein protection by the containers Immediate challenges: More sensitive protein imaging (Coomassie blue has lower threshold of about 10 ng / 200 fmols of streptavidin, which is too low) Differentiation of streptavidin-decorated nanostructures (no gel shift on 12% PA) Down the road: Protease digest conditions to digest streptavidin away (Proteinase K just fine?)

15. Don’t Worry, Be Happy Now!