Eur. J. Biochem. 233, 631-643 (1995) 0 FEBS 1995

Solution structure of the Na’ form of the dimeric guanine quadruplex [d(G3T4G3)I2 Max A. KENIRY’, Gary D. STRAHAN’, Elisabeth A. OWEN’ and Richard H. SHAFER‘

’ Research School of Chemistry, The Australian National University, Canberra, Australia * Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, USA

(Received 6 Aprilll2 June 1995) - EJB 95 0550/2

The solution structure of the DNA quadruplex formed by the association of two strands of the DNA oligonucleotide, d(G,T,G,), in NaCl solution has been determined by ‘H two-dimensional NMR tech- niques, full relaxation matrix calculations and restrained molecular dynamics. The refined structure incor- porates the sequences 5’-G1,G2AG3AT4AT5AT6AT7AG8,G9AG10A-3’ and 5’-G11,G12AG13AT14AT15A. T16AT17AG18,G19,G20A-3‘ (where S and A denote syn and anti, respectively) in a three-quartet, diago- nal-looped structure that we [Strahan, G. D., Shafer, R. H. & Keniry, M. A. (1994) Nucleic Acids Res. 22, 5447-54551 and others [Smith, F. W., Lau, F. W. & Feigon, J. (1994) Proc. Nut1 Acad. Sci. USA 91, 10546- 105501 have described. The loop structure is compact and incorporates many of the features found in duplex hairpin loops including base stacking, intraloop hydrogen bonding and extensive van der Waals’ interactions. The first and third loop thymines stack over the outermost G-quartet and are also associated by hydrogen bonding. The second and the fourth loop thymines fold inwards in order to enhance van der Waals’ interactions. The unexpected sequential syn-syn deoxyguanosines in the quadru- plex stem appear to be a direct consequence of the way DNA oligonucleotides fold and the subsequent search for the most stable loop structure. The implications of loop sequence and length on the structure of quadruplexes are discussed.

Keywords: G-quartet ; quadruplex ; solution structure ; telomere; two-dimensional NMR.

Repetitive guanine-rich regions of DNA are found at the ter- mini of chromosomes (Zakian, 1989; Blackburn, 1990, 1991, 1994) and at or proximal to the promoter regions of genes (Ev- ans et al., 1984; Kilpatrick et al., 1986). The addition and re- moval of non-coding telomeric structures at the end of chromo- somes at defined times in the cell cycle enables replication to occur without the loss of the chromosome termini after each cycle (Blackburn, 1990, 1991) and may also assist in the posi- tioning and organization of chromosomes in the nucleus (Funab- iki et al., 1993). An understanding of the role of telomere DNA may have important implications for the problems of cell aging and cancer. Interest in guanine-rich DNA segments is not just confined to telomeres. Guanine-rich segments have been impli- cated in the association of two copies of the RNA genome in the mature HIV (human immunodeficiency virus) virion and have also been shown to repress the transcription of genes in their immediate vicinity (Sundquist and Heaphy, 1993). Further biological relevance is provided by the recent identification of proteins specific for DNA quadruplexes (Liu et al., 1993). This has in turn aroused interest in the DNA structural elements that are recognized by these proteins.

Correspondence to R. H . Shafer, Department of Pharmaceutical Chemistry, School of Pharmacy, Box 0446, University of California, San Francisco, CA 94143, USA

Abbreviations. HIV, human immunodeficiency virus ; DIPSI, decou- pling in the presence of scalar interactions ; DQFCOSY, double- quantum-filtered two-dimensional correlation spectroscopy; NOESY, two-dimensional Overhauser exchange spectroscopy ; P. E. COSY, primi- tive exclusive correlation spectroscopy ; ROESY, rotating frame Over- hauser enhancement spectroscopy ; TOCSY, total correlation spec- troscopy.

There have been several NMR and X-ray studies of DNA quadruplexes. Detailed structures have been determined for sin- gle-stranded, double-stranded and four-stranded quadruplexes (Wang and Patel, 1993a,b; Schultze et al., 1994a,b; Wang et al., 1994). All of the reported four-stranded structures have their DNA strands aligned parallel to each other, with each of the nucleotides in the anti conformation (Aboul-ela et al., 1992; Wang and Patel, 1992; Kang et al., 1992; Gupta et al., 1993; Laughlan et al., 1994). Folded structures are different, as they must have some strands aligned in an antiparallel fashion, which in turn requires some of the guanine nucleotides to adopt the syn conformation in order to form a G-quartet. In all but one of the reported structures of folded quadruplexes, both single and double stranded, sequential deoxyguanosines strictly alternate between syn and anti conformations about the glycosidic bond (Kang et al., 1992; Smith and Feigon, 1992, 1993; Schultze et al., 1994a,b; Wang and Patel, 1993a). The exception is the hairpin dimer quadruplex formed by two folded strands of d(G,T,G,), the first reported structure to exhibit sequential syn- syn guanine nucleotides (Smith et al., 1994; Strahan et al., 1994).

