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=Process=
 
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[[Image:Project Outline Header.jpg | center]]
<a href=http://openwetware.org/wiki/Biomod/2011/Harvard/HarvarDNAnos:Designs><img src="http://openwetware.org/images/3/3c/Design.jpg" width=150px></a>
 
<a href=http://openwetware.org/wiki/Biomod/2011/Harvard/HarvarDNAnos:Methods><img src="http://openwetware.org/images/a/ac/Methods.jpg" width=150px></a>
 
<a href=http://openwetware.org/wiki/Biomod/2011/Harvard/HarvarDNAnos:Results><img src=http://openwetware.org/images/4/47/Hdna_results.jpg width=150px></a>
 
<a href=http://openwetware.org/wiki/Biomod/2011/Harvard/HarvarDNAnos:Protocols><img src="http://openwetware.org/images/3/3a/Protocols.jpg" width=150px></a>
==Objective==
<a href=http://openwetware.org/wiki/Biomod/2011/Harvard/HarvarDNAnos:Tutorials><img src="http://openwetware.org/images/2/24/Tutorials.jpg" width=150px></a>
Our goal is to create DNA origami containers that can load, hold, and release cargo. To do this, we must:
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*Design and fold robust [[#Container Designs | three dimensional origami featuring enclosed interiors]] with optimized volumes
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*Design and implement [[#Opening/Closing and Loading/Solubilization Mechanisms | opening/closing mechanisms and loading/solubilization mechanisms]] for our containers that allow us to controllably:
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:#Load various forms of cargo by attaching it to the inside of a container and then closing the container
==Lab Notebooks==
:#Solubilize this cargo without leakage to the exterior of the container
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:#Open our container, releasing our cargo
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<a href="http://openwetware.org/wiki/Biomod/2011/Harvard/HarvarDNAnos:LabNotebook_Nick"><img src="http://openwetware.org/images/7/73/Notebook_nick.jpg" width=150px></a>
 
<a href="http://openwetware.org/wiki/Biomod/2011/Harvard/HarvarDNAnos:LabNotebook_Sherrie"><img src="http://openwetware.org/images/e/ed/Notebook_sherrie.jpg" width=150px></a>
==Designing our Containers==
<a href="http://openwetware.org/wiki/Biomod/2011/Harvard/HarvarDNAnos:LabNotebook_Evan"><img src="http://openwetware.org/images/8/86/Notebook_evan.jpg" width=150px></a>
 
<a href="http://openwetware.org/wiki/Biomod/2011/Harvard/HarvarDNAnos:LabNotebook_Shwinn"><img src="http://openwetware.org/images/d/de/Notebook_shwinn.jpg" width=150px></a>
===Sphere===
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*Using caDNAno, we have [[Biomod/2011/Harvard/HarvarDNAnos:Documents#Spherical Container Design | replicated the sphere]] featured in the [http://www.sciencemag.org/content/332/6027/342.full recent Science paper by Han et al.] so that we can generate the exact same staple sequences published by Han et al.
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*Now we need to implement [[#Opening/Closing and Loading/Solubilization Mechanisms | opening/closing and loading/solubilization mechanisms]] for the sphere
==[[Biomod/2011/Harvard/HarvarDNAnos:Presentations | Presentations]]==
*We also need to cap the holes (approximately 2-3 nm in diameter) at the poles of the sphere, or figure out how to exploit these holes
==[[Biomod/2011/Harvard/HarvarDNAnos:caDNAnoFiles | Repository of caDNAno Files]]==
*We are in the process of creating "SphereCAD," a computer program that synthesizes our caDNAno file and 3D models in order to streamline correlation between theta/phi, bp position, and whether that bp position points into or out of the sphere. This will help in the design process of O/C and L/S mechanisms.
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:[[Image:Sphere_Image.jpeg | 300x300px]]
:3D model of our sphere, created in Autodesk Maya
 
===Box with Lid===
*We have designed a [[Biomod/2011/Harvard/HarvarDNAnos:Documents#Box Container Design | box with double layer walls, lid with perpendicular helices and a double hinge]]
**We have designed one [[Biomod/2011/Harvard/HarvarDNAnos:Documents#2011-06-17 | O/C mechanism for this box]], which utilizes parallel locking handles and an [[#Azobenzene | azobenzene strand]] or azobenzene-sequence-with-toehold lock
::[[Image:Perkons8.jpg | 300x300px]]
::Schematic of Box with Lid with Parallel Locking Mechanism, to be locked by azobenzene-sequence strand
:*This design's strands have been ordered
*We now should implement [[Biomod/2011/Harvard/HarvarDNAnos:LabNotebook Evan#Day 2 (2011-06-07) | compression (see second image)]] so that extra force must be applied in order to tightly seal the box, minimizing fluctuations of the lid
*We should also explore other [[#Opening/Closing and Loading/Solubilization Mechanisms | O/C mechanisms]]
[[Image:2011-06-20_figure1.jpg | 500px]]
*We need to implement a [[#Opening/Closing and Loading/Solubilization Mechanisms | L/S mechanism]]
*We should also consider designing a box that has a lid with parallel helices so that the jagged interface between box and lid (due to crossover positions) will be complementary


