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| DNA tethers have some potential drawbacks, in the first place the lack | | DNA tethers have some potential drawbacks, in the first place the lack |
| of rigidity in comparison to globular protein domains. We reasoned that | | of rigidity in comparison to globular protein domains. We reasoned that |
| we could use the same idea of ZFP-based DNA origami add-ons also for | | we could use the same idea of ZFP-based DNA origami add-ons also for the |
| creation of vertical stacks. | | creation of vertical stacks. |
| </span><br style="font-family: Arial;"> | | </span><br style="font-family: Arial;"> |
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| For this purpose DNA origami is modified at selected sites with the | | For this purpose DNA origami is modified at selected sites with the |
| same type of double stranded ZFP binding sites perpendicular to the | | same type of double stranded ZFP binding sites perpendicular to the |
| plane of DNA origami as in protein add-ons. We developed two approaches
| | DNA origami plane as in protein add-ons. We developed two approaches |
| to protein tethers based on twin ZFP binding domains and ZFP-protein | | to protein tethers based on twin ZFP binding domains and ZFP-protein |
| heterodimeric tethering domain. | | heterodimeric tethering domain. |
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| affinity. Formation of stacks requires the precise stoichiometry of | | affinity. Formation of stacks requires the precise stoichiometry of |
| each component in the reaction mixture as the excess of ZFP protein | | each component in the reaction mixture as the excess of ZFP protein |
| tethers could be inhibitory to the interaction, while the lower ration | | tethers could be inhibitory to the interaction, while the lower ratio |
| would result in missing tethers. One solution would be to first | | would result in missing tethers. One solution would be to first |
| functionalize one DNA origami plane with excessive amounts of twin ZFP | | functionalize one DNA origami plane with excessive amounts of twin ZFP |
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| <br style="font-family: Arial;"> | | <br style="font-family: Arial;"> |
| <span style="font-family: Arial;">Tethering two faces of | | <span style="font-family: Arial;">Tethering two faces of |
| DNA origami could be also achieved by the incorporation of different | | DNA origami could also be achieved by the incorporation of different |
| protein heterodimerization domains. In contrast to twin ZFP protein | | protein heterodimerization domains. In contrast to twin ZFP protein |
| tethers two different proteins have to be prapared. However the | | tethers two different proteins have to be prapared. However the |
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| <tr> | | <tr> |
| <td style="text-align: center;"><img | | <td style="text-align: center;"><img |
| style="width: 890px; height: 334px; font-family: Arial;" | | style="width: 890px; height: 295px; font-family: Arial;" |
| alt="This is image of beach house" title="Beach house" | | alt="" title="" |
| src="http://3.bp.blogspot.com/_RpexwBHz31E/TFdIX3QeS7I/AAAAAAAABDA/OoXLHmkbdYw/s1600/Kuefu+Rendering+E+FINAL.jpg"></td> | | src="http://openwetware.org/images/8/8b/Heterodimerizacija_FINALE.png"></td> |
| </tr> | | </tr> |
| <tr style="font-family: Arial;"> | | <tr style="font-family: Arial;"> |
| <td style="text-align: justify;"><span | | <td style="text-align: justify;"><span |
| style="font-weight: bold;">Figure 6: Protein tethers of DNA | | style="font-weight: bold;">Figure 6: DNA origami tethering based on protein heterodimerization.</span> (a) Schemes depict two elements (recombinant proteins in the form of ZFP-solubility tag-dimerizing domain) necessary for stacking of DNA origami planes. (b) After functionalizing each layer seperately and equimolarly mixing the two, stacks are formed. In case of smaller ligands acting as dimerizing partners, such nanoarchitectures could be disassembled by adding excessive amounts of a free ligand to the sample.<br><br></td> |
| origami based on protein heterodimerization. </span>Lorem | |
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| consectetur adipiscing elit. Maecenas eu massa velit. Donec sed risus
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| lacus, ac sollicitudin enim. Proin iaculis sollicitudin felis non
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| congue. Nulla massa odio, facilisis at dictum sed, pretium ut elit.<br><br></td>
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| </tr> | | </tr> |
| </tbody> | | </tbody> |
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Vertical stacks
DNA origami rectangles based on a single-stranded M13 DNA have
dimensions around 100 nm (Rothemund, 2006). Extension into the third
dimension created variable shapes such as different geometrical objects
(Shih, 2004) and a box (Andersen, 2009), weaving the compact 3D objects
from several connected layers (Douglas, 2009). 3D shells were also
produced by bending dsDNA (Han, 2011). Two dimensional tiling by
stitching DNA origami rectangles was reported (Endo, 2010). In general
3D DNA origami design has so far relied mostly on shaping a single M13
DNA strand, which restricted to some extent more complex functional
applications of bionanomolecular structures.
