J. Am. Chem. Soc. 1997, 119, 59-69

J.Am.Chem.Soc.1997,119,59-6959

IntrusionofCounterionsintotheSpineofHydrationinthe

MinorGrooveofB-DNA:FractionalOccupancyof

ElectronegativePockets

MatthewA.Young,?B.Jayaram,?andD.L.Beveridge*,?

ContributionfromtheDepartmentofChemistryandMolecularBiophysicsProgram,Wesleyan

UniVersity,Middletown,Connecticut06459,andDepartmentofChemistry,IndianInstituteof

Technology,HauzKauz,NewDelhi110016,India

ReceiVedFebruary12,1996.ReVisedManuscriptReceiVedAugust27,1996X

Abstract:AsequenceoforderedsolventpeaksintheelectrondensitymapoftheminorgrooveregionofApT-richtractsofthedoublehelixisacharacteristicofB-formDNAwellestablishedfromcrystallography.Thisfeature,termedthe“spineofhydration”,hasbeendiscussedasastabilizingfeatureofB-DNA,thestructureofwhichisknowntobesensitivetoenvironmentaleffects.Nanosecond-rangemoleculardynamicssimulationsontheDNAduplexofsequenced(CGCGAATTCGCG)havebeencarriedout,includingexplicitconsiderationof?4000watermoleculesand22Na+counterions,andbasedonthenewAMBER4.1forcefieldwiththeparticlemeshEwaldsummationusedinthetreatmentoflong-rangeinteractions.ThecalculationssupportadynamicalmodelofB-DNAclosertotheBformthananypreviouslyreported.Analysisofthedynamicalstructureofthesolventrevealedthat,inoverhalfofthetrajectory,aNa+ionisfoundintheminorgroovelocalizedattheApTstep.Thisposition,termedhereinthe“ApTpocket”,wasnotedpreviously(Lavery,R.;Pullman,B.J.Biomol.Struct.Dyn.1985,5,1021)tobeofuniquelylownegativeelectrostaticpotentialrelativetootherpositionsofthegroove,aresultsupportedbythelocationofaNa+ioninthecrystalstructureofthedApUminiduplex[Seeman,N.;etal.J.Mol.Biol.1976,104,109)andbyadditionalcalculationsdescribedhereinbasedoncontinuumelectrostatics.TheNa+ionintheApTpocketinteractsfavorablywiththethymineO2atomonoppositestrandsoftheduplexandiswellarticulatedwiththewatermoleculeswhichconstitutetheremainderoftheminorgroovespine.ThisresultindicatesthatcounterionsmayintrudeontheminorgroovespineofhydrationonB-formDNAandsubsequentlyinfluencetheenvironmentalstructureandthermodynamicsinasequence-dependentmanner.TheobservednarrowingoftheminorgrooveintheAATTregionofthed(CGCGAATTCGCG)structuremaybeduetodirectbindingeffectsandalsotoindirectmodulationoftheelectrostaticrepulsionsthatoccurwhenacounterionresidesintheminorgroove“ATpocket”.TheideaoflocalizedcomplexationofotherwisemobilecounterionsinelectronegativepocketsinthegroovesofDNAhelicesintroducesaheretoforemostlyunappreciatedsourceofsequence-dependenteffectsonlocalconformational,helicoidal,andmorphologicalstructureandmayhaveimportantimplicationsinunderstandingthefunctionalenergeticsandspecificityoftheinteractionsofDNAandRNAwithregulatoryproteins,pharmaceuticalagents,andotherligands.

