Raman signature of the four-stranded intercalated cytosine motif in crystal and solution structures

RamanSignatureoftheFour-StrandedIntercalatedCytosineMotifinCrystalandSolutionStructuresofDNADeoxycytidylatesd(CCCT)andd(C8)†JamesM.Benevides,‡ChulHeeKang,§andGeorgeJ.Thomas,Jr.*,‡DiVisionofCellBiologyandBiophysics,SchoolofBiologicalSciences,UniVersityofMissourisKansasCity,KansasCity,Missouri64110-2499,andDepartmentofBiochemistryandBiophysics,WashingtonStateUniVersity,Pullman,Washington99164-4660ReceiVedDecember13,1995;ReVisedManuscriptReceiVedMarch6,1996XABSTRACT:TheRamanspectralsignatureofthefour-strandedcytosinestructureformedbyintercalationoftwohemiprotonatedandparallel-strandedoligodeoxycytidylateduplexes(so-calledimotif)hasbeenobtainedfromthecrystalstructureofd(CCCT)[Kang,C.H.,Berger,I.,Lockshin,C.,Ratliff,R.,Moyzis,R.,&Rich,A.(1994)Proc.Natl.Acad.Sci.U.S.A.91,11636-11640].IdentificationofRamanmarkersdiagnosticofthecytosinequadruplexiscomplementedbyresultsobtainedinapHtitrationof2′-deoxycytosine-5′-monophosphate(5′-dCMP)toshowthatthespectralfingerprintassociatedwithN3protonationofcytosineisdistinctfromthatofquadruplexformation.TheRamanspectrumthusprovidesadefinitivebasisforevaluatingquantitativelyboththeextentofcytosinequadruplexformationandthedegreeofcytosineN3protonationinDNA.Applicationtoaqueousd(CCCT)andd(C8)demonstratesthatthefour-strandedintercalatedstructureisformedbybothoftheseoligodeoxycytidylatesinaqueoussolution.Whereasboth5′-dCMPandthed(CCCT)quadruplexexhibitamidpointoftitration(apparentpKC)of4.5(0.2at10°C,cytosineprotonationind(C8)isshiftedsignificantlytowardthephysiologicalrange,withpKC)5.8(0.2.ThedifferenceinpKCbetweenthetwoquadruplexesisequivalenttoafreeenergydifferenceof1.7kcal/molat10°C.ThepresentfindingsextendthelibraryofRamanconformationmarkerstodeoxycytidylateresiduesinthenoveliquadruplex.ThesignificanceoftheseresultsforprobingsolutionconformationsoftelomericDNAsequencesisalsoconsidered.Nucleicacidpolymorphism,whichisdependentuponbasesequenceandlocalmolecularenvironment,isanessentialstructuralpropertyofthegeneticmaterial.Recentinvestiga-tionshavefocusedonthecapabilityofgeneregulatoryproteins(Steitz,1990)andspecificcounterions(Cheng&Pettitt,1992)toaltertheglobalstructureofDNA.Thepreciseinterplaybetweenionicenvironmentandnucleicacidconformationisofparticularinterestandimportance,ashasbeenillustratedinthenovelquadruplexstructuresoftelo-mericDNAsequencesandinthevarietyoftriplexstructuresaccessibletopurine/pyrimidinesequences.RecentsurveyshavebeengivenbyWilliamson(1994),LeroyandGue´ron(1995),andFrank-KamenetskiiandMirkin(1995).Anintriguingfeatureofmanymultistrandednucleicacidsisthecapacitytoundergolargeconformationalchangeswithrelativelysmallchangesinlocalconcentrationsofprotonsormetalcations.Forexample,inthecaseofguaninequadruplexesoftelomericDNA(Sen&Gilbert,1990),switchingbetweenalternativequadruplexesinsolutioncanbeinducedbyrelativelysmallchangesinconcentrationofNa+andK+ions(Miuraetal.,1995).Similarly,nucleicacidpolymorphismcandependsensitivelyuponsolutionpH.ConformationalswitchingofDNAunderthecontrolofsolutionpHistheresultofprotonation(ordeprotonation)ofspecificbasesites,whichcanbeinferredfromtheapparentbasepKvalues.Amongthefourbases,thepKvalueclosesttoneutralityandmostlikelytobeofbiologicalsignificanceisthatofcytosine.ThepKgoverningcytosineN3proto-nation(pKC)isnominallyabout2.5unitsbelowneutralpHbutcanbeelevatedtothephysiologicalrangewhenthebaseisincorporatedwithinanucleicacidsecondarystructure(Inman,1964).WhilecytosineN3protonationeliminatesWatson-CrickpairingandthereforedestabilizestheBDNAduplex,itcanenablealternativepairingschemestocom-pensateinpartforthelossofWatson-Crickpairs.Anexampleispairingbetweenprotonatedandunprotonatedcytosinestoyieldthehemiprotonatedbasepair,(C‚C)+,depictedinFigure1A.Stabilizationofsecondarystructureby(C‚C)+pairsmaybeespeciallyimportantfornucleicacidsequencesrichincytosineresidues.Aparallel-strandedduplexstabilizedby(C‚C)+pairswasfirstproposedbyLangridgeandRich(1963)onthebasisoffiberX-raydiffractionstudiesofpoly(rC).Thesalientfeaturesofthisstructurehavebeenconfirmedbybothinfrared(Hartman&Rich,1965)andRamanspectroscopy(Chou&Thomas,1977).Othersecondarystructuremodelsincorporating(C‚C)+pairshavebeendescribedrecently(Robinsonetal.,1992;Robinson&Wang,1993;Jaishree&Wang,1993;Liuetal.,1993;Wang&Patel,1994).Amongthese,thefour-stranded(quadruplex)intercalatedstructureformedbyoligodeoxcytidylatesequencesandtermedtheimotif(Gehringetal.,1993;Leroyetal.,1993)isofparticularinterest.Intheiform,twohemiprotonated†ThisispaperLXintheseriesRamanSpectralStudiesofNucleicAcids.SupportbyNIHGrantGM54378isgratefullyacknowledged(G.J.T.).*Authortowhomcorrespondencemaybeaddressed.‡UniversityofMissourisKansasCity.§WashingtonStateUniversity.XAbstractpublishedinAdVanceACSAbstracts,April15,1996.5747Biochemistry1996,35,5747-5755