Synthesis of RNA by In Vitro Transcription

In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those ... Advertisement CoronavirusResources 2011 SynthesisofRNAbyInVitroTranscription Authors: BertrandBeckert 1 , BenoîtMasquida 2 BertrandBeckert 1 , BenoîtMasquida 2 showmoredetails Series:MethodsInMolecularBiology>Book:RNA Protocol | DOI:10.1007/978-1-59745-248-9_3 Affiliations: DepartmentofCellularandMolecularMedicine,ThePanumInstitute,UniversityofCopenhagen,Copenhagen,Denmark ArchitectureetRéactivitédel’ARN,UniversitédeStrasbourg,CNRS,IBMC,Strasbourg,France less Accessenabledvia:AnInstitution PDF Fulltext Relatedarticles Abstract Invitrotranscriptionisasimpleprocedurethatallowsfortemplate-directedsynthesisofRNAmoleculesofanysequencefromshortoligonucleotidestothoseofseveralkilobasesinμgtomgquantities.Itisbasedontheengineeringofatemplate …more Invitrotranscriptionisasimpleprocedurethatallowsfortemplate-directedsynthesisofRNAmoleculesofanysequencefromshortoligonucleotidestothoseofseveralkilobasesinμgtomgquantities.Itisbasedontheengineeringofatemplatethatincludesabacteriophagepromotersequence(e.g.fromtheT7coliphage)upstreamofthesequenceofinterestfollowedbytranscriptionusingthecorrespondingRNApolymerase.Invitrotranscriptsareusedinanalyticaltechniques(e.g.hybridizationanalysis),structuralstudies(forNMRandX-raycrystallography),inbiochemicalandgeneticstudies(e.g.asantisensereagents),andasfunctionalmolecules(ribozymesandaptamers). less Figures(0)&Videos(0) Keywords Techniques: InVitroTranscription,Template-directedSynthesis,PCR,Size-exclusionChromatography,Autoradiography,Electrophoresis Others: T7RNApolymerase, templatepurification Citations(61) Recentcitations: MichaelHD’Souzaetal.,2022,NucleicAcidsResearch CameronWebbetal.,2022,MolecularPharmaceutics ZhichengZuoetal.,2022,TheCRISPRJournal Relatedarticles Basedontechniques AnEfficientSystemforLet-7MicroRNAandGW182Protein-MediatedDeadenylationInVitro MarcR.Fabian etal., 2011, SpringerProtocols EpithelialSodiumChannelinPlanarLipidBilayers BakhromK.Berdiev & DaleJ.Benos , 2006, SpringerProtocols InVitroTechniquesforStudiesofHIV-1PromoterActivity FabioRomerio & DavidM.Margolis , 1999, SpringerProtocols EmpiricalValidationofOverlappingViruslncRNAsandCodingTranscriptsbyNorthernBlot MehmetKara & ScottA.Tibbetts , 2021, SpringerProtocols FRETAnalysisofRNARibonucleicacid(RNA)–ProteinInteractionsUsingSpinachAptamers LauraGerhard & SvenHennig , 2021, SpringerProtocols Seemore AnEfficientSystemforLet-7MicroRNAandGW182Protein-MediatedDeadenylationInVitro MarcR.Fabian etal., 2011, SpringerProtocols EpithelialSodiumChannelinPlanarLipidBilayers BakhromK.Berdiev & DaleJ.Benos , 2006, SpringerProtocols InVitroTechniquesforStudiesofHIV-1PromoterActivity FabioRomerio & DavidM.Margolis , 1999, SpringerProtocols