信号适体的设计策略及应用

ISSN100727626CN1123870PQChineseJournalofBiochemistryandMolecularBiology20061022(10):787793,3(,350025).,,..,,,().,,2.,..;;;;;;;;;Q54DesignStrategiesandApplicationsofSignalingAptamersLIUFeng2Wei,LANXiao2Peng3(PeoplesLiberationArmyCenterofLaboratoryMedicine,FuzhouGeneralHospital,ClinicalMedicineSchoolofFujianMedicalUniversity,Fuzhou350025,China)AbstractSignalingaptamersareaptamerprobesthatcouplemolecularrecognitiontosignaltransduction.Aptamersdonothavesignalingcapabilitieswhentheyareisolatedfromrandom2sequencelibrariesandcanbeengineeredintosingalreportersbyrationaldesignbasedpost2selectionmodifications.Theexistingrationaldesignstrategiescanbedividedintotwogroups:labledandlabled2freesignalingaptamers.Differentsignalingstrategiesofusingonefluorophorelabeledaptamersbasedonfluorescencepolarizationassay,duallabeledaptamersbasedonfluorescenceresonanceenergytransfersuchasmolecularbeaconaptamers,structure2switchingsignalingaptamersandinsitulabeledaptamers,orotherlabeled2freeaptamersincudingchimericaptamerapproach,substitutionapproach,light2switchcomplexapproachandaptamer2polymerconjugateapproacharereviewed.Invitroselectionapproachforgeneratingaptamersthatcanbeimmediatelytransformedintoeffectivesignalingprobeswithouttheneedforfurtheroptimizationisalsodescribed.Thesesingalingaptamerscanbeausefultoolforthedevelopmentofbiosensorsandhomogeneousassaysespeciallyinrealtimeprotienrecognitionandquantitationwhichdonotrequiremultipleimmobilisationorwashingsteps.Keywordsmolecularrecognition;signaltransduction;designstrategy;labledsignalingaptamers;labled2freesignalingaptamers;fluorescencepolarization;fluorescenceresonanceenergytransfer;invitroselection;biosensor;realtimeprotienrecognitionandquantitation:2006204224,:20062082083Tel:[phone];Fax:[phone];E2mail:[e-mail]:April24,2006;Accepted:August8,2006SupportedbyMilitaryMedicineScienceandTechnologyResearchFoundationofPLA(No.06G038)3CorrespondingauthorTel:13805013386;Fax:[phone];E2mail:[e-mail](aptamer)DNARNA,(systematicevolutionofligandsbyexponentialenrichment,SELEX)().,[13],,.,,,.,..,.3[4]:1).,.2).,,(highthroughputscreening,HTS).3)..ATP,..,.,.,.1,(2),.(fluorescentsignalingaptamers)(monochromophorelabeling)(bischromophorelabeling).1.1,.(fluorescencepolarization,FP).,.,,;,,,.(monochromophorelabeling),,,.,.,,,.McCauley[5].,,(Fig.1,791).,,.Jhaveri[6]RNA,,,ATP,ATP(Fig12).ATP,2.,ATP,,.Fig.2Invitroselectionsinglefluorophorelabellingaptamer[4]:F(fluorophore),.,,Yamana[7](bis288722pyrene),.1.2(bischromophorelabeling).P,,.11211,,(molecularbeaconaptamers,MBA),,,.:(fluorescenceresonanceenergytransfer,FRET)2(Fig13,791).,,,.,,,.2.2,,,.,,.B2DNA,,518nm[8].,Ratiometric,[9].,,,.Yamamoto[10]HIVTatRNA2,2,(,,DABCYL).Tat,2,,;Tat,,,,Tat,(Fig14,791).,.X,(G2),,(Fig15).Li[9].15,412,115,.112pmolPL.Fig.5Molecularbeaconaptamerstrategyforanalyzingthrombin[9]:F(fluorophore);:Q(quencher);:thrombin11212,Nutiu[11](structure2switchingsignalingaptamers).Fig16,3:FDNA(5)QDNA(3)MAP(:,FDNA).MAPQDNA,FDNA.,3,98710:Fig.6Structure2switchingsignalingaptamerstrategy[11]:F(fluorophore);:Q(quencher);:Target,.22,QDNA,,,.,,.,,,.,Nutiu[12]ssDNA,.SELEXssDNA(primerbindingdomains,PBDs).ssDNAPBDs,ssDNA3,(Fig17,)().52ssDNA,ssDNA.,ssDNA,,.P1P2DNA,PBDs.P1P2:,ssDNAdsDNADNA,P1P2PBDs,PBDs;,P1P2FDNA,BDNAQDNA,,P1P2,BDNA,.11213(fluorescamine,FCM),.22,,.,,.,222,(Fig18).Edward[14]ATP,.Fig.8Insitulabelingapproach:F1(acceptor);:F2(donor);:Target2X,,RNA,.(labled2freesignalingaptamers).2.1(chimericsignalingaptamer),,.,[15].(Fig19).2.2StojanovicLandry[16]35(diethylthiotricarbocyanineiodide).,,(2600molPL),09722Fig.1Effectofthrombinbindingonfluorophoremobilityforfluorescencepolarizationanisotrophymeasurements[5]760nm(Fig110).-,.2.3,,,Fig.3Workingmechanismofthefluorophore2quencher2labeledMAB[4]Fig.4Molecularbeaconaptamerstrategyforanalyzingtheviralprotein(Tat)[10]Fig.7Invitroselectionofstructure2switchingsingalingaptamers[13].L1(DNAlibrary),BDNA(biotin2containingDNA),P1,andP2arehybridizedandimmobilizedonavidin2coatedbeads..Aptamerscapableofswitch2structuresfromtheduplexwithBDNAtothecomplexwiththetargetareisolatedbytargetelution..TheselectedsequencesareamplifedbyPCRusingP2andP3asprimers..ThesensestrandisisolatedPAGEpurificationfollowingatreatmentwithNaOH.TheresultedDNAmoleculesareagainannealedtoBDNA,P1,andP2foranewroundofselection.Thereverseprimercontainedaribonucleotide(R)atits32endtocreateachimericantisensestrandpronetoNaOHmediatedcleavage,whichpermittedtheisolationofthesensestrandbygelelectrophoresis19710:Fig.9Chimericaptamerapproach[13]:F(fluorophore);:TargetFig.10Substitutionapproach[17]:Dye;:Target(Fig111).Fig.11Light2switchcomplexapproach[13]:Duplexbindingdye;:TargetJiang[18]([Ru(phen)2(dppz)]2+)IgE2BB(PDGF2BB),100pmolPL110nmolPL10pmolPL.Zhou[19]DNATOTO([Ru(phen)2(dppz)]2+)PDGF2BB,100pmolPL.2.42ssDNA,2,(Fig112).HoLeclerc[20],fmolPL.G2,G2,,2.3Fig.12Aptamer2polymerconjugateapproach[13]:Cationicpolymer;:Target.,,2DNAPRNA;,,.,;,,,..,,,,,,.,,.,,.,.,,.,,,.(References)[1]CoxJC,RajendranM,RiedelT,etal.Automatedacqui