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Study_8

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    Study

300ChineseChemicalLettersVo1.14,No.3,PP300303,2003

    ;http:Hwww.imm.ac.coumal/cc1.html

    ;StudyOUTriplexDNAbyUseofMolecular”LightSwitch”Complex

    ;ofRu(phen)2(dppx)’.

    ;LianShengLING,ZhiKeHE,FangCHEN,HuaShahZHANG,YunEZENG

    ;DepartmentofChemistry,WuhanUniversity,Wuhan430072

    ;InstituteofChemistry,theChineseAcademyofSciences,Beijing100080 ;Abstract:AnewmethodforthestudyoftriplexDNAisestablishedaccordingthefluorescence ;enhancementofmolecular’’LightSwitch’’complexofRu(phenh(dppx)2+whenitintercalateinto

    ;triplexDNA.B@causethefluorescenceintensityofRu(phen)2(dppx)2+bondedtotriplexDNAis ;inthecasehigherthanthatbondedtoduplexDNAincenainrangeofDNAconcentration,the ;methodismuchmoresensitivethanothermetho~reportedpreviously. ;Keywords:Ru(phen)2(dppx)2+,molecular”LightSwitch”,triplexDNA.

    ;StudiesonthestructuraldiversityOfDNAhavedrawnmuchattentioninrecentyears. ;Felsenfeldeta1.firstproposedthestructureoftriple-helixRNAandsuccessfully ;synthesI’ZedakindoftriplexRNA.Mirkinandcoworkers2discoveredatriplexDNA

    ;(I-I-DNA)inanacidsolutionofplasmid.Inthesameycar,Dervaneta1.’attacheda

    ;man-madesingle.strandedTAtorealgeneDNAandlocallyformedakindof ;interstrandtriplex.Tl1iScouldbeusedtoinciseduplexDNAinaprecisellq~nnel”)

    ;showinghighpotentialforapplicatiOIlSasagenecontroller,man-maderestriction ;endonuclease.andpreventingDNAfromcombiningwithproteineta1.Researchon ;three.strandedDNAisbecominganattractivefieldinbiochemistryandmolecular ;biology.Itisurgenttoestablishanefficientandsensitivemethodtostudyontriplex ;DNA.Baia1.4’’developedamethodaccordingtotheincreasingfluorescenceofEB

    ;whenbondedtotriplexDNA.Butthismethodwaslimitedbecausethefluorescenceof ;EBbondedtoduplexDNAishigherthanbondedtotriplexDNA.Molecular”Light

    ;Switch”isakindOfcomplexthatdoesnotluminescenceinwaterbutemitsin

    ;nonaqueoussolventsorinthepresenceOfDNA.InOurpreviousstudy,’’LightSwitch’’

    ;complexofRu(phen)2(dppx)(phen=l,10-phenanthroline,dppx=7,8-dimethy- ;ldipyrido3,2.a:2,3-(3phenanthroline)hasbeenusedtothedeterminationofduplex ;DN.Herehaveestablishedanewfluorescencemethodtostudythetriplex ;constitutedbyd(A)12andd(T)12byuseofRu(phen)2(dppx).Comparedwiththe ;methodofEB.thismethodhastwoadvantages:highersensitivityandthefluorescence ;enhancementofRu(pben)2(dppx)bondedtotriplexDNAishigherthanthatbondedto ;

    ;LianShengLINGeta1.

    ;duplexDNAincertainrangeofDNAconcentration.

    ;Expedmental

    ;301

    ;Doubledistilledwaterwasusedtoprepareallsolutions.Unlessstated,allthe ;chemicalswereofanalytical-reagentgradeorbetter.Oligodeoxyrinucleotides ;AAAAAAAAAAAA(d(A)l2),TTTTTTTTTTTT(d(T)l2),were

    ;GIBCOBRLCo.Themolarextinctioncoecientsare

    ;l0L?M’?cm-,(d(T)l2)=1.116X10L-M”l-cm”,respectively.

