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
Overview Project goal Last week’s progress
Test AscI digest Microcon filtration [Mg2+] and [oligo] titration
This week’s goals
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
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: 0 0 0 50 500 0 50 500digestion time: 0 0 12 12 12 24 24 24 (minutes)
18% PA, 1.5hr @ 120V, SYBR gold staining
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+ unpurified successive flow-throughs flowthrough
scaffold retentate retentate
Image courtesy Millipore
2% agarose, 10mM MgCl2, 1hr @ 60V, EtBr
[Mg], [oligos]: PEG experiments
PEG titration performed for each set of folding conditions
Results: Oligos were not cleared in any of the 0-8%-final PEG precipitations
pellet pellet etc…supern. supern.
[MgCl2] - 10mM (final) 20mM (final) 30mM (final)
[oligos] 100nm (total) X X X
- 600nm (total) X X X
2% agarose, 10mM MgCl2, 1hr @ 60V, EtBr
[Mg], [oligos]: Microcon and PEG
Lane Trial Purification
1 30 nM oligos, 10 mM MgCl2 unpurified
2 30 nM oligos, 10 mM MgCl2 PEG pellet
3 30 nM oligos, 10 mM MgCl2 Microcon retentate
4 30 nM oligos, 20 mM MgCl2 Unpurified
5 30 nM oligos, 20 mM MgCl2 PEG pellet
6 30 nM oligos, 20 mM MgCl2 Retentate
7 30 nM oligos, 30 mM MgCl2 Unpurified
8 30 nM oligos, 30 mM MgCl2 PEG pellet
9 30 nM oligos, 30 mM MgCl2 Retentate
10 1 Kb+ ladder n/a
11 p7308 (44 nM) n/a
12 600 nM oligos, 10 mM MgCl2 Unpurified
13 600 nM oligos, 10 mM MgCl2 PEG pellet
14 600 nM oligos, 10 mM MgCl2 Retentate
15 600 nM oligos, 20 mM MgCl2 Unpurified
16 600 nM oligos, 20 mM MgCl2 PEG pellet
17 600 nM oligos, 20 mM MgCl2 Retentate
18 600 nM oligos, 30 mM MgCl2 Unpurified
19 600 nM oligos, 30 mM MgCl2 PEG pellet
20 600 nM oligos, 30 mM MgCl2 Retentate
1 3 5 7 9 11 13 15 17 19 2 4 6 8 10 12 14 16 18 20
• Results: PEG seems to be more efficient at removing oligos• Nanostructures in PEG seem to be damaged, gel shifting
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
This week’s goals
Continue filtration/purification trials
AscI digest of folded nanostructure