The main motivation for the idea of creating vertical stacks of DNA
origami is the possibility to combine two or more differently-modified
DNA origami breadboards, (e.g. metalized or covered with bound
conductive carbon nanotubes), which is not possible using single
continuous thread. Precise positioning of DNA origami layers could be
achieved using tethers connecting two neighboring DNA origami layers.
Vertical stacking of DNA origami layers could be accomplished using
either DNA or proteins as tethers. The vertical order of DNA origami
layers in the stack could be designed at will using interacting tethers
on each face of the DNA origami layers. The common approach of DNA and
protein tethers is that they both include replacement of conventional
staples with their extended versions, resulting in either
single-stranded or double-stranded DNA extensions which point
perpendicularly to the plane of DNA origami to directly interact or
serve as a protein docking site, respectively.
This type of vertical stacks may lead to <a
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appnanoelectronics">nanoscale
electronic applications</a>, elaborated in the Discussion.
Stacking could additionally be used to create masks for the formation
of 3D patterns similar as in photolithography. Vertical stacking can
increase the density of integration, which is an approach that is
actively pursued in the development of 3D integrated circuits.
DNA tethers
The idea of DNA tethers is to use unique complementary single stranded
DNA sequences protruding perpendicularly from the DNA origami. Their
positions on the opposing face of the other DNA origami stacking
partner are placed at the mirrored positions (Figure 4). Implementation
of this idea requires rational selection of complementary staple
extensions and some additional modifications (described in Methods) as
the tethering segments protrude from different faces of DNA origami
rectangles. By introducing such staples so that they project out of the
DNA origami perpendicularly in both directions, multiple (in theory
infinite) DNA origami stacks could be formed. Precise positioning of
two DNA origami layers above each other requires introduction of
several tethers and their asymmetric arrangement to prevent formation
of staggered overlays.
<tbody>
</tbody>
<img
style="font-family: Arial; width: 800px; height: 385px;" alt=""
src="http://openwetware.org/images/e/e9/VerticalstackDNAtethers.png"> |
| Figure 4. Vertical DNA origami
stacks based on single-stranded DNA tethers. DNA origami
planes are modified independently using staple strands with ssDNA
extensions protruding perpendicularly into solution.
|
Protein tethers
DNA tethers have some potential drawbacks, in the first place the lack
of rigidity in comparison to globular protein domains. We reasoned that
we could use the same idea of ZFP-based DNA origami add-ons also for the
creation of vertical stacks.
For this purpose DNA origami is modified at selected sites with the
same type of double stranded ZFP binding sites perpendicular to the
DNA origami plane as in protein add-ons. We developed two approaches
to protein tethers based on twin ZFP binding domains and ZFP-protein
heterodimeric tethering domain.
Bifunctional - twin
ZFP based approach
Here, the tethering molecule is composed of two distinct ZFPs each
recognizing origami attachment site on a different DNA origami layer.
This chimeric protein fusion, bridged with a solubility domain (MBP),
is prepared in recombinant form in bacteria. Similarly to DNA tethering
approach one could in theory expect formation of a long vertical stack
by the addition of two different ZFP attachment sites on the opposing
faces of DNA origami
<tbody>
</tbody>
<img
style="font-family: Arial; width: 890px; height: 298px;" alt=""
src="http://openwetware.org/images/d/d8/ProteintetheringFINAL.png"> |
| Figure 5: Protein-based tethering
of DNA origami planes with twin ZFP chimeras. (a) Basic
element for such protein mediated tethering comprises a solubility
enhancing domain (MBP) flanked by two distinct ZFPs. (b) Numerous
recombinantly produced twin ZFP chimeras stabilize DNA origami planes
at defined distance locking down the stack.
|
Introduction of several
protein tethers is expected to significantly increase the stability of
such stacks due to the cooperative interactions between several
neighboring binding sites, therefore even ZFPs with weaker affinities
could be used for the stacking purpose. The expected affinity of six
neighboring ZFP interactions should exceed the biotin/streptavidin
affinity. Formation of stacks requires the precise stoichiometry of
each component in the reaction mixture as the excess of ZFP protein
tethers could be inhibitory to the interaction, while the lower ratio
would result in missing tethers. One solution would be to first
functionalize one DNA origami plane with excessive amounts of twin ZFP
chimeras, remove the surplus of proteins and then titrate the system
with the second DNA origami plane.
