Biomedical Engineering Reference
In-Depth Information
SP1 Protein
Gold Nanoparticle
dsDNA
iol Group
FIGURE 1.4
Schematic. of. a. 1D. nanostructure. of. a. 6His-SP1-GNP-DNA. tri-block. conjugate.. The. basic. components. are. the.
6His-SP1.protein,.GNPs,.dsDNA.molecules,.and.thiol.groups.placed.at.different.places.on.the.DNA.structure..
Through.thiol.gold.connection.the.system.can.create.complex.structures.
complementary.ssDNA.determines.the.position.of.the.thiol.groups.along.the.dsDNA.tem-
plate..These.positions.can.be.varied.and.controlled.by.the.speciic.choice.of.the.sequence.
length.and.overlap.
The.ssDNA.sequences.used.to.form.the.dsDNA.wires.in.this.experiment.were.80-mers.
long.. The. various. sequences. had. an. overlap. with. partially. complementary. sequences.
with. thiols. at. the. 5'. ends.
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. The. sequences. were. designed. to. form. long. DNA. templates.
with.thiol.groups.repeated.periodically.every.22,.54,.and.80.bp.and.their.multiplications.
(Figure.1.4).
The.dsDNA.templates,.at.equimolar.concentration.(100.nM).of.sense.and.partially.com-
plementary. antisense. oligomers,. were. mixed. and. annealed.. The. mixture. was. heated. to.
95°C.for.4.minutes,.and.then.incubated.at.70°C.for.10.minutes..Finally,.the.temperature.was.
slowly.decreased.to.4°C.(0.5°C/minute),.and.kept.at.4°C.for.10.minutes..To.ensure.anneal-
ing,.the.samples.were.analyzed.by.agarose.gel.electrophoresis..Only.after.annealing.could.
the.DNA.band.be.seen.in.ethidium.bromide-stained.gel,.conirming.the.formation.of.the.
laddered.dsDNA.wires..Binding.the.hybrids.to.the.template.dsDNA.nanowires.was.done.
by.mixing.the.6His-SP1-GNP.solution.with.the.dsDNA.solution.and.incubating.at.37°C.for.
3.hours.at.different.concentrations.
Figure 1.5A. shows. an. AFM. topography. image. of. the. formed. 1D. nanostructures.. The.
6His-SP1-GNP. hybrids. are. anchored. to. the. template. DNA. through. the. thiol. groups. in.
predeined.positions..Repeating.the.same.procedure.of.incubation.with.nonthiolated.DNA.
did.not.lead.to.formation.of.the.tri-block.conjugates..Figure 1.5B.and.C.show.wires.and.
junctions.based.on.the.tri-block.conjugates..These.junctions.are.composed.of.several.DNA.
segments. connected. together. by. the. 6His-SP1-GNP. hybrid,. seen. as. a. bright. spot. in. the.
topographic.image.
To. verify. the. existence. of. the. GNP. in. the. 6His-SP1. pore,. EFM. was. performed. on. the.
tri-block. conjugate.. Figure 1.5D. shows. an. EFM. image. of. the. nanostructure. shown. in.
Figure 1.5B.. One. can. see. a. polarization. signal. at. the. positions. of. 6His-SP1-GNP.. This.
proves.that.a.GNP.is.attached.to.the.central.cavity.of.the.protein,.which.is.likely.bound.
to.the.DNA.through.the.thiol.group.serving.as.the.connecting.unit.between.two.DNA.
strands.
