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Chemistry and Biology of Non-canonical Nucleic Acids 1st Edition Naoki Sugimoto

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ChemistryandBiologyofNon-CanonicalNucleicAcids

ChemistryandBiologyofNon-Canonical NucleicAcids

NaokiSugimoto

Author

Prof.NaokiSugimoto KonanUniversity

FrontierInstituteforBiomolecularEngineering

7-1-20

Minatojima-minamimachi 650-0047Kobe

Japan

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Preface xi

1HistoryforCanonicalandNon-canonicalStructuresofNucleic Acids 1

1.1Introduction 1

1.2HistoryofDuplex 1

1.3Non-Watson–CrickBasePair 5

1.4NucleicAcidStructuresIncludingNon-Watson–CrickBasePairs 7

1.5PerspectiveoftheResearchforNon-canonicalNucleicAcid Structures 8

1.6ConclusionandPerspective 9 References 9

2StructuresofNucleicAcidsNow 11

2.1Introduction 11

2.2UnusualBasePairsinaDuplex 11

2.2.1HoogsteenBasePair 13

2.2.2Purine–PyrimidineMismatches 13

2.2.3Purine–PurineMismatches 14

2.2.4Pyrimidine–PyrimidineMismatches 16

2.3Non-canonicalBackboneShapesinDNADuplex 17

2.4BranchedDNAwithJunction 19

2.5Multi-strandedDNAHelices 20

2.6StructuresinRNA 20

2.6.1BasicStructureDistinctionsofRNA 20

2.6.2ElementsinRNASecondaryStructures 21

2.6.2.1HairpinLoop 22

2.6.2.2BulgeLoop 22

2.6.2.3InternalLoop 23

2.6.3ElementsinTertiaryInteractionsofRNA 24

2.6.3.1A-MinorInteractions 25

2.6.3.2RiboseZipper 25

2.6.3.3T-LoopMotif 26

2.6.3.4Kissing-LoopInteraction 26

2.6.3.5GNRATetraloopReceptorInteraction 27

2.6.3.6PseudoknotCrosslinkingDistantStemRegions 29

2.7Conclusion 29

References 30

3StabilityofNon-canonicalNucleicAcids 33

3.1Introduction 33

3.2FactorsInfluencingStabilitiesoftheCanonicalDuplexes 34

3.2.1HydrogenBondFormations 34

3.2.2StackingInteractions 35

3.2.3ConformationalEntropy 35

3.3ThermodynamicAnalysisfortheFormationofDuplex 36

3.4FactorsInfluencingStabilitiesoftheNon-canonicalNucleicAcids 39

3.4.1FactorsInfluencingStabilityofTriplexes 39

3.4.2FactorsInfluencingStabilityofQuadruplex 42

3.4.2.1G-Quadruplexes 42

3.4.2.2i-Motif 44

3.5ThermodynamicAnalysisfortheNon-canonicalNucleicAcids 45

3.5.1ThermodynamicAnalysisfortheIntramolecularTriplexand Tetraplex 45

3.5.2ThermodynamicAnalysisfortheIntermolecularTriplex 45

3.5.3ThermodynamicAnalysisfortheTetraplex 46

3.6Conclusion 48 References 49

4PhysicochemicalPropertiesofNon-canonicalNucleic Acids 51

4.1Introduction 51

4.2SpectroscopicPropertiesofNon-canonicalNucleicAcids 51

4.2.1EffectofNon-canonicalStructureonUVAbsorption 51

4.2.2CircularDichroismofNon-canonicalNucleicAcids 53

4.2.3NMRSpectroscopy 56

4.2.4OtherSpectroscopicCharacteristicsofNon-canonicalNucleicAcids 57

4.3ChemicalInteractionsonNon-canonicalNucleicAcids 59

4.3.1Hydration 59

4.3.2CationBinding 61

4.3.3pHEffect 62

4.3.4ChemicalModification 63

4.4ChemicalPlatformontheNon-canonicalStructures 64

4.4.1SpecificityofaLigandtoNon-canonicalStructures 64

4.4.2FluorescencePlatformofNon-canonicalStructures 67

4.4.3InterfaceBetweenProteinsandNucleicAcids 68

4.5PhysicochemicalPropertyofNon-canonicalNucleicAcidsinCell 69

4.5.1MolecularCrowdingConditionthatReflectsCellularEnvironments 69

4.5.2EffectsofCrowdingReagentsonNon-canonicalNucleicAcid Structures 70

4.5.3QuantificationofPhysicalPropertiesofNon-canonicalStructuresin CrowdingCondition 71