Introduction

ThestructureofDNAiswell-knowntobesensitivetosolvent

effects,1,2andtheenergeticsandthermodynamicsofligand

bindingtoDNAinvolveimportantcontributionsfromdehydra-

tionandcounterionrelease.3-5Nosingleexperimentalmethod

ormeasurementcanprovideacomprehensivedescriptionof

thisaspectofthesystem,exceptingcrystallographyintherare

casesinwhichallsolventinthesystemisordered;6thus,

theoreticalstudiesmayprovideavaluablesourceofinformation

onsolvent.Aseriesofnewnanosecond-levelmolecular

dynamics(MD)simulationsontheDNAduplexofsequence

WeslyanUniversity.IndianInstituteofTechnology.XAbstractpublishedinAdVanceACSAbstracts,December1,1996.(1)Saenger,W.PrinciplesofNucleicAcidStructure.;Springer-Verlag:NewYork,1984.(2)Westhof,E.;Beveridge,D.L.InHydrationofNucleicAcids.;Westhof,E.,Beveridge,D.L.,Eds.;CambridgeUniversityPress:Cam-bridge,England,1989;Vol.5,pp24-136.(3)Olmsted,M.C.;Bond,J.P.;Anderson,C.F.;Record,M.T.,Jr.Biophys.J1995,68,634-647.(4)Misra,V.K.;Sharp,K.A.;Friedman,R.A.;Honig,B.J.Mol.Biol1994,238,245.(5)Record,M.T.,Jr.;Anderson,C.F.;Lohman,T.M.Q.ReV.Biophys.1978,11,103-178.(6)Berman,H.M.Curr.Opin.Struct.Biol.1991,1,423-427.??d(CGCGAATTCGCG)arepresentedherein.TheMDmodelincludesexplicitconsiderationofcounterionsandwaterandisbasedonanewlydevelopedempiricalforcefield7andanimprovedtreatmentoflong-rangeinteractions.8,9Analysisofthedynamicalstructureprovidesinformativenewdetailsonthemoleculararrangementofsolvent.TheresultsarefoundtobeconsistentwithcorrespondingresultsfromMonteCarlo(MC)simulationandnonlinearPoisson-Boltzmann(PB)calculationsandsuggestapossiblereinterpretationofthecrystallographicallyorderedsolventpeaksintheminorgrooveofDNAknownasthe“spineofhydration”.BackgroundThepreferentialstabilityoftheright-handedAandBformsofDNAiseffectedbyanincreaseinwateractivityfrom76to92%inaDNAfiberandfromBtoZbyanincreaseinsaltconcentrationinsystemsofGC-richsequences.1Aphenom-(7)Cornell,W.D.;Cieplak,P.;Bayly,C.I.;Gould,I.R.;Merz,K.M.Jr.;Ferguson,D.M.;Spellmeyer,D.C.;Fox,T.;Caldwell,J.W.;Kollman,P.A.J.Am.Chem.Soc.1995,117,5179.(8)Cheatham,T.E.,III;Miller,J.L.;Fox,T.;Darden,T.A.;Kollman,P.A.J.Am.Chem.Soc.1995,117,4193.(9)York,D.M.;Darden,T.A.;Pedersen,L.G.J.Chem.Phys.1993,99,8345.

S0002-7863(96)00459-3CCC:$14.00©1997AmericanChemicalSociety

60J.Am.Chem.Soc.,Vol.119,No.1,1997

enologicalmodelforcounterionsaroundDNAwasproposedsome30yearsagobasedontheideaofcounterioncondensation(CC),5,10inwhichastablefractionofcounterions,essentiallyindependentofsaltconcentration,remainsassociatedincloseproximitywiththeDNA.ForamonovalentcounterionsaroundalinechargemodelofpolyanionicDNA,CCtheorypredicts76%oftheionstobewithin10ÅoftheDNAsurface(17Åfromtheeffectivehelicalaxis)andtheradius,i.e.,the“Manningradius”,definestheboundaryoftheCCshell.

