Biomedical Engineering Reference
In-Depth Information
There. are. several. good. recent. reviews. on. DNA. origami. (Endo. and. Sugiyama. 2009,.
Kuzuya. and. Komiyama. 2010,. Lo. et. al.. 2010,. Nangreave. et. al.. 2010,. Shih. and. Lin. 2010,.
Tørring.et.al..2011),.so.this.section.will.focus.on.practical.details.for.researchers.who.do.
not. have. much. experience. with. DNA. nanostructures.. The. most. important. detail. is. that.
DNA. origami. are. quite. accessible. for. the. neophyte.. While. the. design. of. new. origami.
is. challenging,. reproducing. or. modifying. a. published. origami. is. a. reasonable. starting.
point..Woo.and.Rothemund.(2011).contains.sequences.for.a.simple.60.×.90.nm.rectangular.
origami.(irst.published.in.2006).that.has.been.redesigned.to.remove.torsional.strain..The.
staple.sequences.needed.to.fold.this.origami.design.(or.any.other.published.design).can.be.
purchased.from.a.variety.of.specialized.DNA.synthesis.companies..The.staple.strands.are.
stable.for.years.if.stored.properly,.and.they.can.easily.be.modiied.at.the.time.of.synthesis.
to. incorporate. luorescent. dyes. or. reactive. functional. groups. such. as. azides,. primary.
amines,.or.thiols..The.origami.themselves.assemble.in.good.yield.and.are.easy.to.image.
by.AFM..Typical.assembly.conditions.involve.mixing.2.nM.template.with.20-50.eq.of.each.
staple. strand. followed. by. a. 2. h-long. annealing. step. from. 95°C. to. room. temperature.. If.
desired,.the.origami.can.be.separated.from.the.large.excess.of.staple.strands.by.centrifugal.
iltration.using.a.Micro-con.centrifugal.ilter.device.(MWCO.50,000;.Ke.et.al..2008)..Agarose.
gel.separation.is.sometimes.effective.(Bellot.et.al..2011)..However,.good.images.of.origami.
can. be. obtained. even. from. the. crude. reaction. mixture.. Mica. is. the. standard. substrate.
because.of.its.ease.of.preparation..We.have.used.both.prepared.mica.AFM.substrates.(e.g.,.
from.Ted.Pella.Co.).and.sheets.of.mica.sold.as.windows.for.wood.stoves..The.mica.is.ixed.
to.the.AFM.sample.stub.with.double-sided.sticky.tape,.and.another.piece.of.tape.is.used.to.
peel.off.the.top.layers.of.the.mica,.revealing.an.atomically.lat.and.clean.surface.ready.for.
use..Place.5.μL.of.the.crude.DNA.origami.solution.(inal.concentration.of.origami.around.
1-5.nM).on.the.mica,.add.20.μL.of.the.annealing.buffer,.let.it.stand.for.20.min,.rinse.with.
several.drops.of.buffer,.blow.dry.with.iltered.nitrogen,.and.image.in.air.
Most. DNA. origami. are. annealed. thermally.. However,. there. are. also. several. chemical.
approaches.for.annealing.DNA.origami.that.might.be.useful,.for.example,.if.the.precursor.
oligonucleotides. contain. thermally. unstable. modiications.. Formamide. is. the. main. tool;.
this. solvent. lowers. the. melting. point. of. duplex. DNA. by. about. 0.6°C. per. %. formamide.
present.. Both. a. standard. M13. mp18. rectangle. and. a. 3D. six-helix. bundle. protein. were.
assembled. by. mixing. the. viral. template. strand. with. excess. staple. strands. in. a. buffer.
containing. 85%. formamide. and. dialyzing. the. formamide. out. over. a. period. of. 1-24.h..
Rectangular.origami.assembled.in.satisfactory.yields.and.folding.quality.within.1.h,.but.
the.3D.structures.required.>8.h.in.the.denaturant.annealing.process.to.assemble.as.well.
as.they.did.in.a.2.h.thermal.annealing.process..Urea.has.also.been.used.as.the.chemical.
denaturing.agent.(Jungmann.et.al..2008).
Although. DNA. is. not. particularly. useful. as. an. electronic. material. (but. see. Deng.
and. Mao. 2004),. the. DNA. origami. can. be. used. very. effectively. to. pattern. non-DNA.
components.(Seeman.and.Belcher.2002,.Xiao.et.al..2002,.Yan.et.al..2003b,.Lin.et.al..2007,.
Bui. et. al.. 2010).. Origami. can. be. derivatized. with. 2-6.nm. resolution. in. programmed.
locations..Probably,.the.easiest.derivatization.method.is.to.purchase.staple.strands.that.
are.labeled.with.biotin..Biotin.reacts.readily.with.streptavidin.to.produce.a.bump.that.
is. apparent. to. AFM. (although. groups. of. 3-5. bumps. are. more. reliably. imaged).. The.
streptavidin.can.itself.be.targeted.by.other.biotinylated.species.for.further.derivatization..
A.range.of.conjugation.chemistry.that.can.be.carried.out.on.assembled.DNA.origami.
was. explored. by. Gothelf's. group. and. is. summarized. in. Figure. 1.4. (Voigt. et. al.. 2010)..
This. study. was. important. both. because. it. demonstrates. use. of. several. orthogonal.
derivatization. methods. and. because. it. shows. that. DNA. origami. that. are. supported.
