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Methods in Molecular Biology 2518 James Chappell Melissa K. Takahashi Editors Riboregulator Design and Analysis M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series wasthefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols fromtheseriesareindexedinPubMed. Riboregulator Design and Analysis Edited by James Chappell Biosciences Department, Rice University, Houston, TX, USA Melissa K. Takahashi Department of Biology, California State University, Northridge, Northridge, CA, USA Editors JamesChappell MelissaK.Takahashi BiosciencesDepartment DepartmentofBiology RiceUniversity CaliforniaStateUniversity,Northridge Houston,TX,USA Northridge,CA,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2420-3 ISBN978-1-0716-2421-0 (eBook) https://doi.org/10.1007/978-1-0716-2421-0 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2022 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Since the last decade, RNA has become a powerful biomolecule for implementing gene controlandengineeringlivingsystemsfordiscoveryandapplication.Aversatiletoolboxof naturally inspired and entirely synthetic RNA regulatory mechanisms now exists, able to enact regulation through controlling transcription, translation, and nucleic acid modifica- tion.TheseriboregulatorsincludeRNA-onlysystemssuchassmallRNAsandriboswitches, andribonucleoproteinsystemssuchasthosebaseduponCRISPR-Cas.Coupledwiththese advances has been an expansion of synthetic biology applications that seek to use RNA technologies to create diagnostics, construct synthetic gene circuits, create cellular record- ing devices, and control metabolic flux. Along with this, advances in experimental and computational structure characterization techniques are revolutionizing our fundamental understanding of how natural RNA has evolved and empowering our ability to rationally designsyntheticRNAdenovo.Takentogether,RNAisanincreasinglypowerfulbiomolecule forprogrammingcellular functions. The present book, Methods in Molecular Biology, describes the latest advances in ribor- egulatorsandtechniquestostudy,evolve,anddesignthem.Westartwithaseriesofchapters focusedonthedesignandapplicationofsmallRNA(sRNA)regulatorsystemsthatcontrol transcriptionandtranslation.Theseriboregulatorsareprovidingaversatileandincreasingly diverse set of technologies for expression control and advanced applications that include diagnostics andcreationofsynthetic genecircuitry.Next,we describetechniques tocreate switchableriboregulatorsystemscalledriboswitchesthatallowforconditionalcontrolgene expression in response to the binding of user-specified molecular and physical stimuli. The third part of the book highlights recent technologies that leverage RNA-guided CRISPR- Cassystemstoedittheepigenome,controlgeneexpression,andcreatediagnostics.Finally, weendonaselectionofchaptersdescribingcomputationalandexperimentaltechniquesto investigate the sequence-structure-function relationship of RNA systems that can both advancefundamentalunderstandingandrationaldesignofriboregulators. This book is aimed to assist both scientists and engineers interested in the design and application of riboregulators. We would like to thank all authors for contributing to this bookandhopethatitwillhelpdrivefurtherinnovationinriboregulator research. Houston,TX,USA JamesChappell Northridge,CA,USA MelissaK.Takahashi v Contents Preface ..................................................................... v Contributors................................................................. ix 1 RNP-BasedControlSystemsforGeneticCircuitsinSyntheticBiology BeyondCRISPR........................................................ 1 TrevorR.Simmons,AndrewD.Ellington,andLydiaM.Contreras 2 ComputationalDesignofRNAToehold-MediatedTranslationActivators ..... 