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INVESTIGATINGAPTAMERSFORCAPILLARYELECTROPHORESIS AFFINITYASSAYS By DANIELLEDENISEBUCHANAN ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 2001 V Tomyparentsforalltheirloveandsupport. ACKNOWLEDGMENTS IwouldliketothankProfessorRobertT.Kennedyforhisguidanceandsupport duringmygraduatecareer. IwouldalsoliketothankDr.StevenA.Bennerandseveral pastmembersofhisresearchgroupincludingDr.ThomasBattersby,Dr.Petra Burgstallar,andDr.DarwinAngfortheopportunitytocollaboratewiththemonavery interestingscientificendeavor. Iamindebtedtoawonderfulgroupofwomen,Ms.Lisa Kauri,Ms.CameronChurch,Ms.JacinthMcKenzie,Ms.GretchenPotts,andMrs. CharinaParasdeSilva,fortheirfriendship,encouragement,andguidanceoverthepast years. IwouldalsoliketothemembersoftheKennedyresearchgroupfortheir assistanceovertheyears. IwouldliketothankMr.JonThompsonforhiswonderful insightandfriendshipandProfessorMichaelT.Bowserforalltheadviceandsuggestions tohelpmealongtheway. MostnotablyIwouldliketothankmyparents,Charlesand LorraineBuchanan,fortheirlove,support,andencouragementduringsomeveryrough times. Iwouldliketothankmysisters,JulieandMeghan,foralwaysremindingmeof theimportantthingsinlife. AboveallIwouldliketothanktheLordforthepatience, courage,andstaminatoreachtheend. iii TABLEOFCONTENTS page ACKNOWLEDGMENTS iii ABBREVIATIONS vi ABSTRACT ix CHAPTERS INTRODUCTION 1 AffinityAssays 1 Immunoassays 1 CapillaryElectrophoresis 3 Instrumentation 7 Detection 8 CapillaryElectrophoresisImmunoassays 9 Aptamers 16 % 2 DEVELOPINGANONCOMPETITIVEAPTAMERAFFINITYASSAY EMPLOYINGCAPILLARYELECTROPHORESISWITHLASERINDUCED FLUORESCENCEDETECTION Introduction 28 MaterialsandMethods 33 ResultsandDiscussion 35 Conclusions 41 3 INVESTIGATINGTHEEFFECTSOFELECTRICFIELD,COLUMNTIME,pH, ANDBUFFERCOMPOSITIONONAPTAMERCOMPLEXSTABILITY Introduction 51 MaterialsandMethods 53 ResultsandDiscussion 55 Conclusions 63 IV 4 INVESTIGATIONSOFADENOSINEBINDINGAPTAMERSWITHAFFINITY 5 CAPILLARYELECTROPHORESIS Introduction 76 MaterialsandMethods 80 ResultsandDiscussion 82 6 Conclusions 88 PREPARATIONANDEVALUATIONOFPOLYMERICCAPILLARY COATINGSFORPROTEINSEPARATIONS Introduction 103 MaterialsandMethods 107 ResultsandDiscussion 112 Conclusions 123 SUMMARYANDFUTUREDIRECTIONS 137 REFERENCES 140 BIOGRAPHICALSKETCH 147 v ABBREVIATIONS A* freeaptamer Ab antibody Ab* labeledantibody ACE affinitycapillaryelectrophoresis Ag antigen Ag* labeledantigen Ab-Ag antibody-antigencomplex APCE affinityprobecapillaryelectrophoresis BCECF 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein C complex CE capillaryelectrophoresis CEC capillaryelectrochromatography CEIA capillaryelectrophoreticimmunoassay cIEF capillaryisoelectricfocusing CEMSA capillaryelectrophoresismobilityshiftassay CGE capillarygelelectrophoresis CZE capillaryzoneelectrophoresis EDTA ethylenediaminetetraaceticaciddisodiumsalt ELISA enzyme-linkedimmunosorbentassay vi EMSA electrophoresismobilityshiftassay EOF electroosmoticflow HPMC hydroxypropylmethylcellulose i.d. innerdiameter IgE immunoglobulinE