2D-NOESY of DNA: as easy as A, B, Z

TIBS 12-Aprd 1987 consistent with the operation of this btosynthettc pathwa~ in the lung is the observation that this t~ssue has a high concentration of GPC 2°, and that this glycerophosphodtester ~s exported from the hver to the lung 21

References 1 Rooney, $ A (1985)Am Re~ Besptr D~s 131 439-46O 2 Farrel, P M and Avery° M E (1975) Am Rev Resp:r D~ I l I, 657-688 3 Pos~mayer, F Duwc.G and Hahn. M (1977) Can J Bmchem 55,609--617 4 Ide, H and Wemhold. P A (1982)`/ Bml

133 Chem 251.14926-14931 5 Rostow. B , Kunze, D , Rabe, H and Rezchmann, D (1985) Bmchsm Btophys Acta 835, 465--476 6 Infante, J P (1984)FEBSLetl 170, 1-14 7 Strada, P , Acebal C and Arche, R (1985) Mo~ Cell Bmchem 69, 49-54 8 Mason R J andDobbs, L (3 (1980)./ Btol Chem 255, 5101-5107 9 Tsao, F H C (1986) Lep~ds21, 498-502 l 0 0 m r k , J (3, Bleasdale J E , MacDonald P C and Johnston, J M (1980) Btochem Bzophys Res Commun 94, 985-992 II Infanle.J P (1977)Btochem J 167.847-849 12 Infante, J P (1987) FEBS Lett (Ill press) 13 Salerno, D M and Beeler D A (1973) Btochtm Btophys Acta 326. 325-338

14 Stem. O andStem, Y (1969)J Cell Bml 40 461-483 15 Nagley, P and Halhnan. T (1968) Btochtm Bmphys Acta 163,218-225 16 Bjerve, K S (1969)Btochtm Bmphys Acta 296, 549-562 17 Infante, J P (1986)Mol Cell Bmchem 71, 135--137 18 Infante, J P (1985)Med Btol 63 81-87 19 Jacobs, H C . Ikegamt, M , Jobe, A H , Berry, D D and Jones, S (1985) Bmchtm Bmphys Acta 837 77--84 20 Kattaranta, J K (1981)l Chromatogr 206 327-332 21 Robinson. S . $noswell, A M , Runctman, B andUpton. R N (1984)Btochem J 217 399408

EmergingTechniques

2D-NOESY of as easyas A,B,Z

s~th power dependence of the intensity of the croSs-peak on the distance between the spins, ff the protons are less than 3 A apart then a strong cross-peak ,s expected, whde ff the protons are more than 5 A apart then no cross-peak is anJack S. Cohen tmpated Thus, the pattern of crosspeaks ts a d,rect reflection of the conformation of the molecule Although the Two-donenstonal nuclear Overhauser effect spectroscop; (2D-NOESY) Is an excellent method can be used for accurate d,stance technulue to distinguish q~!~_t__,m_velybetween the conformations of polydeoxynucleotutes m geometry calculations ~, the analysis solution Examplesof dzstmctspectralpatterns obt,~ned w~h A, B, and Z forms of DNA are descnbed here provides an unambiguous ~ustrated basis for qualltatwe dlst,nctton between different DNA conformatmns in soluAlthough several conformations of purme position, or halogeno substitution tion it should be noted that cross-peaks DNA were early defined by X-ray dlf- tn the 5-pynm~dme position, destabilize fractmn methods, It was assumed for the B form The commonly employed arising from through-bond spin-spin (]) many years that only the B form was optical spectrgscoptc methods of circular couplings give nse to two-dimensional found m the cell Tlus was because It IS dtchrolsm (CD), infrared (IR), ultra- correlated spectra (2D-COSY) A major the predominant form m high humidity, violet (UV), and Raman spectroscoptes, difference tn the preparation of 2Dand sperm heads showed a diffuse B- as well as 31p nuclear magneuc resonance COSY and NOESY experiments =s the form pattern However, as well as B (NMR) spectroscopy, cannot unambiguProton NMR Chern~cal Shift forms, both Z and A forms have been ously define structures tn solution "-.= Two-dtmensmnal HMR techmques found m X-ray crystallographic struc°° are now established as excellent methods tures of ohgodeoxynucleotldes, in addition, the spectroscopic observation of for the analysis of molecular structure m transltmns between distinct forms of solution = The two-dimensional nuclear polydeoxynucleotldes =n solutmn has Overhauser effect (2D-NOE) techmque resulted in some amblgmty as to the has proved to be particularly valuable for conformatmnal analysis-' Proton-proactual conformation of DNA m solutmn Several extrinsic factors, such as level of ton 2D-NOE spectra are symmetrical Z hydratton, Ionic strength, temperature, (Fig I), with a proton chenucal shift c specific hgands, and supercolhng, may (frequency) scale on each axas Seen "% influence the B - Z equthbnum, for from above as contour plots, each peak example There are also several mtnnstc on the dmgonal (or the projection onto factors, such as base sequence and cova- either axis) arises from a proton m the lent chemical modification of the base molecule, whde off-diagonal peaks anse Fig SchemaUc representa,on of a 2D NOE~Y Lontour plot. showing Ihreeproton signals along the or backbone, that can promote a gwen from dipolar cross-relaxatmn between diagonal (filled circles) and Ihe swnmetrtcal offprotons (spins) that are close to each conformation For example, it has been diagonal cross-peaks (open orcles, at the mtersecreported that amino substitution In the 2- other in space These so-called cross- uon of honzontal and ~ertwal hne~) that anse from peaks c~n be related to the pa=r of cross- cross-relaxauon between protons pazr~ I-2 and 1--3 relaxln~ spins from which they denve by thin are adlacent an space The absence of a cross J S Cohen ts at the Chmcal Pharmacology Branch. drawing horizontal and vemeal lines on peak from Iwo proton stgnals ¢such as 2-~ arro~) Na~onal Cancer insatute. NIH. Bethesda. MD the plot (Fig I) Due to the reciprocal rod:cares that the prolons are far apart 20892. USA