The thymine-rich loops in these structures assume two basic orientations. An X-ray analysis of the crystalline form of (G4T4GJ2 revealed the thymine loop to be aligned along the edge of the neighbouring G-quartets (Kang et al., 1992). In con- trast, the same DNA sequence forms a quadruplex in solution with the thymine loops aligned diagonally across the neighbor- ing G-quartets as determined by NMR methods (Smith and Fei- gon, 1992, 1993; Schultze et al., 1994b). These two loop config- urations are illustrated in Fig. 1. Similar variations in loop or- ganization have been found for single-stranded quadruplexes

632 Keniry et al. (ELM J. Biachem. 233)

n n

A B C Fig. 1 . Schematic models of representatives of the classes of possible folded-dimer quadruplexes (A) antiparallel-stranded edge-looped model (B) diagonal-looped model (C) alternative diagonal-looped model, created by reversing the orientation of one of the strands with respect to the other. Completely \hndcd polygon\ repiesent \\I? deoxyguanowes, polygons with clear surface\ represent m i deoxyguanomes

(Wang and Patel. 1993a; Schultze et al.. 1994a; Wang et al., 1993). The thymine residues in many of these quadruplexes show little or no mobility on the NMR time scale (Wang et al., 1994; Wang and Patel, 1993a). The stability of these various structures results from quartet stacking interactions (Strahan et al., 1994), quartet-loop stacking interactions and hydrogen bond- ing within the thymine-rich loops (this work).

Here, we present the three-dimensional structure of the quad- ruplex formed by the association of two strands of d(G,T,G,) in the presence of Na ' , determined by two-dimensional NMR and rertrained molecular dynamics. In addition to the sequential syrz deoxyguaninosines reported previously (Smith et al., 1994; Stra- han et al., 1994) we confirm that the structure is diagonal-looped and possesses three G-quartets. As discussed i n our previous study (Strahan et al.. 1994), several factors may contribute to the stability of this unique quadruplex conformation, including the effect of base stacking interactions between the G-quartets and the loop thymines. In addition we present evidence for thy- mine-thymine hydrogen bonds which may also enhance the sta- bility of the loop conformation.

MATERIALS AND METHODS

Sample synthesis and preparation. The oligonucleotide d(G ,T,G J was sjrzthesized and purified as described previously (Scaria et al., 1992) and desalted by dialysis. The quadruplex was formed by first dissolving approximately 7 mg DNA into 0.4 in1 10 mM Na2P0, in 90% H20/10% D,O then following the change in the iniino/aniino region of the NMR spectrum at 20°C on titration with NaCl. The quadruplex was completely formed at a Na ' concentration of 65 mM.

NMR spectroscopy. All NMR spectra were recorded on a Varian VXR 500s spectrometer running at 500.1 MHz and pro- cessed on a SUN 10/30 workstation using Varian VNMR soft- ware. NMR2 (Tripos) and software that was developed in house. Two-dimensional phase-sensitive nuclear Overhauser exchange (NOESY) spectra (80-ms, 130-ms and 250-ms mixing times) in 100% D 2 0 were acquired at 20°C in the pure absorption mode by the method of States et al. (1982) employing 2048x2 com- plex points in the t,dimension and 400x2 complex points in the t , dimension. The spectral bandwidth was 4500 Hz and the recy- cle time was 3.5 s. For NOESY spectra in 90% H20/10% D,O at 2°C (170 ins mixing time). the first pulse of the standard NOESY sequence was replaced by a composite solvent suppres- sion pulse (Davis and Wiinperis. 1989). The spectral bandwidth of the latter experiment was 8500 Hz, there were 2048x2 cotn- plex points in t2, 350x2 complex points in r , , and the recycle

time was 3 s. A homonuclear correlation spectrum was obtained using a total correlation spectroscopy (TOCSY) pulse sequence utilizing a decoupling in the presence of scalar interactions (DlPSI) mixing pulse of duration 80 ms, a field strength of 5.5 kHz and spectral bandwidth of 4500 HL. A rotating frame Overhauser enhancement spectroscopy (ROESY) spectrum was acquired by the technique of Kessler et al. (1987). The field strength of the spin-lock was 5 kHz, the mixing time was 250 ms, the recycle time was 4 s and the spectral bandwidth was 5000 Hz. The double-quantum-filtered two-dimensinal correla- tion spectroscopy (DQFCOSY), ROESY and TOCSY spectra were acquired with 204XX2 complex points in r2 and 400x2 complex points i n t , . All the NOESY, DQFCOSY, TOCSY and ROESY spectra were zero-filled to 2048 real points in tZ and 1024 real points in t , . A 60° shifted sinebell was used in both tZ and t , except when quantification was required and a 90" shifted sinebell was used. Baselines were corrected by a two-dimen- sional version of a baseline correction routine first proposed by Pearson (1 977).

The primitive exclusion (P. E.) COSY spectrum was ac- quired by the technique of Bax and Lerner (1988) employing a spectral width of 5000 Hz. 2048x2 complex points in t2, 4 0 0 x 2 t , increments, 48 scans/increment and a 1.9-s recycle time. The spectrum was zerofilled to 4096X 1024 complex points and apodized with a 45" shifted sinebell in both dimensions. J,,?,, J,,?,,, CJ,,,, CJ,,,,, and ZJ,,,,. were estimated as described by Bax and Lerner (1988).

NMR data quantification. Two-dimensional data sets were processed as described above and cross-peaks were integrated using Varian VNMR software and by NMR2 software from NMRi (Tripos). Resolution and quantification were enhanced in those regions where peaks were overlapped by employing the Maximum Likelihood method (MLM) in the NMR2 software package. We found that by using conservative values of line- sharpening factors, reliable cross-peak volumes could be ob- tained with the MLM algorithm as long as overlap was not se- vere.