4.5.4Non-canonicalStructuresUnderMimickingOrganelleEnvironment 72

4.5.5InsightfortheFormationofNon-canonicalNucleicAcidsinCells 73

4.6Conclusion 75 References 75

5Telomere 79

5.1Introduction 79

5.2StructuralPropertiesofTelomere 79

5.2.1StructuresofTelomere 79

5.2.2StructuralPropertiesofHumanTelomericG4s 81

5.2.3StructureofRepeatsofHumanTelomericG4s 84

5.3BiologicalRelevanceofTelomereG4 86

5.3.1TelomeraseActivity 86

5.3.2TelomeraseRepeatedAmplificationProtocol(TRAP)Assay 89

5.3.3AlternativeLengtheningofTelomere(ALT)Mechanism 89

5.4OtherNon-canonicalStructuresRelatedtoTelomereRegion 89

5.4.1Telomerei-Motif 89

5.4.2TelomereRNA 90

5.5Conclusion 92 References 93

6Transcription 95

6.1Introduction 95

6.2TranscriptionProcess 96

6.2.1TranscriptionInitiation 96

6.2.2TranscriptionElongation 98

6.2.3TranscriptionTermination 99

6.3TranscriptionProcessPerturbedbyCertainSequencesofDNAand RNA 101

6.4TranscriptionProcessPerturbedbyNon-canonicalStructuresofDNA andRNA 103

6.5Conclusion 110 References 110

7Translation 113

7.1Introduction 113

7.2RNAsInvolvedinTranslationMachinery 113

7.3GeneralProcessofTranslation 117

7.3.1TranslationInitiation 117

7.3.2TranslationElongation 119

7.3.3TranslationTermination 119

Contents

7.4RNAStructuresAffectingTranslationReaction 121

7.4.1ModulationofTranslationInitiationinProkaryotes 121

7.4.2ModulationofTranslationInitiationinEukaryotes 123

7.4.3RNAStructuresAffectingTranslationElongation 126

7.4.4RNAStructuresAffectingTranslationTermination 130

7.5Conclusion 133

References 134

8Replication 137

8.1Introduction 137

8.2ReplicationMachineries 137

8.3ReplicationInitiation 138

8.3.1MechanismofActivationofReplicationOrigins 138

8.3.2ActivationControlofOriginsbyG4s 139

8.3.3ControlofTimingofReplicationInitiationbyG4s 142

8.4DNAStrandElongation 142

8.4.1MechanismofDNAStrandElongation 142

8.4.2ImpactofG4andi-MotifFormationsonDNAStrandSynthesis 144

8.4.3RelationshipBetweenG4andEpigeneticModification 145

8.4.4ExpansionandContractionofReplicatingStrandInducedbyHairpin Structures 147

8.5TerminationofReplication 148

8.6ChemistryoftheReplicationandItsRegulation 148

8.6.1CellularEnvironments 148

8.6.2ControlofReplicationbyChemicalCompounds 150

8.7Conclusion 151 References 152

9Helicase 155

9.1Introduction 155

9.2FunctionandStructureofHelicases 155

9.3UnwindingofNon-canonicalDNAStructuresbyHelicases 158

9.4G4HelicasesinGeneExpressions 162

9.5G4HelicasesinReplication 163

9.6G4HelicasesinTelomereMaintenance 164

9.7RelationtoDiseasesbyLossofG4Helicases 165

9.8InsightintoSpecificPropertiesofActivitiesofG4HelicaseUnder CellularConditions 165

9.9Conclusion 167 References 167

10DynamicRegulationofBiosystemsbyNucleicAcidswith Non-canonicalStructures 171

10.1Introduction 171

10.2TimeScaleofBiologicalReactions 171

10.2.1CellCycle 172

10.2.2CentralDogma 172

10.2.3DynamicStructuresofNucleicAcids 174

10.3ProcessesintheCentralDogmaAffectedbyDynamicsofNucleicAcid Structures 176

10.3.1EpigeneticRegulationCausedbyChemicalModificationofDNA 176

10.3.2Co-transcriptionalFormationofMetastableRNAStructures 178

10.3.3Co-transcriptionalTranslationandTranscriptionAttenuation 180

10.3.4Co-transcriptionalLigandBindingandGeneRegulation 181

10.3.5TranslationElongationandCo-translationalProteinFolding 183

10.4Conclusion 184 References 185

11CancerandNucleicAcidStructures 189

11.1Introduction 189

11.2DetailMechanismofCancer 189

11.2.1CancerIncidence 189

11.2.2TheRelationshipBetweenGenesandCancer 192

11.3Non-canonicalStructuresofNucleicAcidsinCancerCells 192

11.3.1StructuralCharacteristicsofNucleicAcidsinCancerCells 192

11.3.2Non-canonicalStructuresPerturbGeneExpressionofCancer-Related Genes 195

11.4RolesofNon-canonicalStructuresofNucleicAcidsinCancer Cells 197

11.4.1MonitoringofNon-canonicalStructuresinCancerCells 197

11.4.2RegulationofGeneExpressionsbytheNon-canonicalStructuresin CancerCells 198

11.5Conclusion 199 References 199

12NeurodegenerativeDiseasesandNucleicAcidStructures 203

12.1Introduction 203

12.2ProteinAggregation-InducedNeurodegenerativeDiseases 203

12.3DNAShowsKeyRoleforNeurodegenerativeDiseases 205

12.4RNAToxicPlaysaKeyRoleforNeurologicalDiseases 210

12.5Conclusion 212 References 212

13TherapeuticApplications 215

13.1Introduction 215

13.2OligonucleotideTherapeutics 216

13.2.1AntisenseOligonucleotide 216

13.2.2FunctionsofAntisenseOligonucleotideTherapeutics 217

13.2.3ChemicalModificationsinTherapeuticOligonucleotides 220

13.2.3.1BackboneModifiedOligonucleotides 220

13.2.3.2RiboseModifiedOligonucleotides 221

13.2.3.3OligonucleotideswithUnnaturalBackbone 221

13.2.4OligonucleotideTherapeuticsOtherThanAntisense Oligonucleotide 223

x Contents

13.2.4.1OligonucleotideTherapeuticsFunctioningThroughRNA Interference 224

13.2.4.2OligonucleotideTherapeuticsFunctioningThroughBindingto Protein 224

13.3Non-canonicalNucleicAcidStructuresasTherapeuticTargets 224

13.3.1TraditionalAntibioticsTargetingStructuredRegionofRNAs 225

13.3.2StrategiesforConstructingTherapeuticMaterialsTargetingStructured NucleicAcids 226

