Biomedical Engineering Reference
In-Depth Information
of.amino.acids.onto.each.of.a.set.of.a.few.hundred.scaffold.protein.structures.taken.from.
the.Protein.Data.Bank..This.matching.step.is.extremely.computationally.intensive.and.is.
made.possible.by.a.distributed.network.of.volunteers.who.provide.access.to.their.personal.
computers.through.a.project.called.Rosetta@home.(Das.et.al..2007)..The.set.of.theozyme/
scaffold.matches.is.narrowed.down.through.further.computational.and.intuitive.iltering..
Finally,.around.100.candidates.are.synthesized.and.tested.for.activity.
The.irst.enzymes.designed.using.this.method.were.retro-aldolases.that.break.a.bond.in.
a.substrate.not.found.in.nature.(Jiang.et.al..2008).and.an.enzyme.that.performs.the.Kemp.
elimination,.a.reaction.for.which.no.natural.enzyme.is.known.(Rothlisberger.et.al..2008)..
Both.of.these.reactions.break.bonds,.and.therefore.bind.only.one.substrate.molecule..An.
enzyme.that.catalyzes.the.Diels-Alder.reaction,.which.combines.two.substrate.molecules.
into.one.product,.followed.shortly.(Siegel.et.al..2010)..The.Rosetta.enzyme.design.procedure.
has.been.described.in.detail.and.is.available.for.use.in.any.laboratory.(Richter.et.al..2011).
Eventually,.it.is.hoped.enzyme.design.will.incorporate.artiicial.active.sites.into.de.novo.
scaffolds.designed.to.be.optimal.for.the.active.site.chemistry.and.the.application.environ-
ment..In.an.early.example,.a.binary-patterned.helix.served.as.a.designed.scaffold.for.an.
artiicial.enzyme..The.32-residue.peptide.catalyzed.its.own.replication.by.the.ligation.of.
two. 16-residue. peptides. corresponding. to. its. own. N-. and. C-terminal. halves. (Lee. et. al..
1996;.Saghatelian.et.al..2001)..The.peptide.was.designed.to.fold.into.a.helix.presenting.a.
binding.face..When.bound.to.the.catalyst,.the.chemically.activated.termini.of.the.substrate.
peptides. were. perfectly. oriented. for. ligation,. reducing. the. entropic. cost. of. the. reaction..
Functional.group.preorganization,.in.which.the.folding.energy.of.the.protein.scaffold.bal-
ances.the.cost.of.ixing.the.substrate.and.key.active.site.residues.in.orientations.optimal.
for.the.desired.reaction,.is.one.of.the.most.important.catalytic.strategies.used.by.natural.
enzymes,.and.is.promising.target.for.mimicry.by.artiicial.enzymes.
3.8 Beyond Proteins—Artificial Ribozymes
So.far,.we.have.reviewed.the.major.class.of.artiicial.enzymes:.those.composed.of.protein..
However,.another.important.class.of.biological.enzymes.is.not.protein-based.but.is.instead.
based. on. nucleic. acids.. These. are. termed. ribozymes. or. deoxyribozymes,. depending. on.
whether.they.are.composed.of.RNA.or.DNA..Here,.we.will.focus.only.on.artiicial.ribozymes.
In.the.early.1980s,.Cech.and.Altman.independently.discovered.that.RNA.molecules.not.
only.carried.genetic.information.but.also.had.catalytic.properties.(Guerrier-Takada.et.al..
1983;. Kruger. et. al.. 1982).. This. breakthrough. discovery. was. recognized. with. the. Nobel.
Prize.in.chemistry.in.1989..Since.then,.there.have.been.increasing.efforts.to.create.artiicial.
ribozymes.with.novel.catalytic.properties.
The. irst. artiicial. ribozyme. was. published. in. 1990. (Robertson. and. Joyce. 1990). and.
consisted.of.an.RNA.molecule.evolved.and.selected.in.vitro.to.speciically.cleave.single-
stranded. DNA. (as. opposed. to. the. parental. ribozyme. that. cleaved. RNA. substrates).. The.
general.methodology.to.create.artiicial.ribozymes.(Ellington.and.Szostak.1990;.Tuerk.and.
Gold. 1990). consists. of. synthesis. of. a. DNA. molecule. with. constant. and. random. regions,.
ampliication.by.PCR,.and.in.vitro.transcription.to.produce.the.initial.random.RNA.pool.
that.will.be.used.for.the.irst.round.of.selection.during.the.artiicial.evolution.experiment..
Unlike. proteins,. nucleic. acid-based. enzymes. can. be. directly. ampliied. and. sequenced,.