NMR data analysis - distance restraints. lnterproton dis- tances were estimated from the cross-peak intensity by employ- ing the MARDIGRAS program (Borgias and James, 1988, 1990). The version of this program used here provides reliable estimates of distances involving methyl protons (Liu ct al., 1992). Where the cross-peaks from symmetry related protons in the loops of each strand could not be resolved in the NMR spectrum, the cross-peak intensity was divided by two and equal intensity was assigned to each symmetry related pair of protons. This was justified by the observation that in every case where cross-peaks from symmetry-related pairs of protons were re-

Keniry et al. ( E m J . Biockeni. 233) 633

solved, the intensities of the cross-peaks were the same within experimental error. The pseudo-energy force field for the NOE- derived distance restraints has the form of a flat-well with para- bolic sides. The width of the flat part of the well was derived from the upper and lower distance bounds that were obtained from the program MARDIGRAS, and was dependent on the agreement of the experimental and converged MARDIGRAS cross-peak intensity and on the signal-to-noise ratio (Borgias and James, 1990). Where the difference between the upper and lower distance bounds output by MARDIGRAS was less than 0.03 nm, this difference was replaced by 0.03 nm.

In addition to the distance bounds between proton pairs cal- culated from the NOESY cross-peak intensity by MARDI- GRAS, we have included distance bounds that can be reasonably deduced from relative cross-peak intensities and cross-peak line- widths. Following the procedure described by Kim et al. (1992), we have included a set of experimentally derived distance re- straints that are designed to restrict the conformations sampled by the backbone during restrained molecular dynamics to physi- cally reasonable structures. A short summary of the distance re- straints and a description of the experimental verification fol- lows.

For all residues in the anti conformation, except T 6 and T16, we found the sum of the H4’ Jcouplings (i.e. LT: J,,, = JHILH3. + J,,~,,,,+ JH4,.HS-+ J,,,-,,) to lie in the range 6-9 Hz, the intensity of the H8/H6-HS’, H5” cross-peaks to be less than the intensity of the H8/H6-H1’ cross-peaks and the intensity of the H2‘-HS’, HS” cross-peaks to be less than the intensity of the H2’-H1’ cross-peaks. The combination of these observations limits the range of the dihedral angle ;J (OS‘-C5’ C4’-C3’) of G3. T4, TS, T7, G9, G10, G12. G13, T14, T15, T17, G20 to 60-C40” (Kim et al., 1992). Since CHARMm lacks flat-well potentials for tor- sion angles, these bounds were converted to distances. Hence, we assigned flat-well distance bounds of 0.228-0.270 nm for the H4’-HS’ and H4’-HS” distances of the G3, T4, TS, T7, G9, G10, G12, G13, T14, T15, TI7 and G20 nucleotides. This analy- sis could not be used for residues in the syn conformation or for T6 and T16. Residues in the syn conformation are expected to have weak H8/H6-HS’, H5” cross-peaks for this range of y an- gles. The T6 and T16 residues show sufficiently strong H6-H5’, H5” cross-peaks to preclude restriction of the y angles of these two residues during restrained molecular dynamics. As the in- tensity of each H8/H6-H5’/H5” cross-peak for G3, T4, T7. G9, C10, G12, G13, T14, T17, G20 is less than any of the H6/H8- HI ’ cross-peaks (excluding the six bases in the syn conforma- tion) a minimum bound of 0.34 nm was assigned to all sequen- tial and non-sequential H8/H6-H5’, H5” distance bounds for these residues. Similar analysis of the H2’, H2”-HS’, H5” cross- peaks justified a minimum distance of 0.35 nm for all sequential and non-sequential H2’, H2”-H5’, HS” distance restraints except for the H2’, H2”-HS’, H5” distances of G9 and G19 and the H2’-H5’, H5” distances of T4 and T14.

The ZJF13, (CJ,,, = JHic,12, + JH3,.,,*,, + J,,,,,,, + JI’-Hi,) was determined from the P. E. COSY spectrum to be in the range 10- 14 Hz indicating that the maximum possible H3’-P coupling constant is in the range 2-6 Hz. Although this range for J,-,,, corresponds to multiple values of the dihedral angle E , the result- ing H3’-P distances fall within 0.280-0.325 nm (Kim et al., 1992). Hence this distance range was utilized to restrain the H3’- P distance. Linewidth analysis of the HS’, HS”-H8/H6, Hl’, H3’, H2’ cross-peaks in a 200-ms mixing time NOESY spectrum re- vealed that no cross-peak has a linewidth greater than 30 Hz thereby constraining the HS’-P and HS”-P distances to bounds of 0.265-0.340 nm.

Since there was no evidence for local mobility, all close dis- tance contacts (< 0.45 nm) in the refined models were investi-

gated and repulsive restraints were created for all contacts that did not correspond to a cross-peak in the 250-ms NOESY spectrum by assigning a lower distance bound of 0.45 nm and an upper distance bound of 2.0 nm to the flat-well.

Hoogsteen hydrogen bonding within the G-quartets was maintained for all guanines by applying distance restraints be- tween the heavy atom and the hydrogen of the donor acceptor pair. A distance restraint of 0.1 90 t 0.1 nm was set for the gua- nine N7 to guanine H22 and the guanine 0 6 to guanine HI distances.

For the purpose of brevity, we refer to all distance restraints not derived from MARDIGRAS as non-MARDIGRAS re- straints. They remained the same through all cycles of the refine- ment. In all, there were 232 restraints derived from MARDI- GRAS, 24 distance restraints to maintain hydrogen bonding, 87 to maintain the correct backbone conformation and 21 0 repul- sive restraints to maintain fidelity with the recorded NOESY spectra.

NMR data analysis - dihedral-angle restraints. Estimates of J,,,,,, ZJ,,,,, ZJ,,,,, ZJ,,,-from the P. E. COSY spectrum demonstrated the dominant conformational state of all deoxyri- bose sugars was S type. Consequently, dihedral angle restraints on v, (04’-Cl’-C2’-C3’) and v2 (CI’-C2’-C3’-C4’) were applied to maintain deoxyribose puckers approximately in the S confor- mational state.