Theprincipalsourceofexperimentaldataonsolvationatthemolecularlevelcomesfromx-raycrystallography.11TheDNAdodecamerduplexd(CGCGAATTCGCG)wasfoundtocrystal-lizeasahydrateinaBformofDNAthatcorrespondscloselytotheWatson-Crickdoublehelix,withawell-definedmajorandminorgroovestructure.12,13Thecrystallographicanalysisassumedallsolventpeakstobefull-occupancywatermolecules.Althoughthefractionofwaterthatturnedouttobecrystallo-graphicallyorderedwasonly?25%ofthetotal,aninterestingandprovocativefeaturewasnoted:asequenceoforderedsolventpeaksintheelectrondensitymapoftheminorgrooveregionofAT-richtracts.Thiselementofstructurehasbeentermedthe“spineofhydration”.14TheoriginalspineofhydrationwaspostulatedtobespecifictoATtracts,wheretheN3atomofadenineandtheO2atomofthyminearereadilyavailableashydrogenbondacceptorsininteractionswithwatermolecules.TheN2donorgrouponguaninewasthoughttodisruptthespine,14buthydrationintheCpGregionofthedodecamerwasblockedinthecrystalbyaspermineionandbyhelix-helixpackingandthuscouldnotbedirectlyobserved.TheminorgroovespinehasbeenobservedinanumberofsubsequentB-formcrystalstructuredeterminations.15,16NMRexperimentshavealsobeenreportedtodetectitspresence.17-19Theoreticalcalculationsofvaryingdegreesofrigorhaveinvestigatedthemolecularnatureofthespine20-22andotherfeaturesofDNAhydration,23andthepossibilitythatthespinemayextendintoGpCtractswasnoted.AnearlyMDstudyofasolvatedB-DNAsystemwascarriedoutonthesequenced(CGC).24SequencesrichinA-TbasepairsareknowntopreferentiallystabilizeandpossiblyrigidifytheBformofDNA,withthespineprovidingapossibleexplanation.Anumberofminorgroove-bindingdrugsarealsoknowntopreferentiallypositionthemselvesaboutA-T-richsequences.25-27NetropsinhasbeenshowntoinduceanAtoBtransitioninDNAbybindingintheminorgroove,28whichalongwiththermodynamic

(10)Manning,G.S.Q.ReV.Biophys.1978,11,179-246.

(11)Dickerson,R.E.;Drew,H.R.;Conner,B.N.;Kopka,M.L.;Pjura,P.E.ColdSpringHarborSymp.1983,47,13.

(12)Drew,H.R.;Wing,R.M.;T.Takano;C.Broka;S.Tanaka;Itikura,K.;Dickerson,R.E.Proc.Natl.Acad.Sci.U.S.A.1981,78,2179-2983.

(13)Dickerson,R.E.;Drew,H.R.J.Mol.Biol.1981,149,761-786.(14)Drew,H.R.;Dickerson,R.E.J.Mol.Biol.1981,151,535-556.(15)Berman,H.M.;Olson,W.K.;Beveridge,D.L.;Westbrook,J.;Gelbin,A.;Demeny,T.;Hseih,S.H.;Srinivasan,A.R.;Schneider,B.Biophys.J.1992,63,751-759.

(16)Schneider,B.;Cohen,D.;Berman,H.M.Biopolymers1992,32,725-750.

(17)Liepinsh,E.;Otting,G.;Wu¨thrich,K.NucleicAcidsRes.1992,20,6549-6553.

(18)Fukuzaki,M.;Umehara,T.;Kurita,D.;Shioya,S.;Haida,M.;Mashimo,S.J.Phys.Chem.1992,96,10087-10089.

(19)Kubinec,M.G.;Wemmer,D.E.J.Am.Chem.Soc.1992,114,8739-8740.

(20)Subramanian,P.S.;Swaminathan,S.;Beveridge,D.L.J.Biomol.Struct.Dyn.1990,7,1161-1165.

(21)Subramanian,P.S.;Ravishanker,G.;Beveridge,D.L.Proc.Natl.Acad.Sci.U.S.A.1988,85,1836-1840.

(22)Chuprina,V.P.NucleicAcidsRes.1987,15,293-311.

(23)Westhof,E.InStructuralWaterofNucleicAcids.;Westhof,E.,Ed.;AcademicPress:NewYork,1990;pp11-18.

(24)Forester,T.R.;McDonald,I.R.Mol.Phys.1991,72,643-660.

Youngetal.

bindingstudies29givescredencetoastabilizingpropensityfortheminorgroovespine.

CrystalstructuresofthedinucleotidesdCpGanddApUwerereportedin1976.30,31(NotethatweadoptthenotationCpG,ApU,orApTwheneverthereisanychanceofambiguitybetweenthedesignationofabasepairstepandaWatson-Crickbasepair.)Thesestructureswereofhigherresolutionthanthatdescribedaboveforthed(CGCGAATTCGCG)dodecamer,andanumberoforderedsolventpeakscouldbeassigned.IndApU,aNa+ionwasfoundtoresideintheprotominorgrooveregion,whereastheNa+http://wendang.chazidian.comveryandPullman32carriedoutcalculationsoftheelectrostaticpotentialofthegrooveregionsofDNAandfoundtheelecrostaticpotentialintheminorgroovetobemarkedlyelectronegativeinthevicinityofATsteps.Thus,experimentalandtheoreticalresultscollectivelyraisedthepossibilityoftheresidenceofcationsintheminorgroovespineinB-DNA.Inacrystalstructurerefinementsuchasthatofd(CGCGAATTCGCG)(?2.3Å),itisunderstandablydifficulttoreliablydistinguishbetweenelectrondensitypeaksduetowaterandthosearisingfrommonovalentcations.

