Volume 14 Number 13 1986
Nucleic Acids Research
Tlennodynamic behaviour of the heptadecadeoxynucleotide d(CGCGCGTlTTCGCGCG) forming B and Z hairpins in aqueous solution L.E.Xodo, G.Manzini, F.Quadrifoglio, G.A.van der Marell and J.H.van Booml
Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, I-34127, Italy and 1Gorlaeus Laboratory, State University of Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands Received 12 May 1986; Accepted 30 May 1986
ABSTRACT
UV and CD data of the parUially self-complmentary hoptadecadeoxynuclotide d(C6CGCGTTTTTC6C6C6), obtained as a (mUon of temperature, salt and strand concentraUon, show that: a) at low NaCI and strand concentraUon Ut oligomw exhibits, on Increing the te rture, a biphasic thml profile which is indicatve of two structural translUons, from dimeric duplex to hairpin and from hairpin to coll; b) the loop stabilizes enthtlpically both B and Z hairpin structue with respect to the onding unconsrind hexamr d(C6C6) by a few Kcal/mol; c) the oligomer undergs a B-Z translUon which appas to be complete, at 0. when induced by NaCi04; by contrat the B-Z traition induced by NaCi does not reh completness vn at salt saturation. The independence of the denaturation temperatue. at high salt conditions, on the oligomer concentration indicates that the Z structur is present also iUnthe hirpin.
INTRODCTmN Oer th pat few years, as the solid-pha synthesis of DNA fragments becam a common practce In mony laboatories (1-4). a get number of studies on the conformation and dynamics of synthetic oligodeoxynucleoUdes hae ben carried out(5-13). Short saqune with the ability to fold back Into hairpin structures hm recenUy received coisiderable attention (14-21). Hairpins ae important s y structures bcuse they represent common retu in both t-NAs and r-As. Moreover it has been demtrated that inverted repet sequnces in superhelical DNA can extrude into cruciform structures (22,23) which seem to be implicated at both traption and replication 1le (24). As part of our invUgation of the conformational dymics of altenatng purine/pyrimidine oligodoxynucleotides, in parUcular with respect to the B-Z transiUon (25-27), we hae recenUy studied a rew sequences which can form hairpin structues chacterizd by a Ioop of unpired nuceotide ridue. In this contribuon we report
thrynmic behaviour in aous solution s well a the ability of the hairpin stem to assue left-h ed conformion in high salt conditions of the hep -caox leoUde d(C6C6C6TTTTTC6CGC6).
a
MATERIALS AM) IMETHODS CG ) was synUeized following a modified solid phe phoaphotriestwr method (1-4). d(CGC6C6T The purity of tw oligmer was checked by polyacrilmide-gel electrophoris (28). The gel (20xl5x0.l cm) contained 20% acylamide, 711 ure, 5OmM TrisiHCI (pH5.3), 1mM EDTA and 0.07% anmonium prulfato. Electrophrsis was carried out at room tempeatur and at constant voltage of 600 V. 30 V/cm. Bromophenol blue dye wa usd a a mwaker. Eltrophor b s wer stained plunging th gel in a solution of 0 I RL Press Umited, Oxford, England.
5389
Nucleic Acids Research stains-all dye dissolved in formaide-water (1:1). The oligomer exhibited clerly a single bod confirming its purity. r profl me aed t 270 nm w recorded using a Absorbnce spectra and adorbae versus te Cary 219 Spectrophotomwter ewpped with a trostatad cuvette holder and a Heae F3 Uthrmstst provded at with a teprt programmer Heske PS 10. Malting ars were obtained by incraing the taI the rate of 0.3 C/min. The oligomer concentration rge from 6 to 36 p1 in single strands (assung an rage extinction coeMcient per bm of 7000 M1cm-1). All the solutions at diffrnt ionic streg contained equal concentrations of TrHsCl (pH7.4) ad NeO or NaC104. Samples wW dgegssed immedialy bfore use by stirring Ut solutions under vecuum. CD spectra wore obtained using a Jesco J500A dhroh equipped with a thermostatad coll holer. The didrograph was connected with a Jasco DP-501 N dt procssor.