13.4Non-canonicalNucleicAcidMaterialsforInducingNon-canonical Structures 230

13.5Conclusion 231 References 232

14MaterialsScienceandNanotechnologyofNucleicAcids 235

14.1Introduction 235

14.2Non-canonicalStructure-BasedNanomaterialsResemblingProtein Functions 235

14.2.1Aptamer 235

14.2.2DNAzyme 238

14.2.3IonChannel 240

14.3ProteinEngineeringUsingG4-BindingProtein 240

14.4RegulationofGeneExpressionbyG4-InducingMaterials 242

14.5EnvironmentalSensing 246

14.5.1SensingTemperatureinCells 246

14.5.2SensingpHinCells 248

14.5.3SensingK+ IoninCells 248

14.5.4SensingCrowdingConditioninCells 249

14.6Conclusion 250 References 250

15FutureOutlookforChemistryandBiologyofNon-canonical NucleicAcids 253

15.1Introduction 253

15.2ExploringPotential:PropertiesofNon-canonicalStructuresinUnusual Media 253

15.3SystemizingProperties:PredictionoftheFormationofNon-canonical NucleicAcidsStructures 259

15.4AdvancingTechnology:ApplicationsofNon-canonicalStructuresTaking ConcurrentReactionsintoAccount 262

15.4.1Co-transcriptionalDynamicsofG-Quadruplex 263

15.4.2Co-transcriptionalFunctionalizationofRiboswitch-LikeSensor 263

15.4.3Co-transcriptionalRNACapturingforSelectionofFunctional RNAs 266

15.5Conclusion 267 References 268

Index 271

Preface

Inchemistryandbiology,oneofthemostimportantandinterestingresearchsubjectsisnucleicacids:DNAandRNA.Thenucleicacidsconsistofverysimplematerials:phosphate,sugar,andorganicbases.Theirstructuresarealsoverysimpleas singlestrandsoradoublehelix,incomparisonwithanotherbiomoleculessuchas proteinsandcarbohydrates;howeverthenucleicacidshaveveryimportantgenetic informationandfunctions.

AsImentionedintheIntroductioninChapter1,thereiscloseto70yearshistoryinnucleicacidresearchafterthediscoveryofthedoublehelixDNAstructure (B-form)asthecanonicalonebyJamesDeweyWatsonandFrancisHarryComptonCrickin1953andchemicalbiologyofnucleicacidsarefacingtonewaspect today,thatis, non-canonicalnucleicacids.Throughthisbook,Iexpectthatreaders understandhowuncommonstructureofnucleicacidsbecameoneofthecommon structuresas non-canonicalnucleicacids thatfascinateusnow.Thisnewresearch fieldfornon-canonicalnucleicacidswillsoonbig-sparkattheinterfaceofchemistry andbiology.

Thisbookiscomprisedof15chapterscoveringvariousaspectsofchemistryand biologyof non-canonicalnucleicacids includingnotonlytheirhistory,structures, stabilities,andpropertiesbutalsotheirfunctionsontranscription,translation, regulation,telomere,helicases,cancers,neurodegenerativediseases,therapeutic applications,nanotechnology,andfutureoutlook.Thisbookisavaluableresource, notonlyforgraduatestudentsbutalsoresearchersinthefieldsofphysical chemistry,organicchemistry,inorganicchemistry,analyticalchemistry,biochemistry,biophysics,structuralbiology,computationalbiology,molecularmedicine, molecularbiology,cellbiology,andnanotechnologyandwhowouldliketolearn moreaboutthepotentialimportantrolesof non-canonicalnucleicacids aswellas canonicalones.

Iwishallreadersenjoythisbookandknowtheimportanceofnotonly Watson–Crickdoublehelicalnucleicacids(B-form)butalso non-canonicalnucleic acids liketriplexandquadruplex.InsteadofHamletbywrittenbyWilliamShakespeare,pleaseanswerthequestion“ToBornottoB,thatisthequestion”inthe researchfieldofnucleicacids.

IamdeeplygratefultomycolleaguesinFIBER(FrontierInstituteforBiomolecularEngineeringResearch),KonanUniversity,fortheirexcellentcontributiontomy

xii Preface writingastheco-authorsatthefollowingeachchapter.TheyareDr.ShuntaroTakahashi(forChapters1,4,5,8,9,14,and15),Dr.TamakiEndoh(forChapters2,7, 10,13,and15),andDr.HisaeTateishi-Karimata(forChapters3,6,11,12,and15), whoseeffortshaveimmeasurablyimprovedthequalityandaccuracyoftheinformation.IamalsodeeplygratefultoMs.MiwaInadafordesigningalotoffigures andMs.KatherineWongandDr.LifenYanginWileyfortheireditingthisbookand encouragingme.

NaokiSugimoto

FrontierInstituteforBiomolecularEngineeringResearch(FIBER) GraduateSchoolofFrontiersofInnovativeResearch inScienceandTechnology(FIRST) KonanUniversity

Kobe,Japan

HistoryforCanonicalandNon-canonicalStructuresof NucleicAcids

Themainpointsofthelearning:

Understandcanonicalandnon-canonicalstructuresofnucleicacidsandthinkof historicalscientistsintheresearchfieldofnucleicacids.

1.1Introduction

Thisbookistointerpretthenon-canonicalstructuresandtheirstabilitiesofnucleic acidsfromtheviewpointofthechemistryandstudytheirbiologicalsignificances. Thereismorethan60years’historyafterthediscoveryofthedoublehelixDNA structurebyJamesDeweyWatsonandFrancisHarryComptonCrickin1953,and chemicalbiologyofnucleicacidsisfacinganewaspecttoday.Throughthisbook, Iexpectthatreadersunderstandhowtheuncommonstructureofnucleicacids becameoneofthecommonstructuresthatfascinateusnow.Inthischapter,I introducethehistoryofnucleicacidstructuresandtheperspectiveofresearchfor non-canonicalnucleicacidstructures(seealsoChapter15).