TheoreticalandcomputationalstudiesoftheionatmosphereofnucleicacidsincludecontributionsfromCCtheoryasdescribedabove,10continuumelectrostaticsinwhichtheenvi-ronmentismodeledasaheterogeneousdielectriccontinuum,33andMDcomputersimulation34inwhichthesolventmoleculesmaybeconsideredexplicitly.Duetothehighdimensionalityoftheproblem,MDstudiesarecomputationallyintensive,requiringseveralhundredhoursof(CRAYC90)supercomputertimeforeachnanosecondoftrajectory.MCsimulation35andPBcalculations36areusefulforthestudyoftheionatmosphereinmodelsinwhichtheDNAisheldfixed.

WehaverecentlyinvestigatedfurtherthedynamicalstructureofDNAandparticularlytheminorgroovesolvationusingimprovedlevelsoftheoreticalandcomputationalmethodology.37TheoreticalstudiesofDNAinanaqueoussolutionenvironmentarecomplicatedbythehighlychargednatureofthesystemandtheconcomitantimportanceoflong-rangeinteractions,theinherentflexibilityoftheDNAmolecule,andthedifficultiesofachievingnumericalstabilizationandreliabilityinasimula-tiononacomplexmixedsolventsystemwithincurrentlyrealizablerunlengths.38,39ArecentproposedimprovementintheforcefieldforMDsimulation7andtheapplicationofthe

(25)Kopka,M.L.;Pjura,P.;Yoon,C.;D.Goodsell;Dickerson,R.E.InStructureandMotion:Membranes,NucleicAcidsandProteins;Clementi,E.,Coronsiu,G.,Sarma,M.H.,Sarma,R.H.,Eds.;AdeninePress:Guilderland,NY,1985;pp461ff.

(26)Kopka,M.L.;Yoon,C.;Goodsell,D.;Pjura,P.;Dickerson,R.E.Proc.Natl.Acad.Sci.U.S.A.1985,82,1376-1380.

(27)Pjura,P.E.;Grzeskowiak,K.;Dickerson,R.E.J.Mol.Biol.1987,197,257-271.

(28)Ivanov,V.I.;Minchenkova,L.E.;Minyat,E.E.;Frank-Kamentskii,M.D.;Schyolkina,A.K.J.Mol.Biol.1974,87,817.

(29)Marky,L.A.;Blumenfeld,K.S.;Breslauer,K.J.NucleicAcidsRes.1983,11,2857-2870.

(30)Seeman,N.C.;Rosenberg,J.M.;Suddath,F.L.;Kim,J.J.P.;Rich,A.,J.Mol..Biol.1976,104,109-144.

(31)Rosenberg,J.M.;Seeman,N.C.;Day,R.O.;Rich,A.J.Mol.Biol.1976,104,145-167.

(32)Lavery,R.;Pullman,B.J.Biomol.Struct.Dyn.1985,5,1021-1032.

(33)Sharp,K.A.;Honig,B.Curr.Opin.Struct.Biol.1995,5,323-328.

(34)Jayaram,B.;Beveridge,D.L.Annu.ReV.Biophys.Biomol.Struct.1996,25,367-94.

(35)Jayaram,B.;Swaminathan,S.;Beveridge,D.L.Macromolecules1990,23,3156-3165.

(36)Jayaram,B.;Sharp,K.A.;Honig,B.Biopolymers1989,28,975-993.

(37)Young,M.A.;Ravishanker,G.;Beveridge,D.L.Biophys.J.,submitted.