Dihedral restraints, designed to maintain flat G-quartets, were used in the early stages of refinement but were omitted during the final restrained-molecular-dynamics runs.

Model building and starting structure. Initially, two mod- els were created. The starting structures for both models were derived from the published coordinates of the edge-looped quad- ruplex (Kang et al., 1992) which were obtained from the Brook- haven Protein Data Bank. A model containing three G-quartets was created by removing the bases at the 5’ and 3’ terminus of each strand, then adjusting the orientation of each base to be either syn or anti in agreement with the experimentally deter- mined orientation. One strand was translated to re-establish Hoogsteen bonding in the G-quartets. These manipulations cre- ated a three-quartet edge-looped model. The three-quartet diago- nal-looped model was created from the edge-looped model by first cleaving the loops at the P-05‘ bonds of residue 8 and residue 18 then translating and rotating the segments containing residues 8-10 and 18-20 so that they were located diagonally opposite the segments containing residues 1-3 and 11 -13, re- spectively. The P-05’ bonds were re-established, the positions of all the guanines were fixed and the loop structure was energy minimized. These models constituted the starting structures for the MARDIGRAS/restrained-molecular-dynamics protocol.

Restrained molecular dynamics. Restrained molecular dy- namics was performed in vucuo using the QUANTA 4.0 package (incorporating CHARMm 22.2) of Molecular Simulations Incor- porated (MSI). The pseudo-energy force field for the NOE- derived distance restraints has the form of a flat-well with parabolic sides. The width of the flat part of the well was de- rived as described above. Details of the restrained-molecular- dynamics protocol have been described previously (Gray et al., 1994)

All three-dimensional graphical visualization and manipula- tion was accomplished with either QUANTA 4.0 or Midasplus 2.0 (Ferrin et al., 1988). Conformational features and helical pa- rameters were analysed by the program CURVES 4.1 (Lavery and Sklenar, 1992). Pairwise rmsd were calculated using a utility from XPLOR 3.1 (Brunger, 1992) while sixth-root residual in- dices and R factors were calculated using CORMA 3.1.

634 Keniry et al. (Eul: J. Biochem. 233)

10.2 1 ‘4 10.4 4 I

11 6

11 8

12 0

8 0 7 6 7 2 6 8 6 4 6 0 5 6 5 2

F1 (PPm)

Fig. 2. Expansion of a two-dimensional NOESY spectrum ( T ~ = 170 ms) of [d(G,T,G,)], at 2°C in 10 mM Na,PO,, 65 mM NaCl 90 % H,0/10 %D,O solution illustrating the HS/H6-H1, H2, H3 cross- peaks. Cross-peaks are assigned as follows: A, G20HX-GXHI; B, G12HX-G9HI; C, G10HX-G1XH1~ D, G3HX-G20H1; E, G13HX- G10H1; F, G9H8-Gl9H1; G, G8HX-GllHl; H, G2HX-GI2Hl; I, G19HXG2Hl; J, GllH8-G3Hl; K, GlXHX-GIHl; L, GlHX-Gl3Hl; M, GlXHX-Gl9HI ; N, G18H8-GIH2,; 0, GXH8-GllH2,; P, T16H1’- G l l H l ; Q, T6Hl’-GlHI; R, T16HI’-GlH2,; S, T6HI’-GlIH2,; T,

The spectrum was acquired with a sweep width of 8500 Hz, 4096 com- plex points, 2x350 t , increments, 160 scanshcrement and a 3-s recycle delay The spectrum was zero filled to 4096x2048 complex points apod- ized with a 60” shifted sinebell in both dimensions.

T14HI’-GI3HI; U, T4HI’-G3HI; V, T14Hl’-T16H3; W, T4Hl’-T6H3.

RESULTS

NMR evidence for quadruplex formation. We have previously shown that two d(G,T,G,) strands come together as a dimer and we have demonstrated that the one-dimensional NMR spectrum in H,O of this dimer in the presence of Na’ at 25 “C has 12 imino resonances (Scaria et al., 1992). After lyophilization fol- lowed by dissolution of the [d(GIT4G3)IZ in D20, only four imino resonances remain after 1 h. These four imino resonances were later shown to belong to the central G-quartet.

NOESY spectra in H,O provide further clues to strand con- formation. Each imino proton resonance has at least one cross- peak to a distinct guanine H8 resonance (peaks A-M in Fig. 2). Each imino proton resonance also has a cross-peak to a hy- drogen-bonded guanine amino resonance and some of the imino resonances have cross-peaks to external guanine amino reso- nances. The presence of well resolved hydrogen bonded and ex- ternal guanine amino resonances is indicative of slow rotation about the C-N exocyclic bond. The exchange with solvent has sufficiently slowed for some guanine amino pairs that broad but intense cross-peaks are observed between the hydrogen-bonded and external amino protons (Fig. 2). Some of the more slowly exchanging amino proton resonances also have cross-peaks to guanine H8 resonances (peaks N and 0 in Fig. 2). There are some imino-imino cross-peaks but none is intense, indicating the absence of any G-T wobble pairs. Taken together, these observa- tions support the presence of three G-quartets each of which is formed by Hoogsteen bonding between four guanines. Sequence specific assignments of the exchangeable protons and non-se- quential NOE contacts are reported below.