1.2HistoryofDuplex

Theopeningofthehistoryofgeneticswasmainlydonebythreeresearchers. CharlesRobertDarwin,whowasascientistofnaturalscience,pioneeredgenetics. Thepropositionofgeneticconceptisindicatedinhisbook OntheOriginofSpecies publishedin1859.Heindicatedthetheoryofbiologicalevolution,whichisthebasic scientifichypothesisofnaturaldiversity.Inotherwords,heproposedbiological evolution,whichchangedamongindividualsbyadaptingtotheenvironmentand bepassedontothenextgeneration.However,thatwasstillaprimitiveideafor thegeneticconcept.Afterthat,GregorJohannMendel,whowasapriestinBrno, CzechRepublic,confirmedthemechanismofgeneevolutionbyusing“factor” inheritedfromparenttochildrenusingpeaplantin1865.Thisdiscoverybecame theconceptofgenetics.Atthealmostsametimein1869asMendel,Johannes ChemistryandBiologyofNon-CanonicalNucleicAcids, FirstEdition.NaokiSugimoto. ©2021WILEY-VCHGmbH.Published2021byWILEY-VCHGmbH.

1HistoryforCanonicalandNon-canonicalStructuresofNucleicAcids

FriedrichMiescher,whowasabiochemistinSwiss,discoverednucleicacidsasa chemicalsubstanceofthegeneidentity.Henamedit“nuclein”(later,itwasnamed “nucleicacid,”whichexistsacidicsubstanceinnucleus)andmadetheopportunity tostudynucleicacidchemistry.However,itwouldbedoubtfulifherealizedthat nucleicacidisthegeneidentity.Afterthat,itwasneededtotakealotoftimeto concludethatthegeneidentityisprovedthechemicalsubstance.

ErwinRudolfJosefAlexanderSchrödinger,whowasagreatphysicist,pioneered togoafterthemysteryofgene.Hepublishedabooktitled WhatIsLife?in1944[1]. Thisbookinvitedthestudyofthegenetomanyresearchers.Hementionedinthe bookthathebelievedagene–orperhapsthewholechromosomefiber–tobean aperiodicsolid,althoughhealsomentionedthatgeneisprobablyonebigprotein molecule.Afterthe1950s,chemistryregardingnucleicacidshadbeendeveloping. OneoftheorganicchemistswasErwinChargaff,whowasaprofessoratColombiaUniversityintheUnitedStatesandborninAustria.Hediscoveredthatfromthe resultofpaperchromatographytargetedtothedifferenttypesofDNA,thenumberof guanineunitsequalsthenumberofcytosineunitsandthenumberofadenineunits equalsthenumberofthymineunits[2].ItiscalledChargaff’srules.Ontheother hand,analysisofthesuperstructureofnucleicacidswasalsoproceeding.Atthe beginningofthe1950s,atKing’sCollegeLondon,theresultsofX-raycrystalanalysis wereaccumulatedbyMauriceHughFrederickWilkins,RosalindElsieFranklin,and others.Finally,basedontheirresult,WatsonandCrickwhoworkedatCavendish LaboratoryinCambridgeandproposedthemodelofdoublehelixstructureofDNA (Figure1.1andseeChapter2),publishedasasingle-pagepaperaboutDNAdoublehelixin Nature issuedon25April1953[3].BydiscoveringDNAdoublehelix structure,Watson,Crick,andWilkinswereawardedtheNobelPrizeinPhysiology orMedicinein1962.

CharlesRobertDarwin(left),GregorJohannMendel(middle),andJohannesFriedrich Miescher(right)

Figure1.1 ThediffractionpatternofthecanonicalDNAduplexanditschemicalstructure.

Source:KingsCollegeLondon.

ErwinRudolfJosefAlexanderSchrödinger(left)andErwinChargaff(right)

MauriceHughFrederickWilkins(left)andRosalindElsieFranklin(right)

JamesDeweyWatsonandFrancisHarryComptonCrick

1.3Non-Watson–CrickBasePair

AlthoughthediscoveryofWatson–Crickbasepairsisfamous,weneedtomake surethatWatsonandCrickinitially“proposed”theirmodel.Moreover,Watsonand Crickwerenotthefirstresearcherswhoproposedthestructureofnucleicacids. ThephysicistLinusPauling,whoearnedtheNobelPrizetwotimesinhiscareer, firstproposedthehelixmodelofnucleicacidswithhisassociateRobertCorey[4]. However,thestructurewasfault:itwasatriplehelixhavingnegativelycharged phosphateslocatedatthecoreofthehelix,whichcouldnotexistinnature.After theproposalofWatson–Crickbasepairs,theracefordeterminationofthehelical structureofDNAhadbeenstartedusingpurineandpyrimidinemonomers.The firstsuchstudywasreportedin1959,whenKarstHoogsteen–anassociateofRobert CoreyatCaltech–usedsingle-crystalX-rayanalysistodeterminethestructuresof cocrystalscontaining9-methyladenineand1-methylthymine,wheremethylgroups wereusedtoblockhydrogenbondingtonitrogenatomsotherwisebondedtosugar carbonsinDNA[5].However,thestructurewasNOTWatson–Crickbasepair,in whichtheadeninebasewasflippedupsidedown.Thedifferentbasepairwaslater namedHoogsteenbasepair(Figure1.2andseeChapter2).Afterthediscoveryof

Watson–Crick basepair

Figure1.2 ChemicalstructuresofbasepairsviaWatson–CrickorHoogsteentypes.