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MinorGrooVeSpineofHydrationofB-DNAJ.Am.Chem.Soc.,Vol.119,No.1,199761Figure1.CalculatedNa+counteriondensityaroundacanonicalBformofthed(CGCGAATTCGCG)duplex,basedonasuperpositionof400ionconfigurationsobtainedfromaCIMCsimulationontheDNAand22Na+ionsinadielectriccontinuum.Detailsofthemethodaredescribedinref36.

62J.Am.Chem.Soc.,Vol.119,No.1,1997Figure2.CalculatedDNA-Na+radialdistributionfunctiong(R)(histograms)andrunningcoordinationfractionNC(R)(solidline)fromtheCIMCsimulation-generatedconfigurationsinFigure1.Thereportedg(R)isthevolume-normalizeddensityofcounterionscountedinconcentriccylindricalshellsofradius“R”measuredfromthecenteroftheDNAhelicalaxis.TheNC(R)ionfractionisnormalizedrelativetothetotalnumberof22ionconfigurationssampledandisnotvolume-

normalized.

particlemeshEwald(PME)summationmethodtothelong-rangeinteractionproblem40haveresultedinmorereliableMDsimulationsonDNA.8,37ThenewMDtrajectoriesdescribedinthisarticlearebasedonthisprotocolandprovideabasisforsomenewpredictionsanddeeperinsightintoDNAsolvation.MCandPBcalculations,carriedoutinitiallytoprovideadditionalinformationontheelectrostaticpotentialandstartingstructuresforsubsequentMDs,provedtoeachcontributeusefuladditionalperspectivesonthesolvation,asdescribedbelow.MonteCarloSimulations.(a)Calculations.MCcalcula-tionsoftheionatmosphereaboutacanonicalBformofDNAphosphateswithwatertreatedasadielectriccontinuumweredescribedpreviously.36,41Inthecurrentproject,weextendedthiscomputationalmodeltothetreatmentofall-atomDNAasdescribedbythechargesandvanderWaalsinthenewAMBERforcefield,7andcarriedoutanMCMetropolissimulationon22mobileNa+counterionsaroundafixedcanonicalformofthed(CGCGAATTCGCG)duplex.TheeffectofsolventwateronDNA-Na+andNa+-Na+interactionswasmodeledwithasigmoidaldielectricscreeningfunction.42ThecalculationsemployedBorn-vonKarmannmass-conservingperiodicitywithinacylindricalcellofheight70Åandaradiusof32Å.(b)Results.TheMCcalculatedcounteriondensityaroundthed(CGCGAATTCGCG)DNAhelixisshowninFigure1.Thisresultprovidesgraphic,independentsupportofthegeneralideaofCCasanticipatedalmost20yearsagobyManning.10Furtheranalysisofthecounteriondistributionrevealssignificantnewdetails.TheDNA-Na+radialdistributionfunction(RDF)g(R)fromtheMC-generatedensembleandthecorrespondingrunningcoordinationnumberNC(R)areplottedinFigure2.The

(38)Beveridge,D.L.;Swaminathan,S.;Ravishanker,G.;Withka,J.M.;Srinivasan,J.;Prevost,C.;Loiuse-May,S.;Langley,D.R.;Dicapua,F.M.;Bolton,P.H.MolecularDynamicsSimulationsontheHydration,StructureandMotionsofDNAOligomers.InWaterandBiologicalMacromolecules;Beveridge,D.L.,Swaminathan,S.,Ravishanker,G.,Withka,J.M.,Srinivasan,J.,Prevost,C.,Loiuse-May,S.,Langley,D.R.,Dicapua,F.M.,Bolton,P.H.,Eds.;1993.

(39)Beveridge,D.L.;Ravishanker,G.Curr.Opin.Struct.Biol.1994,4,246-255.

(40)York,D.M.;Yang,W.;Lee,H.;Darden,T.;Pedersen,L.G.J.Am.Chem.Soc.1995,117,5001.

(41)Jayaram,B.;Swaminathan,S.;Beveridge,D.L.;Sharp,K.;Honig,B.J.Phys.Chem.1990,23,3156-3165.

(42)Hingerty,B.E.;Ritchie,R.H.;Ferrel,T.L.;Turner,J.E.Biopolymers1985,24,427-439.

Youngetal.

Figure3.CalculatedDNA-Na+g(R)partitionedalongtheDNAhelicalaxisinto3.4Åtallregionsindicativeofsinglebasepairstepsinordertodisplaysequenceeffects.ThedataintheindividualpanelsofFigure3sumstothetotalg(R)aspresentedinFigure2.

