Artificial Enzymes - The Nanobiotechnology - page 60

greatly.simplifying.the.selection.protocol..After.a.number.of.selection.cycles,.selected.ribo-
zymes.are.identiied.and.tested.for.the.desired.catalytic.activity.
The.reactions.catalyzed.by.artiicial.ribozymes.are.many.and.range.from.RNA.processing,.
such. as. RNA. cleavage,. ligation,. branching,. phosphorylation,. and. capping,. to. peptide.
bond. formation,. alcohol. oxidation,. and. the. Diels-Alder. reaction. (Silverman. 2009).. The.
creation.of.artiicial.ribozymes.that.are.able.to.form.a.5′.to.3′.phosphodiester.bond.(Bartel.
and.Szostak.1993;.Ekland.et.al..1995;.Ikawa.et.al..2004;.Jaeger.et.al..1999;.Landweber.and.
Pokrovskaya.1999;.Robertson.and.Ellington.1999;.Rogers.and.Joyce.1999).and.true.RNA-
dependent.RNA-polymerases.that.are.able.to.polymerize.a.complete.turn.of.an.RNA.helix.
(Johnston.et.al..2001;.Lawrence.and.Bartel.2003,.2005),.together.with.the.crystal.structure.
of. an. RNA. ligase. (Robertson. and. Scott. 2007),. provided. proof. that. although. no. known.
natural.ribozyme.can.catalyze.the.polymerization.of.RNA,.RNA.can.indeed.catalyze.a.key.
step.required.for.its.own.replication,.supporting.the.“RNA.world”.theory.(Joyce.2007)..In.
addition,.in.vitro.evolution.was.used.to.create.a.cross-catalytic.system.in.which.two.RNA.
ligase.ribozymes.catalyze.each.other's.synthesis.(Lincoln.and.Joyce.2009).
In. nature,. there. is. no. RNA. enzyme. capable. of. catalyzing. the. aminoacylation. of. the. 3′.
terminus.of.tRNA;.this.job.is.instead.done.by.a.family.of.protein.enzymes.called.aminoacyl-
tRNA.synthetases..In.a.series.of.studies.(Bessho.et.al..2002;.Goto.et.al..2008a,b;.Kawakami.
et.al..2008a,b;.Lee.et.al..2000;.Murakami.et.al..2003,.2006;.Niwa.et.al..2009;.Ohta.et.al..2007;.
Saito.et.al..2001),.Suga.and.coworkers.used.repeated.cycles.of.in.vitro.evolution.experiments.
to.create.artiicial.aminoacyl-tRNA.synthetase-like.ribozymes,.termed.“lexizymes,”.that.
are.able.to.synthesize.a.wide.array.of.acyl-tRNAs.charged.with.artiicial.amino.acids.and.
hydroxy.acids..Flexizymes.not.only.support.the.existence.of.a.primitive.translation.catalytic.
system.consisting.of.RNA.molecules.only.but.also.provide.an.artiicial.platform.to.express.
nonstandard.peptides.containing.both.proteinogenic.and.nonproteinogenic.amino.acids.
for.therapeutic.applications.(Goto.et.al..2011;.Morimoto.et.al..2011).
RNA.molecules,.like.proteins,.adopt.deined.three-dimensional.structures.that.delineate.
binding. sites. and. catalytic. centers.. Guided. by. molecular. modeling,. Ikawa. et. al.. used.
known. structural. motifs,. or. modules. that. formed. the. reaction. site. to. construct. an. RNA.
scaffold.into.which.a.random.region.was.inserted.(Ikawa.et.al..2004)..With.this.approach,.
they.were.able.to.create.an.artiicial.RNA.ligase.that.accelerated.the.reaction.10 6 -fold.over.
the. uncatalyzed. reaction.. The. artiicial. ribozyme. exhibited. a. higher. product. yield. than.
previously.reported.RNA.ligases,.suggesting.that.most.of.the.RNA.machine.was.properly.
folded.in.a.catalytically.active.way.
The. traditional. approach. to. ribozyme. development. is. to. use. an. in. vitro. selection.
method,.in.which.repeated.rounds.of.selection.are.preformed.and.evolution.occurs.in.a.
stepwise. manner.. However,. analogous. to. the. PACE. system. discussed. in. Section. 3.2. for.
protein-directed.evolution,.an.alternative.is.to.use.continuous.evolution,.which.can.occur.
hundreds.of.times.more. quickly. (Wright. and.Joyce. 1997).. Joyce. and.coworkers. were. the.
irst.to.continuously.evolve.catalytic.RNA,.and.they.were.able.to.apply.it.to.two.types.of.
RNA. ligases. (Voytek. and. Joyce. 2007;. Wright. and. Joyce. 1997).. Despite. the. eficiency. and.
power.of.this.method,.it.is.extremely.limited.in.the.type.of.reactions.that.may.be.catalyzed.
since.it.requires.a.ribozyme.with.a.suficiently.fast.reaction.rate.(Voytek.and.Joyce.2007).
In. the. traditional. in. vitro. selection. process,. there. is. no. room. to. select. for. advanced.
enzymatic. properties. such. as. multiple. turnover,. since. selection. is. generally. based. on.
formation.of.a.covalent.linkage.between.the.RNA.and.a.substrate.labeled.with.a.“capture”.
tag. (Silverman. 2009).. One. way. of. getting. around. this. limitation. is. to. use. IVC. strategies.
(Tawik. and. Grifiths. 1998),. in. which. the. sequence. of. the. ribozyme. (genotype). and. its.
catalytic.activity.(phenotype).become.“linked”.within.individual.droplets.in.a.water-in-oil.