Hoogsteen basepair

6 1HistoryforCanonicalandNon-canonicalStructuresofNucleicAcids

Hoogsteenbasepairs,manyresearcherslookedforWatson–Crickbasepairs.However,onlyHoogsteenbasepairswereidentified.In1973,AlexanderRichfirstdiscoveredWatson–CrickbasepairsinthecocrystaloftheAUandGCdinucleoside phosphatecomplex[6].Andsoonafter,RichardE.Dickerson,whotookoverthe Pauling’slab,firstsolvedthesingle-crystalstructureofaDNAdodecamerusing heavyatomX-raycrystallographyin1980[7].Ittakesmorethan20yearsafterthe discoveryofWatson–Crickbasepairs.TheseresultssuggestthatWatson–Crickbase pairstendedtostablyformundertheconstraintofthehelicalstructureofnucleic acids,whereasHoogsteenbasepairsforminotherstructuralconditions.Therefore, therearecanonicalstructurescomposedbyWatson–Crickbasepairsintheduplex structures.Ontheotherhand,non-canonicalstructuresincludenon-Watson–Crick basepairssuchasHoogsteenbasepairs.

LinusPauling(left),RobertCorey(middle),andKarstHoogsteen(right)

AlexanderRich(left)andRichardE.Dickerson(right)

1.4NucleicAcidStructuresIncluding Non-Watson–CrickBasePairs

BehindtheextensiveeffortstoidentifytheduplexstructureofWatson–Crickbase pairs,Hoogsteenbasepairswerealsofoundinthestructureofnucleicacidsinthe 1960s.FelsenfeldandRichexplainedhowpoly(rU)strandsmightassociatewith poly(rA)-poly(rU)duplexestoformtriplexes[8].FromthechemicalshiftofNMR, theyidentifiedevidencefortriplexformationviaprotonatedG–C+ Hoogsteen basepairsatcytosineN3inapoly(dG)-poly(dC)complexwithdGMPatlow pH[9].In1962,itwasfoundthatshortguanine-richstretchesofDNAcould assumeunusualstructures[10].Thediffractionstudiesofpoly(guanylicacid)gels suggestedthatiffourguanineswerecloseenoughtogether,theycouldformplanar hydrogen-bondedarrangementsnowcalledguaninequartets(G-quartets).Witha stackofafewG-quartets,atetraplexstructureisformedcalledasG-quadruplex (seeChapter2).Inthecrystalstructure,Hoogsteenbasepairsofpolynucleic acidswerefirstfoundintRNAstructure[11].InthestructureWatson–Crickbase pairsformedthesecondarystructureoftRNA,whereasHoogsteenbasepairs supportedthetertiarystructure.NotonlyHoogsteenbasepairsbutalsoother typesofnon-Watson–CrickbasepairswerefoundintRNAstructures.Thetertiary structureofnucleicacidsisimportantespeciallyfornon-codingRNAs,which donotcodegeneticinformation.Thelandmarkofresearchofnon-codingRNA isthediscoveryofribozyme(ribonucleicacidenzyme)byThomasRobertCech in1982[12].Ribozymescatalyzechemicalreactionsaswellasproteinenzymes. Laterstructuralstudiesrevealedthattherearealotofnon-Watson–Crickbase pairstoproducetheactivecoreofenzymaticreactionofribozymes.Therefore, non-canonicalWatson–CrickbasepairsincludingHoogsteenbasepairshavebeen thoughtofasatoolforthetertiarystructureofnucleicacidsexceptforduplexes.

ThomasRobertCech

Withtheprogressofstructuralanalysistechnologyinthe1990s,Hoogsteen basepairsaregraduallyrevealedtoexistinDNAcomplexeswithlowmolecular weightcompoundsandproteinsaswellastransientlyinWatson–Crick-typedouble helix.Furthermore,anothertypeoftetraplexstructureswasidentifiedfromDNA sequenceenrichedincytosineduetothecrossintercalationsofhemiprotonated cytosine–cytosine(C–C+ )basepairsunderacidicconditions[13].Thistetraplex iscalledasi-motif(seeChapter2).Soonafter,therolesofthenon-canonical structureshavebeengainingattention.Especiallysincethe2000s,researchonthe G-quadruplexstructureformedfromHoogsteenbasepairshasmaderemarkable progress.WhenaG-quadruplexisformedonDNAorRNA,thereactivityofthe proteininvolvedingeneexpressionisaffected(seeChapters6–8).Thismeansthat thecentraldogmaproposedbyCrick–thatgeneticinformationisdetermined centrallybytheflowofreplication,transcription,andtranslation–ishighlycontrolledbytheformationofaG-quadruplexstructure.Ingeneral,ithasbeenthought thattheregulationofgeneinformationexpressionisduetoproteinfunctions. However,thespecificstructureofHoogsteenbasepairscontrolsgeneexpressionso thatthenucleicaciditselfcanfunctionlikeaprotein.Thatis,therolesofnucleic acidsmightbeproperlyusedaccordingtobasepairs:WatsonCrickbasepair = information,non-WatsonCrickbasepair = function.Manysequencesthatcan haveaG-quadruplexstructurearedistributedinthetelomereattheendofthe chromosomeandthepromoterregionoftheoncogeneofthegene.Startingwiththe 2013report,therehavebeenmanyreportsontheformationofG-quadruplexesand i-motifsincells.Thesereportspointoutthattheoncogenemaybeactivatedbythe formation(ordissociation)oftheG-quadruplextocausecancer(seeChapter11). Furthermore,ithasbeensuggestedthatthephase-separatedstructureformedby theaggregationofRNAswithG-quadruplexescontributestoneurologicaldiseases suchasamyotrophiclateralsclerosis(seeChapter12).