MCRDFshowstwoinflectionpoints,oneat6.5Åandanotherat14.9ÅfromtheaxisoftheDNA.Themajorinflectionpointat14.9ÅdefinestheManningradiusforthismodel.Some82%ofthecounterionsarefoundtobecondensedwithinthisradius,ascomparedwithManning’svalueof76%at17ÅbasedonalinechargeofDNA.AnalysisoftheinflectionpointintheMCat5.5Å,whichisfollowedbyasmallplateauregionextendingto?9Å,showsthatitisproducedbycounterionspositionedwithinthemajorandminorgrooveregionsofthedoublehelix.Beyondthispoint,thedistributioncorrespondstocounterionsdistributedalongthebackboneoftheDNAandbeyond.

ToelucidatesequenceeffectsintheDNA-Na+distribution,theMCRDFwasdecomposedintocontributionscorrespondingtoeachbasepair.Figure3showstheresultingsetofMC-calculatedDNA-Na+RDFs,partitionedinto3.4Åhighcylindersalongthelongaxisofthehelix.Noteparticularlythatthegrooveregion(?4.5-6.5Å)ofthecentralApApTpTtractshowsanotablecounteriondensity.IntheflankingsequencessomedensityisindicatedattheGpCstepsandvirtuallynoneattheCpGsteps.Proximityanalysis43showsthisdensitytobeconcentratedintheminorgroove,consistentwithresultsreportedfromotherlaboratories.44ThelowestenergycounterionconfigurationidentifiedfromtheMCen-sembleofstructuresshowsoneNa+ionlocatedattheApTstepoftheminorgrooveoftheCGCGAATTCGCGsequence,inaconditionofprimarycoordinationwiththecarbonylgroupsofthesuccessivethyminesonoppositestrandsoftheduplex.Thisposition,notedabovetobeanelectronegativeregion,isreferredtohenceforthasthe“ApTpocket”.

ContinuumElectrostatics.(a)Calculations.ThegeneralcharacteristicsofnonlinearPoisson-BoltzmanncalculationsonDNAweredescribedpreviouslybyJayaramandHonig.36Toinvestigatesequenceeffects,nonlinearPBcalculationswere

(43)Mehrotra,P.K.;Beveridge,D.L.J.Am.Chem.Soc.1980,102,4287-4294.

(44)Lamm,G.;Wong,L.;Pack,G.R.Biopolymers1994,34,227-

237.

MinorGrooVeSpineofHydrationofB-DNAJ.Am.Chem.Soc.,Vol.119,No.1,199763

Figure4.Calculatedelectrostaticpotentialofthed(CGCGAATTCGCG)duplexbasedonnonlinearPoisson-BoltzmanncalculationsperformedusingtheDELPHIprogram.Showninredisaplotofthe-5kTisopotentialsurfacewiththeDNAviewedintotheminorgroove(left)andthemajorgroove(right).

carriedoutonthed(CGCGAATTCGCG)duplexusingtheCornelletal.chargesandvanderWaalsparameters.7Solventwaterwasassigneddielectricconstantof80,andthedielectricwassetattwowithintheDNA.Achargegridof1Åresolutionwasconstructedwithinaboxofdimensions65by45by45Å.NonlinearPoisson-BoltzmanncalculationswereperformedusingtheprogramDelphi45,46ontheDNA-counterionsystem,atasaltconcentrationsufficienttoprovideelectricalneutrality,0.145M.Resultsfromthesecalculationscanbesensitivetotheassumedgeometry.

(b)Results.ThePBcalculatedelectrostaticpotentialfortheEcoRIdodecamerisshowninFigure4.ThecentralAATTregionoftheminorgrooveofthemolecule(Figure4a)wasfoundtoexhibitalargenegativepotentialextendingalongthefloorofthegroove.ThesurroundingCpGstepsintheflankingsequenceshowsomeevidenceofelectronegativepatches;however,thesepatchesaremorelocalizedatthebasepairsteplevelthanwasseenfortheAATTregion.Theelectrostaticpotentialofthemajorgroove(Figure4b)showsonlysmall,localizedelectronegativepatchesintheCGregion.TheresultsindicatethattheminorgrooveoftheAATTregionisfavorableforoccupationbycounterionsandareconsistentwiththeresultsdescribedabovefromtheMCcalculations.Together,theMCandPBresultsraisethedistinctpossibilitythatNa+counterionscouldbefavorablysituatedintheAATTregionoftheminorgrooveofB-formDNA.Furtherstudiesbasedonmoleculardynamicssimulationweresubsequentlycarriedouttofurtherexplorethisissue.