EmpiricalValidationofOverlappingViruslncRNAsandCodingTranscriptsbyNorthernBlot MehmetKara & ScottA.Tibbetts , 2021, SpringerProtocols FRETAnalysisofRNARibonucleicacid(RNA)–ProteinInteractionsUsingSpinachAptamers LauraGerhard & SvenHennig , 2021, SpringerProtocols Dot-BlotHybridizationTechnique AlangarIshwaraBhat & GovindPratapRao , 2020, SpringerProtocols DirectCloningofDouble-StrandedRNAs ManliShen etal., 2015, SpringerProtocols ProbingRNAFoldingbyHydroxylRadicalFootprinting MariaCosta & DarioMonachello , 2014, SpringerProtocols SensitiveandSpecificInSituHybridizationforEarlyDrugDiscovery PernilleA.Usher etal., 2014, SpringerProtocols Trans-ActingAntigenomicHDVRibozymeforProductionofInVitroTranscriptswithHomogenous3′Ends MilenaSzafraniec etal., 2012, SpringerProtocols less References Milligan,J.F.,Uhlenbeck,O.C.(1989)SynthesisofsmallRNAsusingT7RNApolymerase.MethodsEnzymol180,51–62. Gruegelsiepe,H.,Schön,A.,Kirsebom,L.A.,Hartmann,R.K.(2005)EnzymaticRNAsynthesisusingbacteriophageT7RNApolymerase,in:(Hartmann,R.K.,Bindereif,A.,SchönA.,WesthofE.,eds.),HandbookofRNABiochemistry.WILEY-VCHVerlagGmbH&Co.KGaA,Germany,pp.3–21. Milligan,J.F.,Groebe,D.R.,Witherell,G.W.,Uhlenbeck,O.C.(1987)OligoribonucleotidesynthesisusingT7RNApolymeraseandsyntheticDNAtemplates.NucleicAcidsRes15,8783–8798. Huang,F.,Yarus,M.(1997)5′-RNAself-cappingfromguanosinediphosphate.Biochemistry36,6557–6563. Jeng,S.T.,Gardner,J.F.,Gumport,R.I.(1990)TranscriptionterminationbybacteriophageT7RNApolymeraseatrho-independentterminators.JBiolChem265,3823–3830. Dunn,J.J.,Studier,F.W.(1983)CompletenucleotidesequenceofbacteriophageT7DNAandthelocationsofT7geneticelements.JMolBiol166,477–535. Brakmann,S.,Grzeszik,S.(2001)Anerror-proneT7RNApolymerasemutantgeneratedbydirectedevolution.Chembiochem2,212–219. Pleiss,J.A.,Derrick,M.L.,Uhlenbeck,O.C.(1998)T7RNApolymeraseproduces5′endheterogeneityduringinvitrotranscriptionfromcertaintemplates.RNA4,1313–1317. Helm,M.,Brule,H.,Giege,R.,Florentz,C.(1999)MoremistakesbyT7RNApolymeraseatthe5′endsofinvitro-transcribedRNAs.RNA5,618–621. Fechter,P.,Rudinger,J.,Giege,R.,Theobald-Dietrich,A.(1998)RibozymeprocessedtRNAtranscriptswithunfriendlyinternalpromoterforT7RNApolymerase:productionandactivity.FEBSLett436,99–103. Price,S.R.,Ito,N.,Oubridge,C.,Avis,J.M.,Nagai,K.(1995)CrystallizationofRNA-proteincomplexesI.Methodsforthelarge-scalepreparationofRNAsuitableforcrystallographicstudies.JMolBiol249,398–408. Schurer,H.,Lang,K.,Schuster,J.,Morl,M.(2002)Auniversalmethodtoproduceinvitrotranscriptswithhomogeneous3′ends.NucleicAcidsRes30,e56. Bevilacqua,P.C.,Brown,T.S.,Nakano,S.,Yajima,R.(2004)Catalyticrolesforprotontransferandprotonationinribozymes.Biopolymers73,90–109. Suydam,I.T.,Strobel,S.A.,Daniel,H.(2009)Nucleotideanaloginterferencemapping.MethodsEnzymol468,3–30. Sousa,R.,Padilla,R.