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