MolecularDynamicsSimulations.(a)Calculations.AseriesofMDsimulationswereperformedontheDNAoligo-nucleotided(CGCGAATTCGCG)duplexwith22anionicphosphategroupsusingtheAMBER4.1suiteofprograms.47EachstrandwasterminatedwithanOHgroup.Thesolvent

(45)Gilson,M.K.;Sharp,K.A.;Honig,http://wendang.chazidian.comput.Chem.1988,9,327-335.

(46)Sharp,K.,Honig,B.,Eds.DelPhi;2.3ed.;BiosymTechnologiesInc.:SanDiego,CA,1992.

(47)Pearlman,D.A.,Case,D.A.,Caldwell,J.W.,Ross,W.S.,Cheatham,T.E.,III,Fergusen,D.M.,Seibel,G.L.,Singh,U.C.,Weiner,P.,Kollman,P.,Eds.AMBER4.1;4.1ed.;UCSF:SanFrancisco,CA,1995.

wascomposedofawaterbathcontaining22mobileNa+counterions.AllcalculationsemployedtheCornelletal.forcefield7fortheDNAandNa+andtheTIP3Pmodelforwater48,49asincorporatedintoAMBER4.1.Aseriesoffourindependentsimulationswerecarriedouttoevaluatesensitivityofthecurrentprotocoltoinitialstartingconditionsandtoassesstrajectorystabilityandconvergence.AllfoursimulationsbeginwithacanonicalBformoftheDNAderivedfromfiberdiffraction.50Thefirstthreedifferonlyintheinitialplacementofthe22Na+ions,asshowninFigure5.TheplacementforthefirstMD,denoted“MCI”,(Figure5a)isthelowestenergyconfigurationofthe22Na+ionsaboutafixedDNAasobtainedfromtheMCcalculationdescribedabove,usingamodificationofthe“MCIONS”program35andhenceforthreferredtoasMCI.ThesecondMDwasinitiatedfromtheplacement“ESI”(Figure5b)obtainedfromtheAMBERCIONprogram.47TheCIONprogramiterativelypositionseachoftheNa+ionsabouttheDNAatthepointofnextlowestelectrostaticpotential.NotetheMCIONScalculationresultsinanNa+ionresidingattheApTstepoftheminorgroove,whereasCIONdoesnot.ThereisalsoanionresidingataGCstepofthe5′flankingsequenceintheMCIstructure.Weobservedthatthelowestenergyconfigurationwithallparticleinteractionstakenintoconsider-ation(asinMCIONS)doesnotcoincidewiththatofiterativesequentialplacementoftheions(i.e.,CION).Theionplace-mentforathirdsimulation,denoted“BSI”,wasconstructedbysimplyplacingtheions6ÅawayfromthephosphorusatomsalongtheO-P-Obisectorofeachphosphategroup.ThebisectorconfigurationalsoservedasthestartingconfigurationforafourthMDinwhichtheDNAatomswererestrainedtotheircanonicalstartingpositionsandonlytheionsandwatermoleculeswereallowedtomove.ThislatterMDcalculationprovidesforadirectcomparisonwiththeMCandPBcalcula-tionsaboutthefixedDNAstructure.

(48)Jorgensen,W.L.J.Am.Chem.Soc.1981,103,341-345.

(49)Jorgensen,W.L.;Chandrasekhar,J.;Madura,J.D.;Impey,R.W.;Klein,M.L.J.Chem.Phys.1983,79,926-936.

(50)Arnott,S.,Campbell-Smith,P.J.,Chandrasekaran,R.,Eds.AtomicCoordinatesandMolecularConformationsforDNA-DNA,RNA-RNA,andDNA-RNAHelices;CRCPress:Cleveland,OH,1976;Vol.2,pp411-

422.