Non-exchangeable proton assignments. Initial assignments of the non-exchangeable protons were made with the aid of NOESY, TOCSY and DQFCOSY spectra. Analysis of the 250-ms NOESY spectrum, described in detail previously (Stra- han et al., 1994), established the aromatic and H1’ proton assign- ments, along with the following unusual sequence of glycosidic bond angles for the two strands: S’-Gl, G2, G3, T4, T5, T6, T7, G8, G9, G10,-3’ and 5’-Gll, G12, G13, T14, T15, T16, T17, G18, G19, G20,-3’. Here the subscripts S and A denote syn and anti conformations, respectively. This sequence was confirmed by a ‘H-”P-HeteroCOSY spectrum (Strahan et al., 1994)

Deoxyribose assignments. P. E. COSY, DQFCOSY and TOCSY spectra were used to assign the H2’, H2” and H3’ reso- nances to particular residues in the quadruplex by correlating these resonances with the sequentially assigned HI’ following standard practice (Hare et al., 1983). Where possible, the H4’, H5’, H5” resonances were assigned in the same way. Results from the DQFCOSY and P. E. COSY experiments for the H3’/ H4’ and HI’/H2’, H2” regions, respectively, are displayed in Fig. 3. While many peaks are weak due to antiphase cancellation in the DQFCOSY spectrum, strong H3’-H4’ cross-peaks can be seen from the G2, T4 and TI4 sugars, due to their large coupling constants (Fig. 3A). We also note that these three residues exhib- ited strong intraresidue Hl’-H4’ NOESY cross-peaks as well (data not shown). There were some H4’, H5’, HS” resonances that could only be assigned by reference to the NOESY spectrum. The H2’ and H2” resonances were stereospecifically assigned by noting that the intranucleotide HI’-”’’ cross-peak is more intense than the HI’-H2’ cross-peak in the two-dimen- sional NOESY spectra. Many HS’ and H5” resonances could not be stereospecifically assigned because of considerable overlap in this region of the spectrum. The non-exchangeable proton chemical shifts are listed in Table 1.

Exchangeable proton assignments. To assign the exchangeable proton resonances, it is first necessary to establish the topology of the quadruplex because the imino and amino resonances can only be identified using interresidue NOE contacts between the guanine H8 protons and the exchangeable protons within the G- quartet. We have already established that the quadruplex brings together two folded strands and has three G-quartets (Scaria et al., 1992). The model with both loops at the same end of the stem can be excluded because several nonsequential NOE be- tween T6 and GI1 and between TI6 and GI were identified (data not shown) and no interloop NOE were observed. Hence, the topology falls into two general classes, each class having two or more members that differ only in the relative orientation of the strands. Representative structures are illustrated in Fig. 1. The edge-looped models with the loops at opposite ends of the stem can be excluded because of the presence of characteristic NOE between non-exchangeable and exchangeable protons ; the HI’ proton of T4 and the H8 proton of G11 are both close to the same amino and imino protons. Likewise, a similar set links TI4 with G1. While several models may fit some of the contacts listed above, only the diagonally looped models can simulta- neously satisfy all of these. Extensive efforts at modelling the edge-looped models using the complete set of NOE resulted in structures that either could not satisfy the restraints and/or had unacceptable backbone dihedral angles.

It is not possible to discriminate between the two diagonal- looped models (Fig. 1 B and C) solely on the basis of the non- exchangeable NOE. They can, however, be distinguished by a combination of NOE from the NOESY in D,O and the NOESY in H,O. The models differ in the arrangement of the bases in

Keniry et al. (EM J. Biochem. 233) 635 __

4.5

T14 I

4.9 - ' 1 i

5.0 -1 -v --O GQJ

5.1 i G19 G12 G2 -

5'4/ 5.6

T16 T16

G12

Q:

. T14 % T14

Fig. 3. Expansions of two COSY experiments of [d(G,T,G,)], in 10 mM Na,PO,, 65 mM NaCl in 100 % D,O solution at 25°C. (A) An expansion of a double-quantum-filtered COSY spectrum displaying some of the H3'-H4' cross-peaks The spectrum was acquired with a spectral width of 4500 Hz, 4096 complex points i n p , 2x400 t , increments, 128 scanslincrement and a 1 5 s recycle time. The spectrum was zero filled to 4096x1024 complex points and apodized with a 30" shifted sinebell in both dimensions. (B) An expansion of a P. E. COSY spectrum displaying the deoxyribose HI' and H2', H2" cross-peaks.

Table 1. Nucleotide proton chemical shifts of strand 1 of the [d(G,T,G,)], quadruplex. Phosphorous chemical shifts were referenced to trimethyl- phosphate (Strahan et al., 1994). n.a., not available.

H1' H2' H2" H3' H4' HS', H5" HUH3 H2/H4 "p Strand Nucleotide HUH6 H5

1 G1 G2 G3 T4 T5 T6 T7 G8 G9 GI0

7.20 5.93 3.27 3.27 5.00 4.35 7.40 5.70 2.48 2.48 5.06 4.14 8.01 5.94 2.72 2.57 4.96 4.47 6.97 1.73 5.47 2.11 1.32 4.45 3.91 7.24 1.54 5.39 1.89 2.08 4.55 3.46 7.17 1.49 5.45 2.30 1.67 4.69 4.45 7.62 1.73 6.28 2.39 2.10 4.53 3.12 7.46 6.16 2.99 3.56 4.97 4.52 7.99 6.11 2.80 2.69 5.13 4.53 8.07 6.54 2.58 2.68 4.75 4.44

3.97, 3.85 4.34, n.a. 4.18, 4.18 4.40, 4.05 3.58, n.a. 4.08, 3.98 2.80, n.a. n.a. 4.19 4.34, 4.31 4.32, 4.32

