Biomod/2011/Harvard/HarvarDNAnos:LabNotebook Adam

"Directives" from Adam
PROJECT PLANNING (please wikify) At this point we can (major accomplishments so far): -fold closed sphere with some reliability -fold open sphere with some reliability -do a "mini-box" tweezer experiment where we open a system with strand displacement So at this point we need to: -get to the point where we can fold & purify closed or open spheres with near 100% reliability --a sub task for this week is gel extraction of sphere bands followed by more AFM -test 3 ways to make transitions between those states: azobenzene, strand displacement, photocleavable spacer --test these both on the sphere and on "mini-box" --observe transitions by BOTH gel and AFM for the sphere ---try to quantitate our ability to make transitions between closed and open -on minibox we need to try to go through multiple close/open cycles, too -load the sphere with cargo --finish AuNP-DNA conjugation protocol and run tests on AuNP-DNA-DNA hybridization

-(optional) get Evan's box to fold or design another one


 * Design:
 * finish minimal designs for sphere box and lattice box and debug with Adam: order these (today/tonight/over weekend)
 * come up with opening and loading and capping mechanisms and sequences for these, including minimal tests (without any origami)
 * design origami inside origami system -- this will require more brainstorming
 * make sphereCAD awesome -- ideally with some minimal GUI
 * learn Illustrator and Maya and make much better diagrams of what we're doing
 * do literature searches on the chemistry we'll be using and make diagrams
 * Experimental:
 * 0) TEM imaging of nanoparticles - introduction to TEM
 * deposit nanoparticles on TEM grid, image
 * do this with different salt conditions to observe aggregation of nanoparticles
 * we'll also get some 3D origami samples from Wei Sun or someone in William's lab and observe those by TEM, to get a feel for the machine
 * 1) Dimerization of thiolated strands by disulphide bond formation, cleavage by DTT, effects of pH, reversibility of the reaction
 * We'll analyze this system by PAGE gel electrophoresis
 * We have two types of thiolated strands:
 * a) short, with 5' thiol
 * b) long, with 5' thiol
 * Things to look at:
 * Oxidative formation of disulphide bonds: effect of pH (should see a transition around pKa of SH group which is around 8.5?)
 * Effect of lowering the pH after bond formation:
 * Cleavage by DTT:
 * 1.5) We'll also order a 5'-thiol strand, a 3' thiol strand and a bridging oligo and attempt to selectively form HETERO dimers (i.e., full length "staples" with disulphide inserts)
 * 1.75) Can we do this in a 1-pot, "multiplexed fashion", where we just add some bridging oligos to our staple pool, spike the pH up to 7.5 or 8.0, wait six hours, and then fold the origami as usual? We can test multiplexed formation of disulphide bonds using oligo-bridges by having a pool with strands of different lengths and/or fluorescent tags, which we can distinguish on a gel.
 * 2) AuNP-oligo conjugation
 * See the protocol attached to my earlier email.
 * We'll do both the "one to several" strands per nanoparticle protocol, and the "many" strands per nanoparticle protocol. This latter could help for dis-aggregation of the nanoparticles in high magnesium buffer if we want to do 1-pot folding. We'll test this hypothesis by repeating experiment 0
 * We also have both long and short thiolated strands: the long strands should allow separation of 1-strand/AuNp, 2-strand/AuNp etc. whereas the short strands do not allow such easy gel separtion
 * Perhaps we'll split into teams of two to run these two slightly different experiments in parallel. Unfortunately the first few days of this protocol are mostly waiting for things to incubate...
 * 3) Nanoparticle linear chain: the basic idea is to make a chain of nanoparticles first a) hybridizing oligos to a long (189-base) ssDNA "ultramer" such that each hybridized oligo displays a handle sequence, and then b) hybridizing on a nanoparticle-linked strand with a domain complementary to the exposed handles... note how this is quite similar to a situation where we fold an origami with exposed handles and then load a AuNP-strand as cargo, which is what we want to do for loading our containers
 * Questions we can ask:
 * does with work with mono-conjugated or multi-conjugated nanoparticles?
 * how many nanoparticles can we "load" onto the ultramer and how close can we pack them?
 * can we strand-displace them off using competing strands?
 * can we see the chains by TEM?
 * can we do it as a 1-pot folding? if not, what is the protocol for 2-pot folding?
 * Specifications for things we already have:
 * Thiolated strand sequence = (5prime_thiol)-GAACTGGAGTAGCACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT = 55-mer
 * Handle sequence = GTGCTACTCCAGTTC
 * Ultramer "origami" = AATGCTACTACTATTAGTAGAATTGATGCCACCTTTTCAGCTCGCGCCCCAAATGAAAATATAGCTAAACAGGTTATTGACCATTTGCGAAATGTATCTAATGGTCAAACTAAATCTACTCGTTCGCAGAATTGGGAATCAACTGTTACATGGAATGAAACTTCCAGACACCGTACTTTAGTTGCATAT
 * We need 6T spacer at least in order to put nanoparticle near the strand.
 * Raw staples from caDNAno
 * 4) DLS characterization of nanoparticles and origami
 * http://en.wikipedia.org/wiki/Dynamic_light_scattering
 * We'll attempt to use this to measure the hydrodynamic radius of our nanoparticle cargo:
 * with no attached strands
 * with 1, 2, .... N attached strands
 * with "many" attached strands
 * reference on DLS of nanoparticles: http://www.sciencedirect.com.ezp-prod1.hul.harvard.edu/science/article/pii/S1386142507006543 see fig. 4 therein. also, we'll do this (all of it) as a function of magnesium concentration, to see exactly when they aggregate - and whether you can put them in the origami folding buffer which has 10 mM magnesium. we'll do a UV-vis spectrum to measure the nanoparticle concentration, and a DLS measurement to get their size distribution and we'll also look at the nanoparticles by TEM... good to get to know your cargo...
 * we'll also try to measure the radius of the origami this way: we'll see if it comes out right for the sphere, or if the spherical origami diffuses differently than would be predicted by naive calculations
 * note: DLS basically measures particle diffusion. how long does it take a 40 nm diameter particle to diffuse a RMS distance of 1 um in water at room temp? how about to diffuse 1 cm? hint: use the stokes-Einstein equation and the attached image, which relates the diffusion constant to the RMS displacement and the time
 * 5) initial origami foldings
 * Evan/Tom box
 * json file
 * staple excel file
 * order excel file
 * staple pools: set of staples, # of staples, concentration of each staple
 * folding mixtures
 * Sphere (using Sherrie's staples, which differ from Han's)
 * json files
 * mismatches rtf
 * Nick's Excel file
 * link to sphereCAD tools
 * link to tutorial for designing on the sphere
 * staple excel file
 * staple pools:
 * folding mixtures
 * 6) latch characterizations
 * 7) latched boxes
 * 8) cargo loading
 * designs
 * nanoparticle loading
 * origami inside origami