QESULTS AND DISCUSSION TermllY induced t ansite Fig.I shows a typical U mal profil obtined recording the variation of the oligomer absorbance at 270 nm with temperatur in 0.1 M NaCI, 0.1 M TrisHCI and at strand concentration of 36 p11. AltUw it may not apper immeiately evident, a biphasic therml profie is gerally prnt, since a major sitso ce a variation at higher te is preceded by a smller but uequvoal one at lower teeraes. This the feature Is best noticed In corresponding derva curves, which ar reported in Fig. 2 for sevral diffrnt oligomer concentraions. These two obsorbace dchnes ar indicaive of two themlly induced trnsitions: the first (from now on roferd to a trasition IA) occurring at lowr te.pratur, is chaactized by a small hyperchromic variaton nd Its semitranslUon tempeature (Tim) incrases with the A
0Q40
S~~~~4
w w s m goi ou
i TrisJC 0.1 1, profil for d(C6CCTTT InTC6C 606) Fig. 1) Abo (at 270 rnm) veus NaCi 0.1IM (pH=7.3) and at strand concentration of 36 jiM. 5390
Nucleic Acids Research oligornr concntration; th scond (from now on rofered to as trnsition 11) is chactrizd by a higer hyperchromic chnp ad its TII'm is indepenet on th oligoe concentration. The revribility of th nwlting process has ban choed recording a few theral profles on cooling the sample at a rat of 0.3/min; trnsition 11 apprd fully rversible ad trition I exhibited a very slidt hysteesis indicating a minor dviation from quilibrium In the proce. The theral behvour ha ben exlored also at diffrnt NoCI
ncncetrons. In the rag 0.01-0.3 tl. The TI,m Increass with tlh Ionic strengt as shown In Fig. 3, becoming hiher than 90C ad thorefore diMcult to dect at NaCI concration higher thn 0.3 M. Tration I is clerly reolved at NeCI concentrations higter thn 0.05 Ml. ad its Tinm s with ionic stngh. Thes edences, In nlogy with simHlw ones described In the recent literatr (14.17). suggest tho following considerdions: a) transiUon 1. being dependnt on the oligomer contraUon, Involes an interstrand process, wheas trnition 11. which is concentraUon Indepndent, rlcts M intra d procs; b) the UV spectrun at 95*C (aftr trastion 11 hd occUrrd) Is clerly Indicat of ut single strandd coil (being almost coincident with the spectrun obtained with the stoichiometric mixtur of the consttuent nmonmersXdte not shown);
-
0
w
5
Tt
Fig. 2) DifferenUal melUng curves of d(C6C6C6TTTTTC6COC6)inTrJs.HCI o.lMNCl o.1M (pH=7.3) four diffrnt stndrconcen-stions: a) 36 j M; b) 26j M; c)16 ja M; d) 6 Ji M . 5391
Nucleic Acids Research
Fig.3) Plot of the emitrnition trnperaure concentraUon of 16 JIM.
Tl.mveru
ln(Ns+) ror d(CGCGCGTTTTTCGCGC6) it sand
with salt concentraUon suggests tht In both ca the c) the Incres of both trnlUon tempra a deity tha the pt tr ition one (29.30). hs hiwer dcr pretrnition struct Thes considerations lad to the IdenificaUon of transion I with that from a duplex structure 02) to a hairpin structur (H). nd of trnsition 11 with the dentuaUon of the hairpin to th coil state (C) (sdche 1):
(D2)
s
~1'
C Sdhm I
5392
(C)
Nucleic Acids Research
Fig.4)Plotofthe reiprocal semitransltion temperature Tg,m vsus In Cp ford(Ce(GC611T1-Tc6CGC) in Tris.HCl 0.1 M. NaCI 0.1 M (pH=7.3). ic Analysis On the bais of the eqilibris illustratedin Schem 1. a thermodynamic nalysis hs been performed in order to esUmfie Uw enthalpic and entropic dcages Invoved In both trslUons. Wlth regard to translUon 11 the m itude of tw hyperchromic effect has allowed a vanl Hoff alysis of the denotation profile on the assumpUon tht Uw rato between Ut coil and hadrpin concentraUons, which is in turn the uilibriwn constnt Kil (C)I(H) , can be suMcenUy pproximted by the quantity (A-AH)/(A C-A) being A the efrecve aso e AH ad AC the rbso es of the hairpin nd of Uw coil form. rspecUvely, at ach gin temperatue in the rag of the transiiUon. The thermodyamic parmeters hae ben obtained by vant Hoff
Therm e
plots ( In K11 versus l/T). Liner plots he been obtained. The men values of the enthalpy an entropy can for transitiUon ni determined on the basis of the denatur on curves at sevral oligome concentrations ae reported in Table 1. In th case of transition I a similar trment of th data has not been prformd, since the hyperchromlc effect invod is too swail. Hower, an estime of the enthipy chag hs ben obtained throughthe anlysisthO dependence of Tlm on Ut oligomr concentration (14). Fig. 4 shows the plot of In Cp versus 1I/Tf an related thermodynrMic parmters arereperted In Table 1. riments cloely show a greater A numr of corniderations can be made about trnsiton 11. The melting theml stability of the hairpin structured heptadeWmr with rept to that of the corr ing duplex of d(CGCGCG) whos dearatson tem ture is concentraUon dependnt but always lower than tht of Uw Faptadecame(31). We ha tried to assess if this stabilization ha an entlpic contribution beyond the entropic one due to the constMint Imposed by the loop. The comparison of th denaturtion ntalpy changes nmeasred for th hairpin (58 *1 KcalInol) ad for the duplex of d(C6C6C6) (51*i Kcal/melX31). indicates Utt tw preence of th loop stabilizes enthalpically Uw hairpin with respect to the 'sten only' by 7 Kcal/mol. Te origin of this contribution could be attributed to: a) the stdking betwen the thymnidine reidue in the loop nd ut nanking cytsin ad guanine bses paird In Uw stem, as aready sn In the ca or th dodecamer 5393
Nucleic Acids Research Table I Thsrm nionmlc data frw d(CD3ITTTTTCC8)
[NoCll
Ollconc
(M) iaM/strand 0.0
Melting tamp.