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134

TIBS 12-April

(a) A-Form

(b) B-Form

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1987

(c) Z-Form

.

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Fig 2 2D-NOESV symmetnzed absorpuon mode contour plots at 270 MHz usmg a rmxmg time of 50 ms (a) A-form DNA, 17ol)(d2NH2A-dT) m 4 ~f NaCI (b) B.form DNA, poly(dA-dT) mO I MNaCI (c) Z-form DNA, poly(dG.dSMeC) m 3m~ MgCt_, The spectra proleeted on theaxes are the one-dtmet~tonal spectra

•clusion of an addmonal mix[]g time [] the latter case, before final data acquisition, that allows the spins of []terest to cross-relax However, COSY crosspeaks will not be observed [] the 2DNOE spectra described here because the base protons be[]g observed are generally s[]glets (without J coupling), and Jcorrelated cross-peaks are not usually observed for slowly tumbhng polymers4 Examples of the 2D-NOESY patterns observed for the A, B, and Z conforma-

ttons of D N A [] solution are shown [] Fig 2 It can clearly be seen that the patterns m each case are quite dlst[]ct The conformatlonal basts for these differences will be briefly described, starting with the first 2D-NOESY pattern reported, for the well-known B form of DNA The conformatlonal charactenstics that define such DNA structures, and that can be defined by such a pattern, are the sugar conformation, the onentatlon of the base ( s y n or a n n ) , and

A zm,3

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Fig 3 Portion of a stogie strand d(A TA) structure m a right-handed B conformation Specific mira and mter-nucleoude mteracnons that could gtve rise to cro~s-relaxatton due to proton distance5 less that ~3 5 A are lomed by broken hnes

the sense of the hehcal rotation (right or left) All of these can be defined by observauon of close juxtaposmons of pmrs of protons m the structure F~gure 2b exhibits a very strong thyrmdme methyl-ademne H8 cross-peak and strong d e o x y n b o s e - H 2 ' , 2 " cross-peaks with base H8 (punne) and H6 (pynm i d • e ) protons s 6 These arise from the correspondmg pattern of close rater-proton interactions (Fig 3), and the H2'e n d o (2E) conformation of the sugar rmg with anti base conformation m the B form (Fig 4b) A sharp contrast was observed in the 2D-NOESY pattern of cross-peaks for poly(dG-dSMeC) m the presence of magnesmm chlonde (5 raM), which Is known to convert to the Z form 6 (Fig 2c) In that case a strong G H S - G H I ' cross peak was observed, and no G H 8 CMe cross peak This results from the onentanon of the guanme base bemgsyn m the Z-DNA confomlatlon 7 (Fig 4c) It is noteworthy that the one-dimensional spectra of the B and Z forms of this polymer show only rumor differences (see spectra projected on the axes of Fig 4), while the two-dimensional spectra are quite disunct There have been several attempts to define the structural requirements that control the formation of the Z form of D N A it had been surmised that since d(A T) polymers do not convert in high salt to the Z form, and d(G C) polymers do, that the 2-am[]o purlne group was responsible for this difference s It was reported 9-ti. mainly on the basis of CD spectroscopy studies, that alternating (d2NH2A-dT)does in fact convert to the Z form m [ugh salt However, 2D-NOE spectra of t[us synthetm polymer gave an unusual pattern of cross-peaks that cot-