1.5PerspectiveoftheResearchforNon-canonical NucleicAcidStructures

Astheregulationofgeneexpressionbythespecificstructureofnucleicacidshas beenclarified,thenextimportantissueisknowingwhatspecificstructuresare formedwhereandwhenincells.Forexample,Hoogsteenbasepairsareaffected bythemolecularenvironmentssuchasions,pH,andwateractivity.Cellsareinan environmentcrowdedwithmolecules,so-calledmolecularcrowding(seeChapters 3and4),andthemolecularenvironmentchangesdependingonthecellcycle[14]. Forexample,thenucleoluscausesachangeinthemoleculardensityinthenucleus byrepeatingformationanddissociationaccordingtothecellcycle.Thisregulates thetimingofactivationofrRNAtranscriptionineachcellcycle,becausethe transcriptionofrRNAspecificallyoccursinnucleolus.Inaddition,theenvironment ofmitochondriaisparticularlycrowded(upto500mgml 1 )butheterogeneous duetolocallyincreasedprotonconcentrationbytheprotongradientrequiredfor ATPsynthesis.Therefore,itisdesirabletodevelopatechnologythatcanpredict

9 physicochemicalpropertyofspecificstructuresduetoHoogsteenbasepairsin eachcharacteristicmolecularenvironment[15].Inaddition,thereisapossibility tomakeanewapproachofdrugdevelopmentthattreatsdiseasesbychangingthe molecularenvironmentsofcells,ratherthantargetinggenesandproteins.

1.6ConclusionandPerspective

AccordingtoPauling’spersonalcommunicationrevealedbytheNobelFoundation’s disclosure,heconsideredWatsonandCrick’sNobelawardtobepremature.Inspite ofhisopinion,theNobelFoundationdecidedtoawardthePrizetoWatsonandCrick. ThismightsuggestthatWatson–Crickbasepairswereverycommonandmeaningful atthattimebutnon-Watson–Crickbasepairswereartifactandmeaningless.Nowadays,non-Watson–CrickbasepairsarebecomingcommonandsignificantasPauling perhapspredicted.Now,thedaywhentheessenceofnucleicacidsbecomesbeyond theconceptofWatsonandCrickiscomingcloser.

References

1 Schrödinger,E.(1944). WhatIsLife?ThePhysicalAspectoftheLivingCelland Mind.Cambridge:CambridgeUniversityPress.

2 Tamm,C.,Hodes,M.,andChargaff,E.(1952). J.Biol.Chem. 195:49–63.

3 Watson,J.D.andCrick,F.H.(1953). Nature 171:737–738.

4 Pauling,L.andCorey,R.B.(1953). Nature 171:346–346.

5 (a)Hoogsteen,K.(1959). ActaCrystallogr. 12:822–823.(b)Hoogsteen,K.R. (1963). ActaCrystallogr. 16:907–916.

6 (a)Day,R.O.,Seeman,N.C.,Rosenberg,J.M.,andRich,A.(1973). Proc.Natl. Acad.Sci.U.S.A. 70:849–853.(b)Rosenberg,J.M.,Seeman,N.C.,Kim,J.J. etal.(1973). Nature 243:150–154.

7 Wing,R.,Drew,H.,Takano,T.etal.(1980). Nature 287:755–758.

8 Felsenfeld,G.andRich,A.(1957). Biochim.Biophys.Acta 26:457–468.

9 Kallenbach,N.R.,Daniel,W.E.Jr.,andKaminker,M.A.(1976). Biochemistry 15: 1218–1224.

10 Gellert,M.,Lipsett,M.N.,andDavies,D.R.(1962). Proc.Natl.Acad.Sci.U.S.A. 48:2013–2018.

11 Robertus,J.D.,Ladner,J.E.,Finch,J.etal.(1974). Nature 250:546.

12 (a)Kruger,K.,Grabowski,P.J.,Zaug,A.J.etal.(1982). Cell 31:147–157. (b)Zaug,A.J.,Grabowski,P.J.,andCech,T.R.(1983). Nature 301:578–583.

13 Gehring,K.,Leroy,J.L.,andGueron,M.(1993). Nature 363:561–565.

14 Nakano,S.,Miyoshi,D.,andSugimoto,N.(2014). Chem.Rev. 114:2733–2758.

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StructuresofNucleicAcidsNow

Themainpointsofthelearning:

(1)Learninteractionsinnucleicacidstructures.

(2)Understandstructurepolymorphismsofnucleicacids.

(3)StudydifferencesinconformationalpropertiesbetweenDNAandRNA.

2.1Introduction

Nucleicacidsarebasicallymoleculeswithahighdegreeofstructuralflexibilityand polymorphicproperty.Phosphatesinnucleicacidsarenegativelychargedandcause electrostaticrepulsionineachphosphatemoiety.Thiselectrostaticrepulsionisdisadvantageousfornucleicacidstoformacompactandorderedstructure.Nucleic acidsformthehigher-orderstructuresbyoffsettingunfavorableentropychangesand electrostaticrepulsionbyinternalinteractionssuchashydrogenbondingandstackinginteractionsandexternalfactorssuchasinteractionsofnucleicacidswithcations andcosolutes.Inotherwords,thecanonicalnucleicacidstructureconsistingofdoublehelixwithWatson–Crickbasepairsisapartofthepossiblestructuralforms,and nucleicacidsformvariousnon-canonicalstructuresdependingontheinternaland externalfactors.Thischaptershowsbasicelementsthatformnon-canonicalnucleic acidstructuresincludingunusualbasepairing,whoseexistencehasbeenrevealed bystructuralanalyses,andtheirpropertiesofthermodynamicstabilities.Detailed analysesofthestabilitiesofnucleicacidstructuresandfactorsthataffectthemare explainedinChapter3.