33 KaiyueWu,ZhaoqingYan,andAlexanderA.Green 3 DesignofRNA-BasedTranslationalRepressors............................. 49 SeonghoHong,DongwonPark,SomaChaudhary,GriffinMcCutcheon, AlexanderA.Green,andJongminKim 4 DesignofRibocomputingDevicesforComplexCellularLogic............... 65 GriffinMcCutcheon,SomaChaudhary,SeonghoHong,DongwonPark, JongminKim,andAlexanderA.Green 5 ComputationalDesignofSmallTranscriptionActivatingRNAs(STARs) ...... 87 BaiyangLiuandJamesChappell 6 DesignandAssemblyofMultilevelTranscriptionalandTranslational RegulatorsforStringentControlofGeneExpression........................ 99 F.VeronicaGreco,TheaIrvine,ClaireS.Grierson,andThomasE. Gorochowski 7 Model-BasedDesignofSyntheticAntisenseRNAforPredictable GeneRepression........................................................ 111 TaeSeokMoon 8 DesignofaToolboxofRNAThermometers ............................... 125 ShaunakSen,AbhilashPatel,andKrishanKumarGola 9 DevelopmentofSyntheticRiboswitchestoGuidetheEvolutionofMetabolite ProductioninMicroorganisms ........................................... 135 MinsunKim,SunghoJang,andGyooYeolJung 10 EfficientMethodtoIdentifySyntheticRiboswitchesUsingRNA-Based Capture-SELEXCombinedwithInVivoScreening......................... 157 JaniceKramatandBeatrixSuess 11 RNADesignPrinciplesforRiboswitchesthatRegulateRNase P-MediatedtRNAProcessing ............................................ 179 AnnaEnder,PeterF.Stadler,MarioMörl,andSvenFindeiß 12 Design,Characterization,andApplicationofTargetedGene ActivationinBacteriaUsingaModularCRISPRaSystem.................... 203 MariaClaudiaVillegasKcamandJamesChappell 13 ReprogrammingTracrRNAsforMultiplexedRNADetection ................ 217 ChunleiJiaoandChaseL.Beisel vii viii Contents 14 HarnessingCRISPR-Cas9forEpigeneticEngineering ...................... 237 RosaS.Guerra-ResendezandIsaacB.Hilton 15 RNAStructurePrediction,Analysis,andDesign:AnIntroductionto Web-BasedTools ....................................................... 253 RaphaelAngeloI.Zambrano,CarmenHernandez-Perez, andMelissaK. Takahashi 16 Single-MoleculeFRETStudiesofRNAStructural RearrangementsandRNA-RNAInteractions............................... 271 EwelinaM.Małecka,BoyangHua,andSarahA.Woodson 17 CotranscriptionalRNAChemicalProbing ................................. 291 CourtneyE.SzyjkaandEricJ.Strobel Index ...................................................................... 331 Contributors CHASEL.BEISEL • HelmholtzInstituteforRNA-basedInfectionResearch(HIRI), Helmholtz-CentreforInfectionResearch(HZI),Wu¨rzburg,Germany;MedicalFaculty, UniversityofWu¨rzburg,Wu¨rzburg,Germany JAMESCHAPPELL • DepartmentofBioSciences,RiceUniversity,Houston,TX,USA; DepartmentofBioengineering,RiceUniversity,Houston,TX,USA SOMACHAUDHARY • BiodesignCenterforMolecularDesignandBiomimetics,TheBiodesign Institute,andtheSchoolofMolecularSciences,ArizonaStateUniversity,Tempe,AZ,USA LYDIAM.CONTRERAS • McKettaDepartmentofChemicalEngineering,UniversityofTexas atAustin,Austin,TX,USA ANDREWD.ELLINGTON • DepartmentofMolecularBiosciences,UniversityofTexasat Austin,Austin,TX,USA ANNAENDER • InstituteforBiochemistry,LeipzigUniversity,Leipzig,Germany SVEN FINDEIß • Bioinformatics Group,DepartmentofComputerScienceand InterdisciplinaryCenter forBioinformatics,LeipzigUniversity,Leipzig,Germany KRISHANKUMARGOLA • DepartmentofElectricalEngineering,IITDelhi,NewDelhi,India THOMASE.GOROCHOWSKI • SchoolofBiologicalSciences,UniversityofBristol,Bristol,UK F.VERONICAGRECO • SchoolofBiologicalSciences,UniversityofBristol,Bristol,UK ALEXANDERA.GREEN • MolecularBiology,CellBiologyandBiochemistryGraduate Program,GraduateSchoolofArtsandSciences,BostonUniversity,Boston,MA,USA; BiodesignCenter forMolecularDesignandBiomimetics,TheBiodesignInstitute,andthe SchoolofMolecularSciences,ArizonaStateUniversity,Tempe,AZ,USA;Departmentof