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

- 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

Discussion

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

Protein & DNA-Staining Control Assays: 3rd Repeat

Thrombin Aptamers

- 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.)

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)

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

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

Lane Contents

1 1kb DNA ladder (10 μL)2 control: +scaffold -oligos (10 μL 23 μM)3 control: -scaffold +3.2.B.core oligos (10 μL 25 μM)

4 expt B -latches (10 μL)5 expt B +latches (10 μL)6 control: -scaffold +3.2.C.core oligos (10 μL 25 μM)

7 expt C -latches (10 μL)8 expt C +latches (10 μL)9 control: -scaffold +6hb.v5 oligos (10 μL 25 μM)10 expt 6hb (10 μL)11 control: +scaffold -oligos (10 μL 23 μM)

Plans for This Week

1. Perform folding experiment with Design 4 once oligos have arrived

2. Perform folding experiment with biotinylated oligos (instead of oligos with thrombin-binding aptamer sites) for both Design 3 and 4 upon arrival of oligos

3. Check success of above folding experiments (EM, hopefully this afternoon)

4. Continue work on Design 2 (hexagonal honeycomb w/ double-ply lid)