2.2UnusualBasePairsinaDuplex

AsinChapter1,thecanonicalstructureofnucleicacidsisdouble-strandedhelix withWatson–Crickbasepairs(Figure2.1),whicharealmostidenticalintheir

Figure2.1 Watson–CrickandHoogsteenbasepairsindoublehelix.Chemicalstructures ofA-T(a)andG-C(b)Watson–Crickbasepairs.ChemicalstructuresofA-T(c)andG-C+ (d) Hoogsteenbasepairs.N3atomofcytosinenucleobaseisprotonated.(e)StructureofDNA duplexconsistingofallA-THoogsteenbasepairs(PDBID:1RSB).(f)StructureofDNA duplexcontainingtwoconsecutiveG-C+ Hoogsteenbasepairs(PDBID:1QN3).TheDNA duplexisbendingduetointeractionofTATA-boxbindingprotein.Nucleobasesformingthe Hoogsteenbasepairsareemphasizeddark.In(e)and(f),hydrogenbondsbetweenthe Hoogsteenbasepairsareshownindashedlines.

geometricanddimensionalarrangementinthehelix.Thesugargroupsareboth attachedtothebasesonthesamesideofthebasepair.ThedistancebetweenC1′ atomsofthesugarsonoppositestrandsisessentiallythesame.Thesegeometric featuresenableanysequenceofWatson–Crickbasepairsfitintotheduplex.On theotherhand,alargenumberofotherarrangementsandhydrogenbonding patternsofbasepairsarepossible,andmanyhavebeenobservedexperimentally suchasusingX-rayandNMRanalyses.X-rayanalysiscandefinetheduplex structures,whichincorporatethenon-Watson–Crickbasepairsandprovidedetails oftheirhydrogenbondingscheme[1].Ontheotherhand,NMRanalysisprovides informationregardingthedynamicsofthenucleicacidconformationsuchas

2.2UnusualBasePairsinaDuplex 13 mismatchedbasepairs,inwhichtransienttautomericandanionicspeciesform Watson–Crick-typehydrogenbonds[2].

2.2.1HoogsteenBasePair

Hoogsteenbasepair(Figure2.1)isoneofthemajornon-Watson–Crickbasepairs thatcanbeseeninseveralcrystalstructuresofduplexescontainingA Tbase pairs.Forexample,thecrystalstructureofAT-richsequencesadoptedparallel andantiparallelstrandedduplexeswithallHoogsteen-typehydrogenbondingin theirA⋅Tbasepairs(Figure2.1)[3].Inthecaseofantiparallelduplexwiththe Hoogsteenbasepairs,theoverallstructurefeaturesoftheduplexsuchasdiameter oftheduplex,numberofbasepairsperturn,andsugarpuckerconformation aresimilartothecanonicalB-typeDNAduplex.Auniquecharacteristicisthat theadeninenucleobasesintheduplexhave syn conformationintheirglycosidic bondangles.Althoughthesamepatternofhydrogenbondingispossible,A-type RNAduplexesdisfavortheA⋅UHoogsteenbasepairbecausetheA-formgeometry disfavorsthe syn conformationintheadeninenucleobaseduetosugar-backbone rearrangementsneededtostericallyaccommodatetheadenine[4].Formation ofHoogsteen-typehydrogenbondingisalsopossiblebetweenguanine(G)and cytosine(C+ ),inwhichN3atomisprotonated.FormationoftransientHoogsteenbasepairsincludingtheG⋅C+ indiversesequencecompositionhasbeen demonstratedbyrelaxationdispersionassayusingNMR(Figure2.1)[5].Itis consideredthattheHoogsteenbasepairsplayrolesinmodulatinginteraction ofproteinsandbiologicalreactionssuchasinductionorrepairofDNAdamage andreplicationofDNAbyalteringthestructuralandchemicalpropertiesofthe duplex[5].

2.2.2Purine–PyrimidineMismatches

Mismatchedbasepairsbetweenpurineandpyrimidinenucleobasesareknownas transitionmismatches.G⋅TandG⋅Umismatchescanformtwohydrogenbonds, whichareusuallyknownastypical“wobble”basepairs,byshiftingthenucleobase geometryfromthatofWatson–Crickbasepairs(Figure2.2).Thesemismatches areoneofthemoststablemismatchesinDNAandRNAduplexes(Tables2.1 and2.2).G⋅UbasepairisfrequentlyobservedinnaturalRNAs.Itisbecausethat bothguanineanduraciltake anti conformationintheirglycosidicbondangles thatarethesamewithWatson–Crickbasepairsandthedistanceandgeometric arrangementofC1′ atomsofthesugarsdonotlargelychangecomparedwiththe canonicalduplex.Thus,thereisnosignificantworseningofbasestackingand littlerperturbationoftheduplexconformation.Adenineandcytosinealsoform twohydrogenbondsassimilarwayastheG⋅T(U)mismatchwhentheadenine nucleobaseisprotonated(Figure2.2).FormationoftheA+ ⋅Cmismatchedbase pairisdemonstratedbyX-raydiffractionanalysisusingdodecameroligonucleotide

