1. Triggered Sequestration with DNA Nanostructures August 21, 2006 iGEM Week 11: Progress Report Tiffany Chan, Katherine Fifer, Valerie Lau, Matthew Meisel

2. Overview EM Images Nanostructure Purification (PEG) Enzyme-based protection assay Streptavidin Beads and Biotinylated Boxes

3. EM Images (snakes on a grid) c5.0 barrel (10 nM), 0.7% uranyl formate Appear to be lining up end to end, probably because of the stain

4. EM Images

5. EM Images: the power of DNA Highly Extensible structures Multimerization - could create dimers, etc by pairing DNA at the ends of two different barrels

6. PEG precipitation progress P S P S U P S P S P S U P S P S P S P - pellet S - supernatant U - untreated

7. PEG precipitation progress  Results  30mM MgCl 2 w/ 1x oligos seems best  PEG purification seems to be working much better  To be continued  double PEG precipitation to purify away more oligos (first trial unsuccessful)  repetition of precipitations to acquire more purified nanostructures and better images

8. Microcon trials w/ SDS SDS was added to the buffer used to rinse Micron tubes 1: 1 kb+ ladder 2: p7308 3: unpurified nanostructures 4: 6hb retentate (did not filter oligos) 5: 6hb retentate, washed w/ 0.1% SDS 6: nanostructure retentate 7: nanostructure retentate, washed w/ 0.01% SDS 8: nanostructure retentate, washed w/ 0.1% SDS 1234567812345678

9. Enzyme-based protection assay Goal: digest away oligo- ligand with AscI 1: 10 bp+ ladder 2: attachment oligo 3: oligo-ligand 4: attachment oligo and oligo-ligand 5: attachment oligo, oligo-ligand, AscI 6: nanostructure w/ inward-facing ligand, AscI 7: nanostructure w/ outward-facing ligand, AscI 8: p7308, AscI 9: nanostructure w/ inward-facing ligand (-AscI) 10: nanostructure w/ no ligand, AscI 12345678910

10. Enzyme-based protection assay (TBE gradient PAGE) Goal: digest away oligo- ligand with AscI 1: 10 bp+ ladder 2: attachment oligo 3: oligo-ligand 4: attachment oligo and oligo-ligand 5: attachment oligo, oligo-ligand, AscI 6: nanostructure w/ inward-facing ligand, AscI 7: nanostructure w/ outward-facing ligand, AscI 8: p7308, AscI 9: nanostructure w/ inward-facing ligand (-AscI) 10: nanostructure w/ no ligand, AscI 12345678910

11. Streptavidin Beads and Biotinylated Boxes 1) Incubate biotinylated boxes with streptavidin beads (magnetic or agarose) 2) Wash away unbound material 3) Elute the bound material by digesting streptavidin with trypsin, proteinase K, or by some other elution measures

12. Streptavidin Beads and Biotinylated Boxes 1 2 3 4 5 6 7 8 9 10 Lane Component 11kb ladder 2p7308 36hb wash 4biotin-oligos wash 5inside-biotin box wash 6outside-biotin box wash 76hb elute 8biotin-oligos elute 9inside-biotin box elute 10outside-biotin box elute

13. Streptavidin Beads and Biotinylated Boxes trypsin elution - NO ELUTE streptavidin elution - NO ELUTE

14. Streptavidin Beads and Biotinylated Boxes Plans: 1) Elute using enzyme-specific buffers 2) Try streptavidin elution using heat or nanobox-denaturing methods, relying on being able to visualize the ssDNA and oligos

15. Future Image folded design 6 Perfect PEG purification Improve protection assays