BiomedicalEngineering,BostonUniversity,Boston,MA,USA CLAIRES.GRIERSON • SchoolofBiologicalSciences,UniversityofBristol,Bristol,UK ROSAS.GUERRA-RESENDEZ • Systems,Synthetic,andPhysicalBiologyPhDProgram,Rice University,Houston,TX,USA CARMENHERNANDEZ-PEREZ • DepartmentofBiology,CaliforniaStateUniversity Northridge,Northridge,CA,USA ISAACB.HILTON • Systems,Synthetic,andPhysicalBiologyPhDProgram,RiceUniversity, Houston,TX,USA;DepartmentofBioengineering,RiceUniversity,Houston,TX,USA; DepartmentofBioSciences,RiceUniversity,Houston,TX,USA SEONGHOHONG • DepartmentofLifeSciences,PohangUniversityofScienceandTechnology, Pohang,Gyeongbuk,SouthKorea BOYANGHUA • DepartmentofMolecularBiologyandGenetics,JohnsHopkinsMedical School,Baltimore,MD,USA THEAIRVINE • SchoolofBiologicalSciences,UniversityofBristol,Bristol,UK SUNGHO JANG • DepartmentofBioengineeringandNano-Bioengineering,Incheon NationalUniversity,Incheon,Korea;DivisionofBioengineering,CollegeofLifeSciences andBioengineering,IncheonNationalUniversity,Incheon,Korea;ResearchCenterforBio Materials&ProcessDevelopment,IncheonNationalUniversity,Incheon,Korea CHUNLEIJIAO • HelmholtzInstituteforRNA-basedInfectionResearch(HIRI),Helmholtz- CentreforInfectionResearch(HZI),Wu¨rzburg,Germany ix x Contributors GYOOYEOLJUNG • SchoolofInterdisciplinaryBioscienceandBioengineering,Pohang UniversityofScienceandTechnology,Pohang,Gyeongbuk,Korea;DepartmentofChemical Engineering,PohangUniversityofScienceandTechnology,Pohang,Gyeongbuk,Korea MARIACLAUDIAVILLEGASKCAM • DepartmentofBioSciences,RiceUniversity,Houston,TX, USA JONGMINKIM • DepartmentofLifeSciences,PohangUniversityofScienceandTechnology, Pohang,Gyeongbuk,SouthKorea MINSUNKIM • SchoolofInterdisciplinaryBioscienceandBioengineering,PohangUniversity ofScienceandTechnology,Pohang,Gyeongbuk,Korea JANICE KRAMAT • DepartmentofBiology,TechnicalUniversityDarmstadt,Darmstadt, Germany BAIYANGLIU • GraduatePrograminSystems,Synthetic,andPhysicalBiology,Rice University,Houston,TX,USA EWELINAM.MAŁECKA • T.C.JenkinsDepartmentofBiophysics,JohnsHopkinsUniversity, Baltimore,MD,USA GRIFFINMCCUTCHEON • BiodesignCenter forMolecularDesignandBiomimetics,The BiodesignInstitute,andtheSchoolofMolecularSciences,ArizonaStateUniversity,Tempe, AZ,USA;DepartmentofBiomedicalEngineering,BostonUniversity,Boston,MA,USA TAESEOKMOON • DepartmentofEnergy,EnvironmentalandChemicalEngineering, WashingtonUniversityinSt.Louis,St.Louis,MO,USA MARIOMO¨RL • InstituteforBiochemistry,LeipzigUniversity,Leipzig,Germany DONGWONPARK • DepartmentofLifeSciences,PohangUniversityofScienceandTechnology, Pohang,Gyeongbuk,SouthKorea ABHILASHPATEL • DepartmentofBioengineering,ImperialCollege,London,UK SHAUNAKSEN • DepartmentofElectricalEngineering,IITDelhi,NewDelhi,India TREVORR.SIMMONS • McKettaDepartmentofChemicalEngineering,UniversityofTexasat Austin,Austin,TX,USA PETERF.STADLER • BioinformaticsGroup,DepartmentofComputerScienceand InterdisciplinaryCenter forBioinformatics,LeipzigUniversity,Leipzig,Germany;Max PlanckInstituteforMathematicsintheScience,Leipzig,Germany;InstituteforTheoretical Chemistry,UniversityofVienna,Vienna,Austria;SantaFeInstitute,SantaFe,NM,USA ERICJ.STROBEL • DepartmentofBiologicalSciences,TheUniversityatBuffalo,Buffalo,NY, USA BEATRIXSUESS • DepartmentofBiology,TechnicalUniversityDarmstadt,Darmstadt, Germany;CentreofSyntheticBiology,TechnicalUniversityDarmstadt,Darmstadt, Germany COURTNEYE.SZYJKA • DepartmentofBiologicalSciences,TheUniversityatBuffalo,Buffalo, NY,USA MELISSAK.TAKAHASHI • DepartmentofBiology,CaliforniaStateUniversityNorthridge, Northridge,CA,USA SARAH A.WOODSON • T.C.JenkinsDepartmentofBiophysics,JohnsHopkinsUniversity, Baltimore,MD,USA KAIYUEWU • MolecularBiology,CellBiologyandBiochemistryGraduateProgram, GraduateSchoolofArtsandSciences,BostonUniversity,Boston,MA,USA;Departmentof BiomedicalEngineering,BostonUniversity,Boston,MA,USA