Figure2.2 Wobblebasepairsinduplexes.ChemicalstructuresofG-T(a),G-U(b),andA+ -C (c)wobblebasepairs.N1atomofadeninenucleobaseisprotonated.(d)StructureofB-form DNAduplexcontainingG-Twobblebasepairs(PDBID:113D).(e)StructureofA-formRNA duplexcontainingtwoconsecutiveG-Uwobblebasepairs(PDBID:433D).(f)Structureof B-formDNAduplexcontainingA+ -Cwobblebasepairs(PDBID:1D99).Nucleobases formingthewobblebasepairsareemphasizeddark.Hydrogenbondsinthewobblebase pairsareshownindashedlines.

strand[7].However,neutralA⋅Cmismatchesareinequilibriumbetweenthe wobbleandreversewobbleforms,eachofwhichonlyformsonehydrogenbond. Thus,A⋅CmismatchismuchlessstablethanG⋅T(U)mismatchinaphysiological condition(Tables2.1and2.2).

2.2.3Purine–PurineMismatches

Purine–purineandpyrimidine–pyrimidinemismatchesareknownastransversion mismatches.ThereareG⋅A,G⋅G,andA⋅Amismatchesinthepurine–purinemismatch.Amongthem,G⋅Amismatchformsrelativelystableunusualbasepairsin bothDNAandRNAduplexes(Tables2.1and2.2).Itishighlypolymorphicdependingonsequencecompositions.InDNAduplexescontainingG⋅Amismatchesthat formtwohydrogenbonds,variouscombinationsof anti and syn wereobservedin theirglycosidicbondangles(Figure2.3).G⋅Gmismatchpotentiallyadoptsabase paringwithtwohydrogenbonds,inwhichtwoguanosinesaresymmetricallyor asymmetricallyorientedwith anti and syn conformationintheirglycosidicbond angles(Figure2.3).WhentheasymmetricG Gbasepairsfaceeachotherinrotation,fourguaninesformasymmetricquartetasdescribedlater(Chapter3).Recent X-raydiffractionanalysesalsodemonstratedpolymorphicfeatureoftheG Gmismatchbyshowingthatwith syn–syn combinationintheglycosidicbondanglesin

Table2.1 Thermodynamicparametersforduplexformationsin1MNaClbyDNA oligonucleotidescontainingmismatchesa)

SequenceXY(kcal(calmol 1 )(kcal(∘ Cat mol 1 )K 1 )mol 1 )10 4 M)

5′ CAAAXAAAG CG64 51837 742 9

3′ GTTTYTTTC GC62 81797 340 8 AT68.01967.240.1 TA58.61686.536.8 GG53 51584 525 6 TG55 61654 425 7 GA52 61564 223 9

GT46 71374 222 3

AG39.91163.918.0

AA36.91073.715.0

CT53 21613 319 1 TC50 01513 217 5

5′ CAACTTGATATTAATA + Mismatch

(35.8)b) (106)b) (2.9)b) (9)b)

(55.3)b) (171)b) (2.3)b) (15)b)

3′ GTTGAACTATAATTAT –102.128912.455.8

3′ GTTGAGCTATAATTAT TG92 626610 149 4

3′ GTTGAACTATAGTTAT TG95 527410 550 5

3′ GTTGAACTCTAATTAT TC98 42869 747 3

3′ GTTGAATTATAATTAT GT91 32649 447 1

3′ GTTGAACCATAATTAT AC90.92658.744.6

3′ GTTGAACAATAATTAT AA92.02679.2646.2

a)Valuesaresummarizedonesinareference[6].

b)Valuesinparenthesisaresignificantlylessaccurateestimatedusingflatlowerbaselinesin theirmeltinganalyses.

thepresenceofchromomycinA3,whichbindsminorgrooveofthemismatched placeandsupportsthestructureanalysis[8].DetailedstructureofA Amismatch israrelydeterminedbyX-raydiffractionanalysis.Itisconsideredthatthemismatch isdynamicallyfluctuatedandnotabletobeaparticularstructuralstate.

Table2.2 FreeenergyincrementsfortandemmismatchesinRNAoligonucleotidesin1M NaCla), b)

Closingbasepair

a)Valuesaresummarizedonesinareference[6].

b)Valuesareincrementsofthestabilities(ΔGo 37 inkcalmol 1 )comparedwithself complementaryduplexconsistingofCGXX′ CGsequence,whereXandX′ arenucleobasesin theleftcolumn.

c)Thesequencehaseitheranunusualconformationormixtureofconformations.

Figure2.3 MismatchedG-AandG-Gbasepairsobservedinnucleicacidstructures.(a)G-A mismatchedbasepairswithvariouscompositionsintheirglycosidicbondangles.(b) Symmetric(left)andasymmetric(right)G-Gmismatchedbasepairs.

2.2.4Pyrimidine–PyrimidineMismatches

Pyrimidine–pyrimidinemismatchescanbecategorizedinunstablemismatches (Tables2.1and2.2).Whenpyrimidinenucleotidesbasepairthroughhydrogen bonds,C1atomoftheirsugarneedstobeincloseposition.Thisdistortsthe duplexbackboneanddestabilizethestructure.However,severalhydrogenbonding patternswithinthepyrimidine–pyrimidinemismatcheshavebeenobserved.T⋅T mismatchadoptsbyinterconvertingtwobasepairingpatterns,bothofwhichform twohydrogenbondswithwobble-likeconfiguration(Figure2.4).C⋅Tmismatch