Biomedical Engineering Reference
In-Depth Information
1.3.2 Long Oligos: Sources and Handling
The. template. strands. for. DNA. origami. are. generally. too. long. (>6000.bp). to. be. prepared.
synthetically.at.reasonable.cost..Template.strands.can.be.cloned.into.an.organism.such.as.
Escherichia coli
.for.replication,.and.it.is.in.principle.possible.to.prepare.large.amounts.of.
template.strand.this.way..However,.due.to.the.expense.of.the.initial.assembly.and.the.tiny.
amounts.of.DNA.needed,.commercially.available.genetic.material.from.various.organisms.
is. normally. used.. Different. strains. of. organisms. have. slightly. different. sequences,. so. if.
a. literature. procedure. is. being. followed,. it. is. important. to. use. the. same. source. for. the.
template. strand.. The. same. handling. precautions. that. apply. to. short. oligos. should. be.
followed.carefully.for.template.strands.
The.M13.mp18.viral.genome.was.the.irst.long.template.used.(Rothemund.2006).and.has.
served. as. raw. material. for. dozens. of. DNA. origami. designs.. M13. mp18. is. a. ilamentous.
virus. with. an. ss. circular. DNA. genome. 7.5.kbp. (1.kbp.=.1000. bp). in. length.. The. DNA. is.
replicated.in.host.cells.via.a.circular.duplex.intermediate.and.depending.on.how.carefully.
the.M13.mp18.is.puriied,.some.duplex.DNA.may.be.present.in.commercial.M13.mp18,.in.
addition.to.the.desired.ss.template..Due.to.the.high.afinity.of.the.complementary.strand.for.
the.template.strand,.the.duplex.does.not.anneal.with.staple.strands..It.looks.like.a.loop.of.
string.with.measured.height.of.1.5-2.nm.dropped.among.the.DNA.origami..Other.ss.DNA.
templates.have.been.used.successfully..Shih.et.al..used.some.duplex.template.sequences.
like.the.4.7.kbp.plasmid.pEGFP-N1.and.a.1.3.kbp.segment.of.DNA.to.demonstrate.folding.
of. 3D. objects.. It. was. necessary. to. denature. the. duplex. template. strand. completely,. cool.
quickly,.and.use.chemical.denaturants.to.control.folding.(Jungmann.et.al..2008,.Hogberg.
et. al.. 2009).. Pound. et. al.. (2009). used. polymerase. chain. reaction. (PCR). to. amplify. four.
native.template.strands.ranging.from.756.to.4808.bp.in.length.for.use.in.DNA.origami;.this.
method.is.clearly.capable.of.generalization.to.other.desired.templates.
1.3.3 Double-Stranded DNA
Duplex.DNA.assembles.and.achieves.its.3D.structure.as.a.result.of.many.low-energy.local.
interactions.. Both. hydrogen. bonding. and. hydrophobic. interactions. contribute. to. duplex.
formation,.with.G-C.base.pairs.contributing.three.H-bonds.each.and.A-T.base.pairs.two.
H-bonds..The.length,.number.of.mismatched.nucleotides,.and.G-C.content.of.a.sequence.
determine. its. melting. point,. which. can. be. experimentally. measured. by. taking. UV-Vis.
melting.curves;.this.experiment.is.a.good.idea.when.synthetic.modiications.are.made.to.a.
nucleotide..For.simple.duplex.DNA,.expected.melting.points.(Figure.1.2).can.be.calculated.
with. good. accuracy. given. the. sequence,. concentration. of. oligos,. and. concentrations.
of. monovalent. and. divalent. cations. (http://www.idtdna.com/analyzer/Applications/
OligoAnalyzer/)..These.melting.points.are.useful.guidelines.for.design.of.duplex.portions.
of. DNA. nanostructures,. such. as. oligos. for. particle. conjugation.. Less. well. understood.
is. how. the. stability. of. any. duplex. DNA. structure. is. affected. by. bending. or. torsional.
distortions,.which.are.often.present.in.DNA.tiles.or.origami.designs.(both.intentionally.
and.unintentionally;.Kim.and.Bathe.2011,.Woo.and.Rothemund.2011).
Interactions. between. single-. and. double-stranded. DNA. are. of. great. importance.
for. the. assembly. and. reconiguration. of. DNA. nanostructures. (Zhang. and. Seelig. 2011)..
Cascades.of.strand-displacement.reactions.have.even.been.used.for.computational.work.
(Qian.et.al..2011)..The.key.is.to.balance.binding.energy.and.kinetics.(Zhang.and.Winfree.
2009).. Thermodynamically,. one. oligonucleotide. that. is. complementary. to,. say,. 20.bp. of.
a. target. can. displace. another. oligonucleotide. that. is. complementary. to,. say,. 15.bp. of. the.
