SupplementaryMethodsAmonovalentstreptavidinwithasinglefemtomolarbiotinbindingsiteMarkHowarth,DanielJ.-F.Chinnapen,KimberlyGerrow,PieterC.Dorrestein,MelanieR.Grandy,NeilL.Kelleher,AlaaEl-Husseini&AliceY.TingGeneral.Biotin(agiftfromTanabeUSA)wasdissolvedinDMSOat100mM.Biotin-adenosinemonophosphate(biotin-AMP)wassynthesizedasdescribedADDINREFMGR.CITE<Refman><Cite><Author>Coleman</Author><Year>2002</Year><RecNum>12</RecNum><IDText>RNA-catalyzedthioestersynthesis</IDText><MDLRef_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>12</Ref_ID><Title_Primary>RNA-catalyzedthioestersynthesis</Title_Primary><Authors_Primary>Coleman,T.M.</Authors_Primary><Authors_Primary>Huang,F.</Authors_Primary><Date_Primary>2002/11</Date_Primary><Keywords>AcylCoenzymeA</Keywords><Keywords>Adenosine</Keywords><Keywords>AdenosineMonophosphate</Keywords><Keywords>BaseSequence</Keywords><Keywords>Catalysis</Keywords><Keywords>chemistry</Keywords><Keywords>CoenzymeA</Keywords><Keywords>Kinetics</Keywords><Keywords>metabolism</Keywords><Keywords>MolecularSequenceData</Keywords><Keywords>ResearchSupport,U.S.Gov&apos;t,Non-P.H.S.</Keywords><Keywords>Rna</Keywords><Keywords>RNA,Catalytic</Keywords><Keywords>SUBSTRATE</Keywords><Keywords>SulfhydrylCompounds</Keywords><Reprint>NotinFile</Reprint><Start_Page>1227</Start_Page><End_Page>1236</End_Page><Periodical>Chem.Biol.</Periodical><Volume>9</Volume><Issue>11</Issue><Address>DepartmentofChemistryandBiochemistry,UniversityofSouthernMississippi,Hattiesburg,MS39406,USA</Address><Web_URL>PM:12445773</Web_URL><ZZ_JournalStdAbbrev><fname="System">Chem.Biol.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>1andstoredlyophilizedat–20ºC.Aliquotsofbiotin-AMPwerepreparedinPBSat1mMandstoredat–20ºCforusewithinamonth.SDS-PAGEwasperformedat200Vwiththegelbox(XCellSureLock,Invitrogen)surroundedbyicetopreventdissociationofthestreptavidinsubunitsduringelectrophoresis.Plasmidconstruction.WildtypecorestreptavidininpET21a(+)(Novagen)w