____

11.69 11.71 11.40 n.a. n.a. 9.79

10.18 11.89 11.79 11.11

10.88, 5.93 9.69, 6.74 -4.18

n.a. n.a. -4.42 -4.93 -4.77 -4.34 -5.80

9.65, 7.39 n.a. 9.13, 6.91 n.a.

n.a. n.a. -3.82

2 G11 7.39 5.84 2.93 2.69 4.96 4.33 3.98, 3.87 12.06 10.69, 6.41 GI2 8.06 5.36 2.33 2.52 5.05 4.28 4.23, 4.18 11.77 10.42, 6.68 -4.51

-2.95 T14 7.04 1.73 5.57 2.24 1.48 4.49 4.00 4.43, 4.09 n.a. -4.84 T15 7.23 1.54 5.43 1.88 2.00 4.53 3.42 3.58, 3.42 n.a. -4.68 TI6 7.10 1.49 5.27 2.29 1.70 4.66 4.40 4.01, 3.94 9.94 -4.26 TI7 7.58 1.69 6.23 2.38 2.09 4.56 3.21 3.05, 2.77 9.69 -5.68 GI8 7.31 6.22 3.06 3.45 4.98 4.34 4.63, 4.19 11.82 9.69, 6.18 -4.32

G20 8.21 6.34 2.47 2.71 4.79 4.34 4.34, n.a. 11.66 -4.42

n.a. n.a. G13 8.05 6.14 2.83 2.61 4.97 4.52 4.18, 4.18 11.58

G19 7.41 5.97 2.81 3.18 5.06 4.43 4.35, 4.35 10.96 9.21, 6.14 n.a.

each G-quartet. For example, in model 1B the base neighbouring G3 in the counterclockwise direction is GI 1, which is in the syn conformation, and in model 1C the base neighbouring G3 in the counterclockwise direction is G20, which is in the anti confor- mation.

Ten interresidue NOE contacts between T4 and G3 enabled us to determine accurately the orientation of T4 in relation to G3. T4 is in the anti conformation and is partially stacked over G3. This places the H1' of T4 within 0.42 nm of G3H1 and

within 0.38 nm of the hydrogen bonded G3NH, amino proton. Consequently, G3H1 can be independently identified by finding an NOE contact between T4H1' and an imino proton. Cross- peaks between T4H1' and G3H1 and between T14H1' and G13H1 are labelled U and T, respectively, in Fig. 2. Once the G3H1 resonance is identified, distinguishing between the two models is a matter of determining whether the guanine H8 that has an NOE to G3H1 is syn or anti. The cross-peak J in Fig. 2 identifies the guanine as a syn base and closer scrutiny reveals

636 Keniry et al. (ELM J. Biochenz. 233)

Fig. 4. Schematic representation of the observed NOE contacts among the loop thymines and between the loop thymines and the neighbour- ing stem guanines. Unbrokcn lines represent NOE that were quantified and included in the MARDIGRAS analysis of the NOESY spectrum. Broken lines represent NOE that were quantified but included in the restrained-molecular-dynamics as reasonable distance bounds ax judged from the peak intensity. Dotted lines represent NOE that were observed in the spectra but not included in the restrained-molecular-dynamics calculations. The latter contacts could only be assigned during the latter stages of the model refinement and the model distance between the proton pair fully agrced with the spectral intensity of the cross-peak.

the guanine to be GI 1. Similar arguments reveal the counter- clockwise neighbour of G I 3 to be G I (G13Hl-GIH8 is labelled L in Fig. 2). Therefore, model 1B has the correct strand orienta- tion. Assignments of the imino protons and some of the amino protons are given in the legend of Fig. 2 and in Table 1.

Chemical-shift trends. As might be expected from a folded quadruplex, there are some unusual chemical shift values, partic- ularly in the loop residues and the stem residues immediately adjacent to the loop. The H2’ resonance of T4, T14, T6, T I 6 and the H4’ resonance of T5, T15, T7, T I 7 experience large upfield shifts, whereas the H2’ resonance of G8, G18 experience large downfield shifts. The same trends are observed in the equivalent protons in [d(G,T,G,)],, strongly suggesting a similar loop structure (Smith and Feigon. 1993). There are also some interesting chemical shift changes among the stem deoxyribose protons. There is a large downfield shift of the H2’ resonance of G I 9 compared to G9, a large upfield shift of the imino proton of G I 9 compared to G9 and a large downfield shift of the HI’ proton of G12 compared to G2. Table 1 also includes ”P chemi- cal shifts, some of which differ from the values typically ob- served for DNA duplexes. These unusual values may arise from the presence of ,gt i deoxyguanosines, ring currents, stern-loop junctions or other features of the folded quadruplex structure.

Intranucleoside and sequential NOE contacts within the thy- mine loop. The important sequential contacts in the guanine stem are described above and elsewhere (Smith et al., 1994; Strahan et al., 1994), the following discussion will concentrate on the internal and sequential NOE contacts in the thymine loop. As this quadruplex is asymmetric, many of the comparable pro- tons of each loop are not magnetically equivalent, enabling an independent probe of the conformation of each loop. Despite the magnetic inequivalence of many protons in the two loops, it is obvious, after inspection of the intensity of the NOESY cross peaks of all comparable proton pairs, that the conformation of the two loops is almost identical. In the description of the se- quential NOE and the section on non-sequential NOE that fol- lows we will describe only the NOE of the T4-T7 loop but analogous cross-peaks are observed in the T14-TI7 loop. A schematic representation of the observed NOE among the loop residues and the adjacent stem guanines is displayed in Fig. 4.

m - G l 3 HI

14 H 4 I

- Q P - - - - - T16H5.5 e r n -- -TI6 H6

T6 H6

Fig. 5. Expanded region of a two-dimensional NOESY spectrum (q,, = 250 ms) of [d(G,T,G,)], in D,O at 20°C illustrating the unusual

