Title: Triggered Sequestration with DNA Nanoboxes: A New Drug Delivery Method
1Triggered 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
2Thrombin-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 4C - ran gel at 15 V for 18 hrs. at 4C
- stained with Coomassie blue for 20 min.
- - results no visible bands on gel
3Protein 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
4Protein 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
5Folding 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 80C
- - 60 cycles wait 2 minutes, decrease 1C
- - hold at 4C
- 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)
6Design 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
7Design 3 More success?
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)
- 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
8Plans 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)