Tc I cTra
TransI Tras ~g
&HVH(b) .AS Kcsl/mol. Tras.
(U.S.) 11 Tran.
76
8
~~76
4
o
0.04
0.1
A VH (a) Koal/mol. Tr
30
el
=
=
=
23
59 57 58
165 160 163
58
165
55
153
0.2
6 16 26 36 6
37
83 82 83 83 87
0.3
5.8
42
87
38 43 46
(e)rrom the concenLroon depenene or T, ; bYrom the shep or nwlUng curvs
d(C6C6TATAC6C6) (17), or b) the suppreion of th frayng en( effect for the C6 base pair next to the loop. With regard to trniton I the enthaipy dc ge (20*2 Kcal/mol) Is mainly attributable to the strcturl dnge in the thymidine stretch. This suggests tht In Ut dimeric duplex the centrl UhymNidw ridus. rather thn consUtuting a bulge, ae likely to be aprecably stacked and possibly paired by hydrogen bonds. CD Data a B-Z trlntion The ability of th heptdbcamer to nsum a left handed Z conformtion in high slt conditio has bon Investigted by recording Ut CD spctM at a nmer of inreasing selt concentrations. Diffent wits hwe been usd to induce th trition. Fig. 5 shows the CD spectra obtained at OC at severI NaClO4 concentrations. The insert shows th CD signl cagn at 253 ad 295 nm s the salt concntrtion is inrased. It is evident that th oligmr udergoes a B to Z transonand that th transition is complete at od 4 M salt. On the cotray th B-Z btnition prompted by NoCI appeas to be not complete. a illustrtd in Fig. 6. even at satwated conditions. An estimato bsd on th known CD spectr of th hexae d(CGC6C6)2 In the two limiL confortions, and tking Into ccount tUw small contribto attrIbutd to the thrddkw rsidu, prvides for the Z structur fraon a valu of oWt 0.6 (A comparison of the A evalues ot d A reducton of th oliUptcdty vau of the Z-form In th prence of the lttr wit). An importat point is to estoblish if the Z structur Is prnt as a bimoecuar duplex or as a hairpin. An nswr to ths qetn was obtand by studyin th offet of the t on th CD spectrm In hn ly satuated Nea solo for two oligmr concnrations
high sat concentraton In NaCI ad NaC104 Is misledin due to th mrw
differng by an oder of mpltude (C190 JIM. C2- JIMl In single strands, repectively). The relative CD I thrmel profiles ae prcally idisti sbl with a coo ve trnson In the rag 70-90C dwith a midpoint at ound OC, In p nt on th oligomr concentration das not dsown). Such a fndng lealy 5394
Nucleic Acids Research
Fig. 5) CD spectra of d(CGCGCGTTTTTCGCGC6) at incraing aounts of NaClO4 in Tris.HCI 0.1 M (pH=7.3) and at strand concentration of 6.9 it M. Insrt shows tw chane in the ellipUcity at 253 nm and 295 rnm a a function of NaCIO4 concentrabon. shows that ts denaturation is an intmoecular proces, alogous to tho one observed at low alt concentraton. The structure tht drgoes U dinturUon proc Is thefore tho hdrpin, or, bottr the heirpins since te CD spectra show tw prene of not much dissimilr amots of both B and Z helices in saturaed NeCI. A smll variOan in the CD signal is foud also in the low tpratur ra (0 to 40TC). Since th 253 nm bad become slighUy more negtive and tw 295 nr one is slighUy less negtive on incring th temperature in this range, this beaour is likely due to emil shin toward B form in the B-Z oquilirium. This flnding is in nt withthe bdwaviour of d(C6G3.4 oligomer, as edenced by NMR (32) and CD
(31) measurements. A qualitetvely similr reult wa obtained when the molting temperature of tw hirpin in 5 M NaCIO4 (th solvnt in which Ut hairpin Is pr mainly In the Z-conformatUon) wa followd by both UV ad CD n bmment at different oligonucotde strand conentratons. Also In this c T11,M Is indsepndent emelUng teperate is m edly depr on th oligomer concentraion and, s expeted, by th helix destOlization effet exerted by the aClo nion (33). 5395
Nucleic Acids Research
Fig.6) CD Spectra of d(CGC6CGTTTTTCGCGC6) at incrasing anounts of NaCI in Tris.HCI 0.1M(pH47.3) and at strand concentraUon of 9 jIM. Insert shows th chang in ellipUcity at 253 nrn and 295 nrn as a function of NaCi concentrsUon.