(1995)AmutantT7RNApolymeraseasaDNApolymerase.EMBOJ14,4609–4621. Ichetovkin,I.E.,Abramochkin,G.,Shrader,T.E.(1997)Substraterecognitionbytheleucyl/phenylalanyl-tRNA-proteintransferase.Conservationwithintheenzymefamilyandlocalizationtothetrypsin-resistantdomain.JBiolChem272,33009–33014. Abstract Invitrotranscriptionisasimpleprocedurethatallowsfortemplate-directedsynthesisofRNAmoleculesofanysequencefromshortoligonucleotidestothoseofseveralkilobasesinμgtomgquantities.Itisbasedontheengineeringofatemplate …more Invitrotranscriptionisasimpleprocedurethatallowsfortemplate-directedsynthesisofRNAmoleculesofanysequencefromshortoligonucleotidestothoseofseveralkilobasesinμgtomgquantities.Itisbasedontheengineeringofatemplatethatincludesabacteriophagepromotersequence(e.g.fromtheT7coliphage)upstreamofthesequenceofinterestfollowedbytranscriptionusingthecorrespondingRNApolymerase.Invitrotranscriptsareusedinanalyticaltechniques(e.g.hybridizationanalysis),structuralstudies(forNMRandX-raycrystallography),inbiochemicalandgeneticstudies(e.g.asantisensereagents),andasfunctionalmolecules(ribozymesandaptamers). less Relatedarticles Basedontechniques AnEfficientSystemforLet-7MicroRNAandGW182Protein-MediatedDeadenylationInVitro MarcR.Fabian etal., 2011, SpringerProtocols EpithelialSodiumChannelinPlanarLipidBilayers BakhromK.Berdiev & DaleJ.Benos , 2006, SpringerProtocols InVitroTechniquesforStudiesofHIV-1PromoterActivity FabioRomerio & DavidM.Margolis , 1999, SpringerProtocols EmpiricalValidationofOverlappingViruslncRNAsandCodingTranscriptsbyNorthernBlot MehmetKara & ScottA.Tibbetts , 2021, SpringerProtocols FRETAnalysisofRNARibonucleicacid(RNA)–ProteinInteractionsUsingSpinachAptamers LauraGerhard & SvenHennig , 2021, SpringerProtocols Seemore AnEfficientSystemforLet-7MicroRNAandGW182Protein-MediatedDeadenylationInVitro MarcR.Fabian etal., 2011, SpringerProtocols EpithelialSodiumChannelinPlanarLipidBilayers BakhromK.Berdiev & DaleJ.Benos , 2006, SpringerProtocols InVitroTechniquesforStudiesofHIV-1PromoterActivity FabioRomerio & DavidM.Margolis , 1999, SpringerProtocols EmpiricalValidationofOverlappingViruslncRNAsandCodingTranscriptsbyNorthernBlot MehmetKara & ScottA.Tibbetts , 2021, SpringerProtocols FRETAnalysisofRNARibonucleicacid(RNA)–ProteinInteractionsUsingSpinachAptamers LauraGerhard & SvenHennig , 2021, SpringerProtocols Dot-BlotHybridizationTechnique AlangarIshwaraBhat & GovindPratapRao , 2020, SpringerProtocols DirectCloningofDouble-StrandedRNAs ManliShen etal., 2015, SpringerProtocols ProbingRNAFoldingbyHydroxylRadicalFootprinting MariaCosta & DarioMonachello , 2014, SpringerProtocols SensitiveandSpecificInSituHybridizationforEarlyDrugDiscovery PernilleA.Usher etal., 2014, SpringerProtocols Trans-ActingAntigenomicHDVRibozymeforProductionofInVitroTranscriptswithHomogenous3′Ends MilenaSzafraniec etal., 2012, SpringerProtocols less References Milligan,J.F.,Uhlenbeck,O.C.