G13H1’ cross-peaks. Sample and spectral details are given in Fig. 2. T6H6-T6H5’, H5”; T16H6-T16H5’, H5”; T4H4’-G3Hl’; T14H4’-

The orientation of T4 with respect to the stem is well deter- mined by 10 sequential NOE with G3. Many of the NOE, such as an intense G3H2”-T4H6 cross-peak and an observable T4Me- G3H8 cross-peak, in combination with the anti conformation of T4, indicate that the T4 base is stacked over the base of G3. There are some unusual NOE of moderate intensity involving deoxyribose and backbone protona that are not normally seen in linear DNA. The T4Hl’-T4H4’ NOE cross-peak is unusually strong, as described above. The T4H4’-G3HI’ (Fig. 5 ) and T4H2”-T5H5’, H5” cross-peaks indicate that the backbone is starting to turn at the T4-T5 junction.

Somewhat normal connectivity continues through to T5, which is also in an anti conformation. There is an intense T4H2”-TSH6 cross-peak, a moderately intense T4H2’-T5H6 cross-peak and a weak T4HI’-T5H6 cross-peak. There is also a T4H6-TjMe contact which, in combination with the other se-

Keniry et al. EL^ J . Biochem. 233) 637

Table 2. Comparison of the sixth-root residual indices R,“ and the standard R factors of [d(G,T,G,)], in the presence of Na’ at 20°C at two mixing times.

Mixing time R,,,,,\,,,,d,,, R,,,,c,rc,,,Ll, R,,,‘,, R;,,,tr,d”, R;,,,,clrc,,,,,,, R;,,,,,,

250 0.26 0.32 0.27 0.043 0.051 0.047 130 0.27 0.41 0.28 0.048 0.061 0.050

10.6 I g 10.8 - 2 11.0

11.2

11.4

11.6

11.8

12.0

12.2

/= - 1 G8-GI9

T6-G3 -

Tl6-G13-0 - 0

43- - 12.0 11.6 11.2 10.8 10.4 10.0 9.6

F1 (wm)

Fig. 6. Expanded region of a two-dimensional NOESY spectrum of [d(G,T,G,)], in 90 % H,0/10 %D,O. Spectral and sample details are given in Fig. 2.

quential contacts and the internal contacts, suggests that the base of T5 is stacked over the base of T4.

The normal HI ’, H2’, H2”-base proton sequential connectiv- ity is broken at the T5-T6 step. Moreover T6 has a very unusual cross-peak pattern ; there are no sequential T5H2’, H2” cross- peaks to T6H6 and even the intranucleoside T6H2’, H2”-T6H6 cross-peaks are very weak, suggesting an unusual glycosidic conformation. There is, however, an observable T5H4‘-T6H6 contact. Also worthy of note are unusually strong T6H6-T6H5’, H5” cross-peaks (Fig. 5) .

The sequential connectivity is restored at the T6-T7 step, as indicated by the presence of T7H6-T6H2” and T7H6-T6H3’ cross-peaks. There are no base-base NOE contacts between T6 and T7, indicating that the two aromatic rings are not stacked over each other. The sequential connectivity is broken again at the T7-G8 step. The only observable NOE contact between T7 and C8 is a weak T7H4’-G8H8 cross-peak. The latter cross- peak, however, served to verify the the strand sequence.

Non-sequential NOE. As mentioned previously, the three- quartet quadruplex has only 5’-syn-anti-3’ and 5’-syn-syn-3‘/5’- anti-anti-3’ sequential connectivity in the stem. This results in the closest non-sequential H8-H8 contact being about 0.85 nm (Strahan et al., 1994). Thus, non-sequential guanine H8-H8 cross-peaks are not expected for this quadruplex. However, sev- eral non-sequential imino-imino proton contacts are expected for this model. Many of these contacts would be too close to the diagonal to be observed, but those contacts that could be defini- tively assigned are shown in Fig. 6. The GSHI-Gl9H1, G3H1- G12Hl and G9Hl-GIlHl distances are all in the range 0.30-

Table 3. The range of RMSD calculated for each residue of the five structures from the simulation period of the final restrained-molecu- lar-dynamics run and the averaged structure of the final 4ps of the simulation period.

Residue RMSD of

all heavy atoms base heavy atoms

nm

GI G2 G3 T4 T5 T6 T7 G8 G9 G10 GI 1 GI 2 GI 3 TI 4 T15 TI 6 T17 G18 GI 9 G20 GI -G20

0.19-0.50 0.27-0.65 0.25-0.41 0.36-0.75 0.33-0.87 0.25-0.78 0.26-0.59 0.31 -0.45 0.44-0.57 0.48-0.89 0.21 -0.39 0.27 - 0.51 0.32-0.55 0.38-0.48 0.23 -0.71 0.23-0.49 0.30-0.61 0.34-0.71 0.29-0.68 0.26-0.90 0.40-0.60

0.22-0.44 0.23-0.46 0.16-0.32 0.30-0.80 0.33-0.58 0.23-0.66 0.24-0 64 0.25-0 ’ 0.29-0 1 ’

0.23-0 48 0.15-0 30 0.27-0.47 0.20-0.48 0.15 -0.55 0.30-0.81 0.16-0.42 0.38-0.66 0.22-0.56 0.18-0.66 0.27-0.71 0.30-0.47

34 nm in the final refined model. Of particular interest are the loop-stem T6H3-G3H1 and T16H3-Gl3HI contacts (labelled T6-G3 and T16-GI 3, respectively, in Fig. 6).