A precise ealuation of th enthalpy change In NaC104 5 tM. associated to transition 11 (from Z-hrpin to coil)
was difricult to obtain due to the smal difference in hyperchromicity betwn the two states. However the r age value over three diffrnt melUng pronies at diffrnt oligonucleoUde strand concentrations has ben found to be 55 ± 2 Kcal/mol. Comparing this value with the ehlpy change associed with th denatusUon of the Z-form duplex dGCGC6C6)2 in 5 tM NaC104 (49±1 Kcal/mol). it tun out that the loop stabilim
enthlpicslly the hairpin also when its stmn is In the Z conformation. CONCLUSION This work. bsd on optical and chirooptical meanswu nts. shows wt the hpleotide d(C6CCGTTTTTCGCGCG) exists in queu solution at low strnd c tration a an equlibrin betwen two structured conformaUons: the dmeric duplex and the monomeric hairpin, not differenUy from lgous oligom reported In he recnt litwetO (14-17.20,21). At 0.1 Mi NaCi the dimer Is prndoinant at lower tempeature and higwer strnd oncitrations and the quilibrium is shifted towads Ut hairpin on incrasWg the tempature and/or decrsing th strand concntrn. The hairpin, in both and Z conformations, peas stabilized by th loop with rect to the duplex of d(C6CGCC ) not only Urough an entropic but also enthalpic contribuUon. Since the trsitlon romn the dimeric duplex to the hairpin proceds 5396
Nucleic Acids Research a cuiform structur, as alredy pdopose (16). work is in progres in our laortory in order to obtain also kinetic dat on this proces by men of T-jum experiments with the aim of getting more information about the en eics of this traition. Van de Sande et .1. (21) hae reconUy provided experimental evidence, meinly on the bsIs of the electrophortic mobility, that the 24-mer sequene n structure and hav wgd that the hairpin is d(C6C6C6C6C6TMTC6C6C6C6C6) is able to assume a maintained also when high salt condiUons promot the B-Z trnsiUon of the double helix stem. The spectroscopic data presnwtd in this peper for the ha de o Uoti d(C6CGCOTTTTTC6COC6) demonstat inded th a of ng stabIlIty Z-stem haIrpin conformation, cobr the vIdences reported elIer (21). It is InterUng to note that the ability of the hairpin to assume a left-handed conformation is influnced by the nture of the co-ion (C104verss Cl), being C104 more effective Utn CW.
probably tiwoug
AKNWLEDhENTS This work has ben carried out with th financil contribuUon of Consiglio Nazionale delle Ricrche and of Ministero della Pubblica Istruzione. REFERENCES
1)6ait.JM.ltthes,H.WD..SinghJM. and Timas.R.C.(1982)J.Qim.Sx.Cmwn..37-40. 2)Eflmov,VA.JRerdatto,S.V. and Chdkhmakhche,0.6.(1982) AxcukAcA*sRs. 10,6675-6694. 3X'llyoshi,K.Jlyake.T.Jozuml.T. d ItatkuraK. (1980) AWMc/A Rks.8.5473-5409. 4an Boom,JI., van der larell,.A.. van Boecel, CAA, Wille, 6. ad Hoyng, C.. F. (1962) Q)Scvl Ew)wircs/sofartOf rGw L edited by H.6.6assen ad A. Lang. p.53-70. 5ePrisco Albergo, D.arky. L. A., relaer, K. J. wnd Turner.DJH.(1961) 5/o*w n 20, 1409-1413. 6Ontal. D. J.. Kozlowskil. S. A.. Marky. L. A. Broke. C.. Rice. J. A.. Itekura. K. ad Breslar. K. J. (1962)
&~oteev>l5y,2 ,426-436.
