Transcript Triggered Sequestration with DNA Nanostructures

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
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Project goal
Last week’s progress
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Test AscI digest
Microcon filtration
[Mg2+] and [oligo] titration
This week’s goals
Project goal
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To create a scaffolded DNA
nanostructure that protects a ligand
from degradation
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When the ligand is presented on the
outside of the nanostructure, we expect
there to be no protection
AscI digest
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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
500
digestion time:
(minutes)
0
0
12
12
12
24
24
24
18% PA, 1.5hr @ 120V, SYBR gold staining
Microcon filtration
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Samples are pipetted
into the Microcon filter
and are successively
diluted and centrifuged
Image courtesy Millipore
1 kb+
unpurified
scaffold
retentate
successive flow-throughs
flowthrough
retentate
Results: relatively
good oligo removal, but
poor nanostructure
yields
2% agarose, 10mM MgCl2, 1hr @ 60V, EtBr
[Mg], [oligos] and More PEG
experiments
2% agarose, 10mM MgCl2, 1hr @ 60V, EtBr
pellet
pellet
etc…
supern. supern.
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[MgCl2]
-
10mM (final)
20mM (final)
30mM (final)
[oligos]
100nm (total)
X
X
X
-
600nm (total)
X
X
X
PEG titration performed for
each set of folding
conditions
Results: Oligos were not
cleared in any of the 08%-final PEG
precipitations
Prespin
Other filtration options
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Microcon
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use more material—is yield loss
constant?
use a detergent to loosen
nanostructures from membrane
(Triton X-100?)
Sepharose spin columns
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large molecules elute first
homemade: Datta et al. (2003), Anal.
Biochem., 317, p. 284.
Clonetech
Load
sample
Centrifuge
(large
molecules
elute first)
Image courtesy Clonetech
This week’s goals
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Continue filtration/purification trials
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AscI digest of folded nanostructure