Protein heterodimer
approach
Tethering two faces of
DNA origami could also be achieved by the incorporation of different
protein heterodimerization domains. In contrast to twin ZFP protein
tethers two different proteins have to be prapared. However the
advantage of the heterodimer approach is that interactions between
heterodimers could be regulated and derivatization of DNA origami with
bound ZFP-interacting domains can be performed separately.
<tbody>
</tbody>
<img
style="width: 890px; height: 295px; font-family: Arial;"
alt="" title=""
src="http://openwetware.org/images/8/8b/Heterodimerizacija_FINALE.png"> |
Figure 6: DNA origami tethering based on protein heterodimerization. (a) Schemes depict two elements (recombinant proteins in the form of ZFP-solubility tag-dimerizing domain) necessary for stacking of DNA origami planes. (b) After functionalizing each layer seperately and equimolarly mixing the two, stacks are formed. In case of smaller ligands acting as dimerizing partners, such nanoarchitectures could be disassembled by adding excessive amounts of a free ligand to the sample.
|
Coiled-coil-dimer based
approach
Coiled-coils are naturally occurring protein domains comprising
intertwined alpha helices in parallel or antiparallel orientation. The
idea here is to genetically fuse each of the two coiled-coil forming
segments to one of the two MBP-ZFP chimeras. Coiled-coil interactions
therefore mediate stacking of DNA origami layers decorated at the
defined positions through the specific ZFP binding domain. Preferably
the interactions between coiled-coil partners are antiparallel
(McClain, 2001), with coiled-coil segment serving as a rigid rod,
separating the two layers.
SH3- SH3 ligand
based approach
SH3- SH3 ligand pair
forms tight and specific interaction and has been used to assemble
protein scaffold in a biosynthetic reaction (Dueber, 2009). We
considered this interaction, even though it is in the sub μM range, for
the formation of a vertical stack. Addition of a soluble SH3 ligand
peptide could be used to regulate the disassembly of the stack.
Similarly to the strategy using coiled-coils, two DNA origami planes
would need to be separately functionalized with the two chimeras and
mixed at equimolar ratios to form a stack.
- Andersen ES, Dong
M, Nielsen MM, Jahn K, Subramani R, Mamdouh W, Golas MM, Sander B,
Stark H, Oliveira CL, Pedersen JS, Birkedal V, Besenbacher F, Gothelf
KV, Kjems J (2009) Self-assembly of a nanoscale DNA box with a
controllable lid. Nature
459: 73-76.
- Douglas SM, Dietz
H, Liedl T, Högberg B, Graf F, Shih WM (2009) Self-assembly of DNA into
nanoscale three-dimensional shapes. Nature 459: 414-418.
- Dueber JE, Wu GC,
Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, Prather KL, Keasling
JD (2009) Synthetic protein scaffolds provide modular control over
metabolic flux. Nature
Biotechnol 28: 753-9.
- Endo M, Sugita T,
Katsuda Y, Hidaka K, Sugiyama H (2010) Programmed-assembly system using
DNA jigsaw pieces. Chemistry
18: 5362-5368.
- Han D, Pal S,
Nangreave J, Deng Z, Liu Y, Yan H (2011) DNA origami with complex
curvatures in three-dimensional space. Science 332:342-6.
- McClain DL, Woods
HL, Oakley MG (2001) Design and Characterization of a Heterodimeric
Coiled Coil that Forms Exclusively with an Antiparallel Relative Helix
Orientation. J. Am.
Chem. Soc. 123: 3151-3152.
- Rothemund PWK
(2006) Folding DNA to create nanoscale shapes and patterns. Nature 440: 297-302.
- Shih WM, Quispe JD,
Joyce GF (2004) A 1.7-kilobase single-stranded DNA that folds into a
nanoscale octahedron. Nature
427: 618-621.
</body>
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