1. iGEM Week 10: Progress Report
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 This week’s goals
Test AscI digest
Microcon filtration
[Mg2+] and [oligo] titration
This week’s goals

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

4. AscI digest
The “attachment oligo” and the “oligo ligand,” with 15 complimentary bps, were briefly incubated at room temperature
The double-stranded construct was then treated with AscI under four different digestion conditions
Results: Digestion for 24 min with 500mU of AscI works well.
25 bp+
10 bp+
10 bp+
25 bp+
milliunits AscI:
digestion time:
(minutes)
18% PA, 120V, SYBR gold staining

5. Image courtesy Millipore
Microcon filtration
Samples are pipetted into the Microcon filter and are successively diluted and centrifuged
Results: relatively good oligo removal, but poor nanostructure yields
Image courtesy Millipore
1 kb+ unpurified successive flow-throughs flowthrough
scaffold retentate retentate
2% agarose, 10mM MgCl2, 60V, EtBr

6. [Mg], [oligos]: PEG experiments
2% agarose, 10mM MgCl2, 60V, EtBr
pellet pellet etc…
supern. supern.
PEG titration performed for each set of folding conditions
Results: Oligos were not cleared in any of the 0-8%-final PEG precipitations
[MgCl2] -
10mM (final)
20mM (final)
30mM (final)
[oligos]
100nm (total) X
600nm (total)

7. [Mg], [oligos]: Microcon and PEG
Lane
Trial
Purification 1
30 nM oligos, 10 mM MgCl2
unpurified 2
PEG pellet 3
Microcon retentate 4
30 nM oligos, 20 mM MgCl2
Unpurified 5 6
Retentate 7
30 nM oligos, 30 mM MgCl2 8 9 10
1 Kb+ ladder
n/a 11
p7308 (44 nM) 12
600 nM oligos, 10 mM MgCl2 13 14 15
600 nM oligos, 20 mM MgCl2 16 17 18
600 nM oligos, 30 mM MgCl2 19 20
Results: PEG seems to be more efficient at removing oligos
Nanostructures in PEG seem to be damaged, gel shifting

8. Other filtration options
Prespin
Load sample
Centrifuge
(large molecules elute first)
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
Image courtesy Clonetech

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