    ;purchasedfromthe

    ;(d(A)l2)=1.830×

    ;Concentrationsw~l’e

    ;calculatedaccordingtotheirextinctioncoefficients.Ru(phen)2(dppx)(BF4)2’3H20was

    ;homemadeaccordingtothereference.whichwasdescribedpreviously.Thestock ;solutionofRu(phen)2(dppx)z(1.0×10mol’L)waspreparedbydissolving9.4nag

    ;Ru(phen)2(dppx)(BF4)2?3H20in100mLwater.ThebuffersolutionofpH=7.0was ;controlledby0.2tool?Tris.HClbuffer.

    ;111efluorescenceintensitywasmeasuredwithaShimadzuRF.5301

    ;spectrofluorometerwithaquartzcell(1cmXlcmcross?section)equippedwithaxenon ;lampanddualmonochrometer.ThepHwasmeasuredwithaModelpHB_4pHmeter ;(ShanghaiLeiciEquipmentFactory,China).Samplescontainingappropriate ;concentrationsofRu(phen)2(dppx)andrelatedoligonucleotidesweremadeupto10mL ;in0.02mol?LTris.HClbuffersolution(pH=7.0.50mmol?LNaC1).111e

    ;fluorescenceintensitywasmeasuredwiththefollowingsettingsofspectrofluorometer: ;excitationwavelength(k:x),448nm;excitationslit(EX),10rim;emission ;wavelength(Zzm),604nm;Emissionslit(EM),10ntr1.

    ;ResuitsandDiscussion

    ;Oligodeoxyrinucleotidesd(A)12andd(T)12wereusedtoformduplexDNA((dA-dT),2) ;andtriplexDNA(dTdT)12,where*denotestheHoogsteenbasepairingandthedash ;denotestheWatson-Crickbasepairing).Thetitrationofd(A)12withd(T)12inthe ;presenceofRu(phen)2(dppx)isshowninFigure1.Thefluorescenceintensity ;increasedwiththeincreasingconcentrationofT)l2.andthefluorescenceintensitywas ;linearwiththeconcentrationofd(T)12untiltheratioofd(T)12:d(A)12reachedl:1. ;regressionequationisI=6.58+2.035x109C(.9994),accompaniedwiththeformation ;ofduplexDNA((dA-dT)12).111efluorescenceintensityincreasedcontinuouslywith ;increaseofd(T)12beforetheratioofd(T)12:d(k)12reached2:l,thenewlinearregression ;equationisI=136.07+1.157×10C=0.9970),whichshowedthetransformationof ;duplexDNA--->triplexDNA(dTdk-dT)12).However,thetitrationofd(T)12with ;d(A)12inthepresenceofRu(phen)2(dppx)2+showednotransformationprocigure2), ;andthetriplexDNAwasformeddirectly.Inthetransformationprocessofduplex ;DNA((dA-dT)12)triplexDNA(dTdk-dT)12),theHoogsteend(T)12thirdstrandliesin ;themajorgrooveofthetemplateduplexDNA,runningparalleltothed(A)12strand. ;Hence,thethirdstrandchangedthebindingmodeormayinhibitthebindingofmajor ;grooveinteractivedrugs”.Thefluorescenceenhancementinthisprocessshowsthat

    ;Ru(phen)~(dppx)mayintercalateinthemajorgrooveofDNA,whichisinaccordance ;

    ;302StudyonTriplexDNAbyUseofMolecular”LightSwitch”Complexof

    ;Ru(phen)(dppx)

;withtheresultsreportedpreviously.ItalsosuggeststhatRu(phen)2(dppx)notonly

    ;intercalatesintheWatson?Crickbasepair,butalsointercalatesinHoogsteenbasepair.

