Biomedical Engineering Reference
In-Depth Information
entire.genome.sequenced.(Fiers.et.al..1976)..Early.attempts.at.protein.engineering.of.MS2.
were.focused.on.genetic.manipulation.and.peptide.insertion.into.the.capsid.protein.(Mastico.
et.al..1993)..One.advantage.of.using.MS2.as.a.nanomaterial.is.that.large.quantities.can.be.
obtained.from.infected.cultures.or.through.expression.in.
E. coli
.(tens.of.mg/L.culture).
The.MS2.phage.is.a.Biosafety.Level.1.pathogen.that.only.infects.
E. coli
.bacteria..It.does.
so.by.initially.binding.to.the.F.pili.(F-bacteria.cannot.be.infected),.after.which.it.injects.its.
genome.into.the.cell..The.assembled.capsid.exhibits.T.=.3.quasi-symmetry.and.is.28.nm.in.
diameter..The.mature.capsid.contains.a.single-strand.of.(+).sense.RNA,.3569.nucleotides.
in.length,.180.copies.of.a.monomeric.capsid.protein.(∼13.kDa),.and.one.copy.of.a.maturase.
protein.. Fully. assembled. with. its. RNA,. the. capsid. has. a. central. void. with. a. diameter. of.
∼7.nm. (Toropova. et. al.. 2008).. It. has. been. shown. that. the. RNA. can. be. hydrolyzed. and.
removed.without.disrupting.the.capsid.structure.(Hooker.et.al..2004)..Removing.the.RNA.
from. the. capsid. increases. the. interior. void. diameter. to.∼12.nm. (Koning. et. al.. 2003).. The.
assembled.capsid.has.a.series.of.32.pores,.∼1.4.nm.wide,.centered.on.both.the.threefold.and.
ivefold.symmetry.axes,.enabling.the.transport.of.small.molecules.in.and.out.of.the.capsid.
structure.(Valegard.et.al..1990).
Self-assembly.of.MS2.requires.the.binding.of.the.capsid.protein.to.a.19.nucleotide.RNA.
hairpin.loop.known.as.the.operator.sequence.(TR)..The.capsid.protein.exists.in.three.con-
formations.(A,.B,.C);.the.difference.between.them.is.in.the.FG.loop.that.connects.the.F.and.
G. β-strands.. The. monomers. associate. into. two. types. of. dimers. (AB. and. CC).. In. solution.
without. RNA. present,. the. protein. dimers. exist. in. a. symmetric. CC. conformation,. where.
introduction.of.viral.RNA.induces.an.allosteric.switch.to.an.asymmetric.AB.conformation..
It.has.been.suggested.that.capsid.assembly.begins.with.an.AB.dimer.binding.to.the.operator.
sequence,.an.interaction.that.inhibits.further.AB.dimer.association.with.RNA,.and.favoring.
additions.of.AB.dimers.already.associated.with.CC.dimers.(Elsawy.et.al..2010)..This.model.
greatly.reduces.the.complexity.for.the.eficient.self-assembly.that.is.observed.in.MS2.
7.4.2 Drug Delivery
Viruses.are.by.their.very.nature,.ideal.systems.for.the.transport.of.molecular.payloads..In.
order.to.replicate,.they.must.deliver.their.genome.across.the.host.membrane.to.the.interior.
of.the.cell..The.highly.ordered.and.repeatable.capsid.structure.has.allowed. scientists.to.
attach. over. 100. ligands. with. a. single. modiication. step.. Multiple. research. groups. have.
envisioned.a.twofold.approach,.whereby.the.interior.of.the.capsid.is.modiied.to.enable.
the.attachment.of.a.therapeutic.payload,.and.the.exterior.is.decorated.with.molecules.for.
targeting.purposes.
Early. engineering. efforts. with. the. MS2. bacteriophage. utilized. chemical. conjugation.
to. link. drug. molecules. to. the. RNA. operator. hairpin. structure. for. loading. of. molecules.
inside. the. capsid.. In. 1995,. Wu. et. al.. reported. conjugating. a. thiol-terminated. operator.
RNA. sequence. to. the. Ricin. A. Chain. protein. (RAC).. When. placed. into. solution. with.
capsid.monomers,.multiple.RNA-RAC.molecules.were.packaged.into.each.capsid..Native.
lysine. residues. on. the. exterior. of. RAC-loaded. capsids. were. subsequently. conjugated. to.
transferrin,.a.targeting.ligand.associated.with.receptor-mediated.endocytosis.(RME).(Wu.
et.al..1995)..Another.study.utilized.chemically.synthesized.operator.sequences.with.5′.or.3′.
extensions.containing.the.nucleotide.analog.5-luorouridine.(5fU,.a.common.therapeutic.
molecule).for.loading.into.MS2.capsids.(Brown.et.al..2002).
Alternative. chemical. approaches. for. loading. materials. on. the. interior. of. the. capsid.
without.the.use.of.the.operator.sequence.have.also.been.developed..For.instance,.native.
tyrosine. residues. (Tyr85). on. the. MS2. capsid. interior. can. be. targeted. for. modiication.