7X)iadrfoglio.F.Jsnzini.6.Dlkelspiel.K. and Crea.R.(1962)AxIucAci**AsRs. 10.3759-376a. 6)Chu,Y.6and Tnoco,I.k. (19B3)&Ow1yvix22,1235-1246. 9Orefer.S.M.DePrisco AlbergoD. and Turner.D.J. (1963)5Aw/ymm22.1 107-1 I31. 10fazakerley,6.V., van der Melare.A., van Boom,Ji. and GuschibaewW.(1964) At/*k/ Acid RAs. 12. a269-6279. 1)Cheng,DJ..,1Kanl.S.,Frechet,D.,Tso,P.OP.,Uesugi,S.,Shida.T. and lkeharJ1.(1964)&iw4qn,w23, 775795.
12)atslD.J.lKolowski.SA..Ikuts,S. and Itakurs,K.(1964) Aio23I,wistr23.3207-3226. 13)Ott.. and Eckstein.F.(1965)8idiskry.24.2530-2535. 14f1aky,LA.Blmnfeld,.S.,zlowski,S. and BreslauerK4J.(1963) 54vmw4/nw22,I247-1257. 15ummers. MI.F.. Byrd, RA.. 6alo. KA.. Samson.CJ.. Zon. 6. ad Egan.W.(1985) Ax/uk Adc/ ARs. 13. 6375-6386. 161cheffler.IL.Elson,El. ad Baldwinl.(196a) J A9i/.36,291-304. 17%Pemmc,D1,Chou,S.,Hare,D.R. and Reid,B.R.(1965)Mce,Acid*sRs. 13,3755-3772. l6Maasnoot,CA.6.Aderuin,SJ., BeRendsnR.6.. Janse. .6JM.. Binnrndljk.TJJ.. Hilbers.C.W.. van der .O Dn 1,115-129. Mael,6A. and van Boom4JI.(1963) JJ1/m5I. 19bdeau4.6. nd 6ilham.P.T.(1985) AtcvAc/s 13.68259-8274. 20ftesnoot, CA.6., de Bruin, SH., Hllbers,C.W., van der MaeI.6A. and van Boom,J.H. (1965) . /nf. /J Sow. 8iinwl. SYZw. hAawtki &w/. id.5.8.767-780. 21)6 Aj1.W. SdcheewelderKJ.I. ad van de Sande.JJ4.(1965)5hoI.Wistry,24.5698-5702. 22)Si n.RR. Nd Pettijhn.DE.(1964) JJb/J /..259.6593-6600. 23)Frank-Kamenetkij,MD. wnd Vologodkiij,A.V. (1964) iWtw,307, 461-462. 24) 6isrerA.(1966)Al&m212.I4B0-1461. 25)Xsedrifoglio,F.Jlanzini,6.YathlndrN. ad Crea,R. (1963) 16th Jerslem Symposium. ZW4: Ahe eciw of /ifeWullmn ed.). Doi'cht. Hollad. p. 61-74. 5397
Nucleic Acids Research 26XifIoglio. F.. Mnzini.6.. Vmr,. t.. Dink*ipisi. K. and Cre. R. (1962) Mof*k AcidsARs9. 21952206.
27XhQdrifrg1io7.s1aim.e.
and
YtMndra.N.(1964)J/AhWJA/. 175.419-423.
2a)Fran.R. and KosterwH.979) AkkAc*Abi.62069-207.
29eCordJ1.T.Jr. nd Lohvn,.TJ.(1978) ia 17.159-166.
[email protected]. ad Lohmn.TI.(1978) aw-tAwAA.,103-178. 31 1.nzini .6.. Xodo.L.E. and (hisfoglio.F. (data to be putblihd) 32Uolak. T. A.. Bor. P.N.. Levy. G.C.. van Boomn.JH. and WangA. H. J. (1984) Akc/Ik Ads Alis. 12. 4625-4635. 33) Von Hippel. HP and Schleich.T.. (1969) Scw, &W sYWiiy of bioiug ma/ wwrio'/uciu Ed. by Timueff and Fman . Vol.2. p.449-457.
5398