T I B S 12 - A p r i l 1987

(a)

135

A-Form

(b)

B-Form

(c)

Z-Form

?

o

'="v.,"z', -,

Fig ,t A single nucleotlde m the (a) A form with the ~EdeoJo,nbose conformanon, (b) B form wah the 'E conformatmn and anh.glyco~Mec base orientation and fc) Z form syn-glycostdtc conformation The close specific mtranucleoltde mteractmns that are different m the three cases, nameh HS-H3' H8-H2' and HS.-HI' are indicated by a broken hne

responded to neither that of a B or Z form (Fig 2a), and this was attributed to an A forml2 The most notable feature of this pattern is a pmr of cross-peaks to the sugar HY proton, which arise from T(Me)-H3' and punne HS--pynmldme H6-H3' proton interactions due to the Y - e n d o (3E) sugar conformation (Fig 4a) However, there was one point of ambiguity in this pattern, namely that both the base protons ]n quesnon (AH8 and TH6) are overlapped in the high salt A-form spectrum (Fig 2a) Consequently it is ~mposs]ble to tell which of the two, or both, nucleotldes are in the 3E conformation In order to clanfy this questmn and confirm the observation of this A form pattern, several other synthetic polymers

were also Investigated This included sequences with 5-halogeno-pynmldlne substitution, which was also expected to favor the Z form It was found that both poly(d2NH2-d5BrU ) and poly(d2NH2d51U) gwe an A-form 2D-NOESY pattern in high salt (Fig 5) Fortuitously, the base protons for the latter sequence were resolved In high salt, thus allowing us to draw the conclusion that both nucleohdes in this sequence are in the 3E

conformation I Thus, a first-order analyms of the conformat,on of a polydeoxynucleonde m solutmn becomes mainly a matter of pattern recognition, m wluch one relates pattern of cross-peaks in the 2D-NOE spectrum (Fig 2) to the closest interproton distances in the conformation

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~

. B

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Fig 5 2D-NOESY comour plot of poly(d2NHJl-dSlU) m 4M NaCi04 M~mg amc was 20 n~ Oiher detads as for Fig 2

(Fig 4) A recent test of this approach has been made with the sequence poly(dA)poly(dr), which had been proposed, on the basis of X-ray diffraction stuches, to ex,s, m a heteronomous conformation,, e ~ t h the A-strand in the A form and the T-strand ,n the B form 14 Two studies have found that the 2D-NOESY pattern observed for this homopolymer corresponds to both strands be,ng m the B form. with only minor differences from the pattern gwen by the alternating d(A-T) copolymer1516 References I Bax. A

and Lerner, L (1986) Science 232

960-967 2 Havel.T F and Wuthnch. K (1985)J Mol Btol 182.281-294 3 Keepers. J W and James T L 0984) J Magn Reson 57 41~-426 4 Welss. M A Ehason.J L andS~a'es.D J 0984) Proc Nail Acad So USA 81 60196023 5 Assa-Munt N and Kearns. D R (1984) Biochenustry 23. 791-796 6 Borah. B Cohen. J S and Bax. A (1985l Biopolymers 24 747-765 7 Wang. A H-J.Qulgley G J Kolpak.F J. Craw[ord. J L. van Boon. J H. van der Marel. G and Rich. A (1979)Nature 282 680686 8 Drew. H R and Dickerson. R E (1981)J Mol Biol 151.535-556 9 Gaffney.B L.Marky L A and Jones R A (1982) Nucleic Acids Res 10 4351-4~61 10 Jown T N . Mclntosh. L P Arndt-Jo~m D J etal (1983)J Biomol SIrutt Dvnam I 21-57 II Howard F B Chen C-W,Cohen J S and Mdes H T (1984)Biochem Biophvs Res Commun 118 848-853 12 Botah B Cohen J S Howard F B and Mdes H T (1985)Biochemmry 24.7456-7462 13 Borah. B Howard. F B.Mdes H T and Cohen J S (1986)Btochemmo'25.746"l-7470 14 Amoit S. Chandrasekharan R Hall I H and Puiglaner L C (1983)NuclewAcMsRes 11 4141-4155 15 Behhng. R W and Kcams. D R (1986) Btochenustry 25 3335-3345 16 Roy. S Borah B Zon G andChoen J S Bmpolymers (m press)