(1989)SynthesisofsmallRNAsusingT7RNApolymerase.MethodsEnzymol180,51–62. Gruegelsiepe,H.,Schön,A.,Kirsebom,L.A.,Hartmann,R.K.(2005)EnzymaticRNAsynthesisusingbacteriophageT7RNApolymerase,in:(Hartmann,R.K.,Bindereif,A.,SchönA.,WesthofE.,eds.),HandbookofRNABiochemistry.WILEY-VCHVerlagGmbH&Co.KGaA,Germany,pp.3–21. Milligan,J.F.,Groebe,D.R.,Witherell,G.W.,Uhlenbeck,O.C.(1987)OligoribonucleotidesynthesisusingT7RNApolymeraseandsyntheticDNAtemplates.NucleicAcidsRes15,8783–8798. Huang,F.,Yarus,M.(1997)5′-RNAself-cappingfromguanosinediphosphate.Biochemistry36,6557–6563. Jeng,S.T.,Gardner,J.F.,Gumport,R.I.(1990)TranscriptionterminationbybacteriophageT7RNApolymeraseatrho-independentterminators.JBiolChem265,3823–3830. Dunn,J.J.,Studier,F.W.(1983)CompletenucleotidesequenceofbacteriophageT7DNAandthelocationsofT7geneticelements.JMolBiol166,477–535. Brakmann,S.,Grzeszik,S.(2001)Anerror-proneT7RNApolymerasemutantgeneratedbydirectedevolution.Chembiochem2,212–219. Pleiss,J.A.,Derrick,M.L.,Uhlenbeck,O.C.(1998)T7RNApolymeraseproduces5′endheterogeneityduringinvitrotranscriptionfromcertaintemplates.RNA4,1313–1317. Helm,M.,Brule,H.,Giege,R.,Florentz,C.(1999)MoremistakesbyT7RNApolymeraseatthe5′endsofinvitro-transcribedRNAs.RNA5,618–621. Fechter,P.,Rudinger,J.,Giege,R.,Theobald-Dietrich,A.(1998)RibozymeprocessedtRNAtranscriptswithunfriendlyinternalpromoterforT7RNApolymerase:productionandactivity.FEBSLett436,99–103. Price,S.R.,Ito,N.,Oubridge,C.,Avis,J.M.,Nagai,K.(1995)CrystallizationofRNA-proteincomplexesI.Methodsforthelarge-scalepreparationofRNAsuitableforcrystallographicstudies.JMolBiol249,398–408. Schurer,H.,Lang,K.,Schuster,J.,Morl,M.(2002)Auniversalmethodtoproduceinvitrotranscriptswithhomogeneous3′ends.NucleicAcidsRes30,e56. Bevilacqua,P.C.,Brown,T.S.,Nakano,S.,Yajima,R.(2004)Catalyticrolesforprotontransferandprotonationinribozymes.Biopolymers73,90–109. Suydam,I.T.,Strobel,S.A.,Daniel,H.(2009)Nucleotideanaloginterferencemapping.MethodsEnzymol468,3–30. Sousa,R.,Padilla,R.(1995)AmutantT7RNApolymeraseasaDNApolymerase.EMBOJ14,4609–4621. Ichetovkin,I.E.,Abramochkin,G.,Shrader,T.E.(1997)Substraterecognitionbytheleucyl/phenylalanyl-tRNA-proteintransferase.Conservationwithintheenzymefamilyandlocalizationtothetrypsin-resistantdomain.JBiolChem272,33009–33014. Figures(0)&Videos(0) Advertisement Citations(61) Recentcitations: MichaelHD’Souzaetal.,2022,NucleicAcidsResearch CameronWebbetal.,2022,MolecularPharmaceutics ZhichengZuoetal.,2022,TheCRISPRJournal Keywords Techniques: InVitroTranscription,Template-directedSynthesis,PCR,Size-exclusionChromatography,Autoradiography,Electrophoresis Others: T7RNApolymerase, templatepurification