==Designing Our Cargo==
===Making chains of nanoparticles using "ultramer"===
[[Image:Nanoparticle Chain.png | 500px]]
*We are currently working on attaching the handles to the ultramer
**We ran a [[Biomod/2011/Harvard/HarvarDNAnos:LabNotebook Evan#Day 12 (2011-06-21)| reaction ladder]] where the first reaction had only the ultramer, the second had the ultramer with the first handle annealed, the second had the ultramer, first handle, and second handle, etc. Here is the [[Biomod/2011/Harvard/HarvarDNAnos:Documents#2011-06-23 | PAGE result]].
*Next, we need to figure out how to attach thiolated DNA strands to gold nanoparticles
===Synthesizing nanoparticles===
*We have made our [[Biomod/2011/Harvard/HarvarDNAnos:LabNotebook Evan#Day 13 (2011-06-22) | own homemade gold nanoparticles]], 5 nm (as per "Preparing Colloidal Gold for Electron Microscopy" by Polysciences, Inc.) and other sizes
*We have used [[Biomod/2011/Harvard/HarvarDNAnos:Documents#Making Gold Nanoparticles | DLS to confirm their size]] and determine their hydrodynamic radius
*Next, we will use TEM to image these nanoparticles, varying salt conditions to observe aggregation
==Providing Functionality to our Designs==
==="One-pot" folding vs. Fold-then-load===
*
*
===Strand Displacement===
[[Image:2011-06-20_figure3.jpg | 500px]]
Disadvantages
*Single strands probably diffuse slowly into sphere
===Photocleavable Spacers===
[[Image:Sphere_pcspacer.jpg | 500px]]
[http://www.idtdna.com/catalog/Modifications/Modifications.aspx?catid=6 From IDT]
===Azobenzene===
[[Image:Liang azobenzene.png | 500px]]
[http://onlinelibrary.wiley.com/doi/10.1002/cbic.200700649/full Liang et al (2008)]
*First order of strands for [[#Box with Lid | Box with Lid]] has lock handles adapted to azobenzene sequence. Two options:
**use actual azobenzene strand to lock, UV to unlock
**use azobenzene-sequence strand with toehold to lock, strand displacement to unlock
*We will first test both mechanisms on a [[Biomod/2011/Harvard/HarvarDNAnos:Documents#2011-06-17_2 | "minimal box"]] which is just a strand of DNA with a central poly T representing the actual box
==Testing the effectiveness of our containers==
===Sphere Design===
# Normal sphere
# Sphere with disulfide on equator and cargo handle staples
# Sphere with strand displacement cargo handle staples, normal strands on equator --> tells us if single strands diffuse into sphere through top and bottom holes
# Sphere with strand displacement equator and cargo handle staples
# Sphere with strand displacement cargo handle staples, disulfide on equator (?)
# Sphere with strand displacement equator staples, disulfide handle staples (?)
# Sphere with restriction enzyme equator staples, disulfide cargo handle staples
# Sphere with azobenzene staples
*NOTE 1: If from (3) we see that single strands do diffuse into the sphere, in the next round of design we can add capping mechanisms to seal off the top and bottom holes of the sphere. (Maybe even create a torus!)
*NOTE 2: To test our program, we eventually need to attach cargo to outside.
===Box Design===
*Bare box without cargo L/S mechanism
**actual azobenzene: F_12X (folds open, lock, then UV release)
**azobenzene sequence with toehold: F_n+ (folds open, lock, then strand displacement release)
**intra-molecular locking (folds locked, then strand displacement release)
*Box with cargo L/S mechanism
===Staple Strands to order for Sphere Design===
#Matching strands on equator
#Matching strands not on equator
#Non-matching strands on equator (from Han's paper)
#Matching on equator (from Han's paper)
#Non-matching strands on equator (from our caDNAno file)
:*Staples with disulfide
:*Staples with strand displacement mechanism
:*Staples with restriction enzyme sequences
:*Staples with azobenzene
#Matching on equator (from our caDNAno file)
===Staple Strand to order for Box Design===
#Core staples to not be manipulated
#Lock staples
#Staples that might be affected when direction of lock staples is changed
#Staples from which cargo-binding mechanism may be attached
==Automation of our processes with CAD Software==
===SphereCADBasic===
*
*
===SphereCAD with standalone GUI===
*
*
==Additional Project Ideas (for another summer!)==
See [[Biomod/2011/Harvard/HarvarDNAnos:Brainstorming#Unexplored Project Ideas (for another summer!) | here]]
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Latest revision as of 21:40, 2 November 2011

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Process

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Lab Notebooks

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Presentations

Repository of caDNAno Files


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