IS internalstandard J 5-(3”-aminopropynyl)-2’-deoxyuridine Kd dissociationconstant LC liquidchromatography LIF laserinducedfluorescence ft electrophoreticmobility MEKC micellarelectrokineticcapillarychromatography MOPS 3-[N-morpholino]propanesulfonicacid MW molecularweight o.d. outerdiameter PCR polymerasechainreaction PEO polyethyleneoxide) pi isoelectricpoint RIA radioimmunoassay SDS sodiumdodecylsulfate SELEX systematicevolutionofligandsbyexponentialenrichment TBE/K Tris,borate,EDTA,andpotassiumchloridebuffer TGK Tris,glycine,andpotassiumphosphatebuffer Tris tris(hydroxyaminomethane) UV ultraviolet Vlll AbstractofDissertationPresentedtotheGraduateSchool oftheUniversityofFloridainPartialFulfillmentofthe RequirementsfortheDegreeofDoctorofPhilosophy INVESTIGATINGAPTAMERSFORCAPILLARY ELECTROPHORESISAFFINITYASSAYS By DanielleDeniseBuchanan August2001 Chairman: RobertT.Kennedy MajorDepartment: Chemistry Noncompetitive CE aptamer assays were developed for thrombin and IgE. BufferandpHeffectswereinvestigatedtodeterminetheeffectoncomplexformation anddetection. IncomparisontophosphateandMOPSbuffers,largercomplexeswere detectedusingatris(hydroxyamino)methane,glycine,andpotassiumphosphate(TGK) pH8.4buffer. Goodresolutionbetweenthecomplexandfreepeakswasachievedinthe TGKbufferfortheIgEaptamerassay;however,bridgingbetweenthecomplexandfree peakswasobservedfortheassayusingtheloweraffinitythrombinaptamer. Detection limitsforthethrombinandIgEaptamercomplexesweredeterminedinTGKas70nM and5nMrespectively. Otherseparationparameterssuchaselectricfieldstrengthand columntimewereinvestigated. Thetimeonthecolumnwasshowntoaffecttheamount ofcomplexdetected;however,electricfieldsintherangeof125-600V/cmshowedno adverseeffectsonthecomplex. IX A DNAaptamer, initially selected forATP, was characterizedforuseasanaffinity stationaryphase. Usingaffinitycapillaryelectrophoresis(ACE),bindingconstantsfor thisaptamerweredeterminedforvariousadenosinecontainingmoleculessuchasAMP, ADP,andATP. Dissociationconstants(Kd)oftheDNA-ligandcomplexwerefoundto besimilarforadenosineandADPbutdifferedfromAMPorATP.Separationparameters suchaspH,temperature,andsaltconcentrationwereoptimizedinordertomaximizethe precisionandminimizetheerrorintheelectrophoreticmobility andbindingconstant measurements. UsingACEthebindingofanotherATPselectiveDNAaptamercontaininganon- naturallyoccurringpositivelychargednucleotide,5-(3"-aminopropynyl)-2'-deoxyuridine (J),wasinvestigated.Themodifiednucleotide“J”wasincorporatedintotheaptamerin orderto enrichthediversity ofthe initial aptamerselectionpool as well aspossibly enhancethebindingaffinityoftheaptamerwiththeATPligand.Thebindingconstantof theJ-baseaptamer(120 pM)wasdeterminedandcomparedtothatofthepreviously characterizedstandardnucleotideATPaptamer(133pM). Noenrichmentinbindingwas measuredwiththemodifiedbaseaptamer. Withimprovementsinaptamerselectionsand furtherdevelopmentofnon-standardnucleotides,aptamerdiversityandfunctionalitywill continuetobeenrichedincreasingaptamerapplicabilityforanalyticaldiagnostictools. x