    ;Figure1Fluorescencetitrationcurveof

    ;d(A)l2(1.52×10-mol?L’)withd(T)l2inthe

    ;presenceofRu(phen)2(dppx)(1.0×10

    ;molL’1.NaCl:50mmol?L’

    ;g

    ;?n

    ;g

    ;gn

    ;g

    ;g

    ;o

    ;l

    ;04812162O242632

    ;Concentrationofd(T)12(10-.mo1.L)

    ;Figure2Fluorescencetitrationcurveof

    ;d(T)12(2.3×10”mol?L’)withd(A)l2inthe

    ;presenceofRu(phen)~(dppx)f1.0×10.6

    ;molL’1.NaCl:50mmol?L’

    ;02468101214

    ;Concentrationofd(A)12(10-.mo1.L’)

    ;Figure3ThebindingratiosofRu(phen)2(dppx)bindingwithtriplexDNA(triangle)

    ;andduplexDNA(circle)

    ;

    ;C

    ;

    ;D

    ;0

    ;C

    ;0

    ;2

    ;0

    ;j

    ;c

    ;D

    ;ConcentrationofA.TorTA’T(~10mol11

    ;Ru(phen)2(dppx):1.0×lO-6molL,NaCl:50mmolL’

    ;Basepairs/Ru(phen)2(dppx).0.Basetriplets/Ru(phen)2(dppx)2+~--~2.0

    ;Toinvestigatethereasonofthefluorescenceenhancementinthetransformation

    ;processofduplexDNA((dA?dT)12)lexDNA(dT’dA-dT)12),thebindingratioof ;tripletverseRu(phen)2(dppx)2’andbasepairverseRu(phen)2(dppx)2Wasstudied.Itis

    ;demonstratedinFigure3thatthebindingratioofbasepairverseRu(phen)2(dppx)2+is

    ;about4.0,whilethatoftripletverseRu(phen)2(dppx)2iSabout2.0,whichmeansthatthe

    ;bindingsiteincreasedintheduplexDNA((dA-dT)l2)triplexDNA(dT*dA-d1r)l2) ;gt,00n00g0

    ;lsIloHoQIloQ?o|0f1oII

    ;IspLloQLl’so-Iof1

    ;

    ;LianShengLINGeta1.303

    ;transformationprocess.AnditsuggeststhattheminorgrooveofHoogsteenbasepair ;providesnewbindingsitesforRu(phen)2(dppx)2whichisconsistentw. ;iththe

    ;fluorescenceenhancementinthistransformationprocess.TheeffectofFe(CN)~on ;thefluorescenceintensityofRu(phen)2(dppx)2triplexDNAindicatesthatRu

    ;complexindeedbindswiththetriplexDNA.

    ;Inconclusion,whenRu(phen)2(dppx)x+bindstotriplexDNA,itnotonly ;intercalatesintheWatson-Crickbasepair,butalsointercdIatesintheHoogsteenbase ;pair.Thus,thefluorescenceintensityofRu(phen)2(dppx)2bondedtotriplexDNAis ;hi~erthanbondedtoduplexDNAincertainrangeofDNAconcentration. ;Ru(phen)2(dppx)isbotterforthestudyoftriplexDNA.

    ;TheauthorsacknowledgethesupportofAlexanderyonHumboldtFoundation,CASK.C.Wong ;post-doctoralResearchaw~’dfundandNaturalScienceFoundationofChinaforpostdoctoral

    ;fellowship,EducationalMinistryofChina,theNationalNaturalScienceFoundationof ;China(29605001),WuhanUniversityanditsAlunmiAssociation.

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    ;3.H.E.Moser,P.B.O~ven,Science,1987,23&645.

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    ;7.L.S.Ling,Z.ICHe,G.WSong,YE.C.Wang,C.L.Bai,X.D.Chen,Shen,Ana1. ;.cta.,21,207.

    ;8.ELin~ln,ABroo,B.Norddn,AmChemSoc.,I,118,2644.

    ;9.M.Arkin,E.D.Stomp,E.Holmin,J.Barton,A.H6rmann,E.J.C.Olson,P.E. ;Barbara,Science,1996,273,475.

    ;10.C.Murphy,M.Arkin,YJenkin,N.D.Gema,H.Bossmann,N.J.Turro,J.K.Barton, ;Science,199l3.2,1025.

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