Biomedical Engineering Reference
In-Depth Information
Attached
(a)
(b)
(c)
Figure 4.5
Strategies. for. cargo. attachment. to. microtubules.. (a). Attachment. of. biotinylated. cargo. to. microtubules. via.
streptavidin. coupling.. (b). Attachment. of. cargo. to. microtubules. via. an. antibody. linkage.. Antibodies. are.
ixed.to.the.microtubule..Target.antigens.present.on.the.cargo.bind.to.these.antibodies.with.high.speciicity..
(c). Use. of. DNA. hybridization. between. a. microtubule-bound. ssDNA. oligonucleotide. and. a. cargo. element.
that.is.functionalized.with.the.complementary.ssDNA.oligonucleotide.strand..By.incorporating.a.geograph-
ically.pertinent.temperature.trigger.into.the.device,.the.dsDNA.molecules.can.be.dissociated.and.the.cargo.
unloaded.
streptavidin..Antibodies.bond.with.high.speciicity.via.noncovalent.hydrogen.bonds.and.
Van der Waal forces
..Their.general.universal.availability,.speciically.if.protein.fusions.are.
used,.means.that.they.are.readily.accessible.as.tools..They.are.typically.used.in.vitro.to.pull.
molecules.out.of.solution.or.to.identify.targets..A.strategy.of.a.“double.antibody.sandwich”.
of.microtubule-antibody-target-antibody-luorophore.has.been.used.(Ramachandran.et.al.,.
2006)..In.this.method,.one.antibody.is.coupled.to.the.microtubule.and.used.to.capture.a.
target. molecule.. A. second. antibody,. which. recognizes. the. target. and. binds. at. a. distinct.
location,.contains.a.luorophore.for.detection..Multiple.antibodies.can.be.used.in.designs.
along.with.multiple.luorophores.to.be.able.to.speciically.capture.and.detect.additional.
targets..Fluorescent.strategies.such.as.luorescence.resonance.energy.transfer.(FRET),.also.
can. be. applied. to. detect. interactions. within. 10.nm. range. (Selvin,. 2000).. One. issue. with.
biotin/streptavidin.binding.is.that.streptavidin.can.bind.to.multiple.biotin.molecules..This.
could.be.viewed.as.beneicial.or.result.in.extra,.unwanted.bonds.such.as.the.binding.of.
two.microtubules.or.two.target.molecules.together..A.solution.to.this.may.be.to.directly.
functionalize.the.microtubules.with.antibodies.by.way.of.covalent.cross-linking..However,.
this.too.requires.a.means.to.dissociate.antibody.and.antigen.to.allow.unloading.
Loading. and. transport. in. a. cell. can. occur. simultaneously,. along. with. building. of. the.
structure.itself,.if.not.regulated..In.nature,.integral.elements.of.afinity.are.used.to.retain.
motor. proteins. in. proximate. vicinity. to. the. microtubule. even. when. it. is. not. correctly.
oriented. for. motility.. Afinity. elements. include. the. highly. charged. C-terminal. tails. of.
α-/β-tubulins. of. microtubules. and. secondary. non-ATP. dependent. MTBDs. such. as. in.
kinesin-14.Ncd.(Wendt.et.al.,.2003)..Brownian.motion.will.also.impact.orientation.of.the.
cargo. near. the. shuttle.. Whether. docking. stations. are. needed. to. preorient. cargo. to. be.
picked.up.will.depend,.in.part,.on.the.application,.such.as.the.use.of.ixed.docking.sites.
in.the.ribosome.for.translating.mRNA.into.a.polypeptide..Cargo.unloading.depends.on.
the. strategy. used. in. attachment.. Strategies. used. in. unloading. may. include. the. use. of.
chemicals. or. ions,. cleaving. enzymes,. changes. in. pH. or. temperature,. or. use. of. incident.
UV-light.to.sever.photosensitive.bonds..This.latter.approach.can.be.done.using.relatively.
small.target.spot.sizes.of.1.μm
2
,.and.would.allow.dropping.of.the.cargo.at.a.desired.loca-
tion.(Hess.and.Vogel,.2001).
