1. Triggered Sequestration with DNA Nanostructures: A New Drug Delivery Method August 14, 2006 iGEM Week 10: Progress Report Tiffany Chan, Katherine Fifer, Valerie Lau, Matthew Meisel

2. Overview Project goal Last week’s progress test AscI digest Microcon filtration [Mg 2+ ] and [oligo] titration This week’s goals

3. Project goal To create a scaffolded DNA nanostructure that protects a ligand from a degradation enzyme When the ligand is presented on the outside of the nanostructure, we expect there to be no protection

4. AscI digest Two ss DNAs (the “attachment DNA” and the “oligo ligand”) with 15-bp complimentary stretch were briefly incubated at room temperature The double-stranded construct was then treated with AscI under four different digestion conditions 25 kb+ 10 kb+ 25 kb+ milliunits AscI:00050500050500 digestion time:00121212242424 (minutes)

5. Microcon filtration Samples are pipetted into the Microcon filter and are successively diluted and centrifuged Results: relatively good oligo removal, but poor nanostructure yields 1 kb+unpurifiedsuccessive flow-throughs scaffoldretentate Image courtesy Millipore

6. More PEG experiments PEG titration performed for each set of folding conditions pelletpelletetc…supern.

7. Other filtration options Microcon use more material—is yield loss constant? use a detergent to loosen nanostructures from membrane (Triton X-100?) Sepharose spin columns large molecules elute first homemade: Datta et al. (2003), Anal. Biochem., 317, p. 284. Clonetech Prespin Load sample Centrifuge (large molecules elute first) Image courtesy Clonetech

8. This week’s goals Continue filtration/purification trials AscI digest of folded nanostructure