Synthesize"RNA"transcriptsthatalsocontainstandardormodifieddNTPsusingthismutantT7RNApolymerase
Generate"RNA"transcriptswithbothrNMP/2´-dNMPorrNMP/2´-modified-NMPcomposition- Createmodified"RNA"transcriptsthatareresistanttoRNaseA-typeRNases
- Useforavarietyofapplicationswhere"RNA"withincorporateddNTPsisbeneficial
T7R&DNA™Polymerase*isarecombinantmutantformofT7RNApolymerase(Y639Fmutant),inwhichTyr-639hasbeenreplacedbyPhe.1,2ThismutationenablesT7R&DNAPolymerasetoincorporate2´-deoxyribonucleosidetriphosphates(dNTPs)intofull-lengthtranscriptsmuchmoreefficientlythanthecorrespondingwild-typeenzyme,1whileretainingthesamecatalyticactivityforincorporationofcanonicalNTPs,andthesamehighpromoterspecificityaswild-typeT7RNAPolymerase.TheABIlityofthismutantenzymetoincorporatecertain2´-modified-dNTPs(includingbutnotlimitedtodNTPshavinga2´-fluorineor2´-aminogroup)enablesefficientinvitrosynthesisoftranscriptscomposedofmixedrNMP/2´-dNMPorrNMP/2´-modified-dNMPfromanyDNAtemplatethatisdownstreamoftheT7RNApolymerasepromoter.Suchmodifiedtranscriptsareusefulformanyapplications.
Note:Completesubstitutionofone2´-dNTP(orone2´-modifieddNTP)foracanonicalrNTPinaT7R&DNAPolymerasereactionresultsinaslightdecreaseinyield.Additionalsubstitutionsof2´-dNTPs(or2´-modified-dNTPs)forcanonicalrNTPswillfurtherreduceyieldofthetranscriptproduced.Substitutionwithallfour2´-dNTPs(or2´-modifieddNTPs)willresultinextremelylowyieldsoftranscriptandisNOTRECOMMENDED.
UnitDefinition:OneunitofT7R&DNAPolymerasecatalyzestheincorporationof1nmolofribonucleosidetriphosphateintoRNAin1hourat37°CunderstandardassayconditionsusingaDNAtemplatewiththeT7promoter.
StorageBuffer:50%glycerolcontaining50mMTris-HCl(pH7.5),0.1MNaCl,1.0mMDTT,0.1mMEDTA,and0.1%Triton®X-100.
5XTranscriptionBuffer:0.2MTris-HCl(pH7.5),50mMNaCl,30mMMgCl2,and10mMspermidine.
References
- Sousa,R.andPADIlla,R.(1995)EMBOJ.14,4609.
- U.S.PatentNos.5,849,546and6,107,037.
*Coveredbyissuedand/orpendingpatents.
