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Triggered Sequestration with DNA Nanoboxes: A New Drug Delivery Method

Triggered Sequestration with DNA Nanoboxes: A New Drug Delivery Method. July 17, 2006 iGEM Week 6: Progress Report Tiffany Chan, Katherine Fifer, Valerie Lau, Matthew Meisel. Thrombin-Aptamer Binding Assay.

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Triggered Sequestration with DNA Nanoboxes: A New Drug Delivery Method

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  1. Triggered Sequestration with DNA Nanoboxes: A New Drug Delivery Method July 17, 2006 iGEM Week 6: Progress Report Tiffany Chan, Katherine Fifer, Valerie Lau, Matthew Meisel

  2. Thrombin-Aptamer Binding Assay • used aptamer 6hbab1 (which binds to a standard 6-helix-bundle nanotube at its 5' end and contains a thrombin aptamer sequence at the 3' end) • - loaded onto a non-denaturing polyacrylamide gel (10% to 20% gradient) at 4°C • ran gel at 15 V for ~18 hrs. at 4°C • stained with Coomassie blue for ~20 min. • - results: no visible bands on gel

  3. Discussion

  4. Protein & DNA-Staining Control Assays • goal: image varying concentrations of thrombin and aptamers using Coomassie blue and EtBr respectively - ran 10-20% polyacrylamide gel run at 25 V for 2.5 h. • - protein section rocked in GelCode Blue Stain for 1 hr.; DNA section rocked in 100 mL Tris-glycine buffer + 10 μL of 10 mg/mL EtBr for 1 hr. • - results: loading dye diffused about 3 mm in all directions into the gel with neither the protein nor the DNA imaged on the gel

  5. Protein & DNA-Staining Control Assays: 3rd Repeat - 12% polyacrylamide gel run at 120V for 15 min. - protein section stained w/ GelCode Blue Stain (Coomassie Blue) for 30 min.; DNA section stained w/ 10μL EtBr in 100 mL Tris-glycine buffer for 30 min. - results: both protein and DNA successfully imaged, w/ protein appearing in distinct bands (implying that there are several different sized molecules in the protein mixture: perhaps the fastest band is thrombin monomer, the second fastest is dimer, etc.) Aptamers Thrombin

  6. Folding of Design 3 • Reagents: • - 9 μL p7308 scaffold = (10 nM)/(44 nM) x 40 μL • - 16 μL oligos (3.2.A) = (100 nM)/(250 nM) x 40 μL • - 4 μL 10x folding buffer (500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl2) • - 11 μL dH2O • total volume: 40 μL • Annealing protocol: • - start at 80°C • - 60 cycles: wait 2 minutes, decrease 1°C • - hold at 4°C • Gel analysis: • - 2% agarose gel supplemented w/ 10 mM MgCl2 • - run in 1x TBE supplemented w/ 10 mM MgCl2 for 30 min at 130 V • - Rocked for 15 min in 1x TBE, 10 mM MgCl2, 100 μg/mL EtBr (forgot to put it in the gel)

  7. Design 3: Successful Gel Analysis • Results: • - ladder, scaffold, and oligos all appear as expected • - folding reaction mixture shows oligo smear (expected) as well as a band that runs slower than the scaffold

  8. Design 3: More success? • Results: • Designs with latches appear to fold in a manner similar to latch-less design • No shift when latch oligos are added • Inconclusive as to whether lid closure is occurring

  9. Plans for This Week • Perform folding experiment with Design 4 once oligos have arrived • Perform folding experiment with biotinylated oligos (instead of oligos with thrombin-binding aptamer sites) for both Design 3 and 4 upon arrival of oligos • Check success of above folding experiments (EM, hopefully this afternoon) • Continue work on Design 2 (hexagonal honeycomb w/ double-ply lid)

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