Biomedical Engineering Reference
In-Depth Information
techniques.such.as.molecular.beacon-based.SELEX.(Rajendran.and.Ellington.2008),.tailored.
SELEX. (Vater. et. al.. 2003),. photo-SELEX. (Jensen. et. al.. 1995),. and. evolution-mimicking,.
algorithm-based. SELEX. (Ikebukuro. et. al.. 2005). have. been. explored. to. improve. aptamer.
selection.. As. aptamer-based. clinical. research. grew. at. an. increasing. pace,. several. new.
selection.techniques.such.as.cell-SELEX.(Shangguan.et.al..2006).and.tissue-SELEX.(Mi.et.al..
2010). were. developed.. Aptamers. from. cell/tissue-SELEX. are. particularly. promising. for.
diagnostic.or.therapeutic.applications.since.they.can.directly.recognize.cell/tissue.surface.
markers..However,.it.is.important.to.note.that.the.iterative.process.of.aptamer.selection.is.
complicated.and.time.consuming..To.overcome.this.problem,.automated.SELEX.(Cox.and.
Ellington.2001,.Hybarger.et.al..2006).and.microarray-based.SELEX.(Knight.et.al..2009).have.
been.studied..It.is.believed.that.the.optimization.of.these.SELEX.methods.will.speed.up.
the.development.of.aptamer-based.research.
2.2.2 Aptamer Truncation
The. full-length. nucleic. acid. aptamers. selected. through. a. SELEX. process. typically. have.
80-100.nucleotides..However,.only.some.of.those.nucleotides.in.a.full-length.aptamer.are.
critical. in. binding. to. the. target. molecule.. A. full-length. aptamer. is. usually. composed. of.
three.functional.regions..The.most.critical.region.that.directly.contacts.the.target.is.usually.
10-15. nucleotides. long. (Gold. et. al.. 1995).. Upon. contact. with. the. target. molecule,. these.
nucleotides. will. fold. into. secondary. structures. such. as. stems,. bulges,. hairpins,. loops,.
or.pseudoknots..Another.region.that.also.plays.an.important.role.in.enhancing.aptamer.
binding. afinity. contains. several. nucleotides. to. support. the. interactions. between. the.
aptamer. and. its. target.. The. nucleotides. in. these. two. regions. that. either. directly. contact.
the. target. or. facilitate. the. binding. are. regarded. as. essential. nucleotides.. In. addition. to.
the.essential.nucleotides,.there.are.several.nucleotides.that.neither.bind.to.the.target.nor.
support. the. binding.. These. nucleotides. are. nonessential,. forming. a. nonessential. region..
After. aptamer. selection,. it. is. desirable. to. perform. aptamer. truncation. to. remove. these.
nonessential.nucleotides.for.several.reasons.
First.of.all,.standard.DNA.or.RNA.synthesis.is.eficient.with.a.reasonable.cost.when.an.
oligonucleotide.is.shorter.than.60.nucleotides..However,.a.full-length.aptamer.selected.from.
the.library.usually.has.80-100.nucleotides.(Yang.et.al..2007)..In.addition,.the.nonessential.
nucleotides. may. negatively. affect. the. aptamer. binding. afinity. due. to. steric. hindrance.
or. intramolecular. interactions.. Furthermore,. in. comparison. to. a. full-length. aptamer,.
a. truncated. aptamer. will. have. less. chance. to. induce. immunoreaction. (Krieg. et. al.. 1995,.
Hornung. et. al.. 2005).. Truncated. aptamers. can. provide. more. lexibility. for. constructing.
aptamer-based.nanostructures.since.they.are.smaller.than.full-length.aptamers.
Currently,.there.are.two.major.methods.to.determine.the.essential.sequences.of.a.full-
length. aptamer.. One. is. experimental. determination. by. massive. synthesis. and. analysis.
of. truncated. sequences. from. the. full-length. aptamer.. The. truncated. sequences. can. be.
generated.through.RNA.transcription.(Lupold.et.al..2002),.enzymatic.ingerprinting.(Dey.
et. al.. 2005),. solid-phase. synthesis. (Marro. et. al.. 2005),. and. so. on.. So. far,. this. truncation.
method. is. the. most. widely. used.. However,. it. is. often. complicated. and. time. consuming.
(Fischer. et. al.. 2008).. In. addition,. for. 2′-luoro-modiied. RNA. aptamers,. the. chemical.
synthesis. of. truncated. aptamers. is. very. costly.. The. other. type. of. aptamer. truncation.
is. based. on. structural. simulation.. After. selection,. secondary. structures. of. the. aptamer.
will.be.theoretically.predicted.by.computer.programs.such.as.mfold.and.RNA.structure.
(Mannironi. et. al.. 1997,. Meli. et. al.. 2002,. Zuker. 2003,. Schuster. 2006,. Jossinet. et. al.. 2007,.
Shangguan. et. al.. 2007).. The. essential. sequence. of. a. full-length. aptamer. will. then. be.
