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1、Oligonucleotidesynthesis1OligonucleotidesynthesisOligonucleotidesynthesisisthechemicalsynthesisofrelativelyshortfragmentsofnucleicacidswithdefinedchemicalstructure(sequence).Thetechniqueisextremelyusefulincurrentlaboratorypracticebecauseitprovidesarapidandinexpensiveaccesstocustom-madeoli
2、gonucleotidesofthedesiredsequence.WhereasenzymessynthesizeDNAandRNAina5'to3'direction,chemicaloligonucleotidesynthesisiscarriedoutintheopposite,3'to5'direction.Currently,theprocessisimplementedassolid-phasesynthesisusingphosphoramiditemethodandphosphoramiditebuildingblocksderivedfromprote
3、cted2'-deoxynucleosides(dA,dC,dG,andT),ribonucleosides(A,C,G,andU),orchemicallymodifiednucleosides,e.g.LNA.Toobtainthedesiredoligonucleotide,thebuildingblocksaresequentiallycoupledtothegrowingoligonucleotidechainintheorderrequiredbythesequenceoftheproduct(seeSyntheticcyclebelow).Theproces
4、shasbeenfullyautomatedsincethelate1970s.Uponthecompletionofthechainassembly,theproductisreleasedfromthesolidphasetosolution,deprotected,andcollected.Theoccurrenceofsidereactionssetspracticallimitsforthelengthofsyntheticoligonucleotides(uptoabout200nucleotideresidues)becausethenumberoferro
5、rsaccumulateswiththelengthoftheoligonucleotidebeingsynthesized.[1]ProductsareoftenisolatedbyHPLCtoobtainthedesiredoligonucleotidesinhighpurity.Typically,syntheticoligonucleotidesaresingle-strandedDNAorRNAmoleculesaround15–25basesinlength.Theyaremostcommonlyusedasantisenseoligonucleotides,
6、smallinterferingRNA,primersforDNAsequencingandamplification,probesfordetectingcomplementaryDNAorRNAviamolecularhybridization,toolsforthetargetedintroductionofmutationsandrestrictionsites,andforthesynthesisofartificialgenes.HistoryTheevolutionofoligonucleotidesynthesissawfourmajormethodsof
7、theformationofinternucleosidiclinkagesandhasbeenreviewedintheliteratureingreatdetail.[2][3][4]EarlyworkandcontemporaryH-phosphonatesynthesisIntheearly1950s,AlexanderTodd’sgrouppioneeredH-phosphonateandphosphatetriestermethodsofoligonucleotidesynthesis.[5][6]Thereact