There are several non-sequential NOE contacts between the loop residues and the stem residues and among the loop thy- mines. These NOE are important for determining the correct conformation of the loop. The lack of even moderately intense intranucleoside NOE between the base proton and the H2’, H2” protons of T6 suggested an unusual conformation for this resi- due. This is further supported by several NOE contacts between the base protons of T6 and the base and deoxyribose protons of GI 1. NOE contacts are detected between T6Me and the H8 and HI’ protons of GI 1, between T6H6 and the H4’ proton of G1 I , and between T6H1’ and the imino proton of GI 1. These contacts serve to locate the T6 base in the vicinity of the base of GI 1. The intense NOE between the imino proton of T6 and the HI’ of T4 (The cross-peak labelled W in Fig. 2) serves to locate T6 near T4. There is an unexpected NOE contact between T7Me and T5H1’. This places T7 in the general vicinity of T5.

Analysis of the two-dimensional NOE data and structure re- finement. The program MARDIGRAS estimates interproton distances by an iterative procedure that applies the complete re- laxation matrix to minimize the deviations between calculated and experimental NOE intensities (Borgias and James, 1988,

638 Keniry et al. ( E m J. Biochern. 233)

Fig. 7. Monoviews of (left) the five structures and the averaged structure of [d(G,T,G,)], from the last 4 ps of the simulation from the final restrained-molecular-dynamic5 run and (right) the averaged structure showing the stacking of the Gl-G13-GlO-G18 G-quartet over the G2-G12-G9-G19 G-quartet (top) and the GZ-G12-G9-G19 G-quartet over the G3-Gll-GS-G20 G-quartet (bottom). The GI-GI 3-GIO-Gl8 plme I \ displayed in cyan. the G2-C12-G9-G19 plane IS di5pldyed in red dnd the G3-Gll-GS-G20 plane 15 displayed in yellow

1990). The initial input to MARDIGRAS consisted of the edge- looped and diagonal-looped quadruplex structures created as de- scribed above.

There are two commonly used indices for the evaluation of structural quality. One of these, the residual index, R, is analo- gous to the crystallographic R factor. James and coworkers (Thomas et al., 1991) have noted that the R factor is dominated by the fit of cross-peaks corresponding to short distances and have introduced a sixth-root residual index, R;. The crystallo- graphic R factor and R; are listed in Table 2 for the final averaged structure. The R and R; factors compare favorably with other recently published structures of DNA duplexes and quadruplexes.

Structural analysis. Five structures were obtained from the last 4 ps of the simulation from the final restrained-molecular-dy- namics run. These five structures and the averaged structure are displayed in Fig. 7. The conformation is well defined at the three G-quartets with the exception of the 3’ terminus of both strands. There is a larger degree of variation at TS, T7 and T15, T17, respectively, compared to the rest of the structure. The range of RMSD variations of all the heavy atoms for each base and nu- cleotide unit and the base atoms for the five simulation struc- tures compared to the averaged structure are presented in Table 3.

Previous studies of the four-quartet analog of this quadru- plex, i.e. [d(G,T,G,)],, have shown that the edge-looped form found in the crystalline state (Kang et al., 1992) has two wide and two narrow grooves whereas the diagonal-looped form found in solution (Smith and Feigon, 1992, 1993) has a wide groove, a narrow groove and two medium grooves. The width of the grooves of the averaged structure of [d(GIT,C,)], was monitored by measuring the nearest phosphorus-phosphorus dis- tance (minus the combined radii of the phosphorus atoms, 0.58 nm). The final structure was found to have three different

groove widths, the width of the wide groove was 1.31 nm, the width of the narrow groove was 0.27 nm and the width of the t v o medium grooves was 0.8 nm.

The three G-quartets are remarkably planar, considering that no restraints for imposing planarity were used in the latter stages of refinement. The central G-quartet has the greatest degree of planarity. Both the upper and lower G-quartets are slightly buck- led due to strain imposed by the loop conformation. G I 0 and G20 are more out of plane than the other bases in the two outer G-quartets. G I 0 is approximately 16” out of plane compared to the other three bases and G20 is similarly 6” out of plane com- pared to the other three bases in its G-quartet. The rise and twist between the two planes that encompass the S’-G,G,-3’ sequence are 0.34 nm and 18”, respectively. The corresponding values of rise and twist for the for the two planes that encompass the 5’- G,G,-3’step are 0.36 nm and 22” [The rise was calculated as the distance between the geometric center? of the respective planes and twist as the angle between the vectors formed from the coor- dinates of C1’ and the geometric center of the respective planes (Schultze et al., 1994b)I. The stacking of the two planes that encompass the S’-G,G,-3’ sequence (the red and yellow G- quartets in Fig. 7) is similar to that of other related quadruplexes (Wang and Patel, 1993a; Laughlan et al., 1994: Schultze et al., 1994b); the five-membered rings of the stacked guanines over- lap. By contrast, there is considerable stacking of both the five- membered and six-membered rings in the two planes that en- compass the S’-G,G,-3’ step (the red and cyan G-quartets in Fig. 7), not surprisingly reminiscent of the adjacent guanines in duplex DNA. Many of these details are similar to those that were previously described for the unrestrained-molecular-dy- namics runs carried out on the quadruplex stem region (Strahan et al., 1994).

The average backbone torsion angles and the range over these angles for the five structures are reported in Fig. X. With the exception of GX and (318, which are at the loop-stem junc-

Keniry et al. ( E m 3. Biochem. 233) 639

Fig.8. Variation of the backbone torsion angles a, /?, y, 6, E and 6, the glycosidic torsion angle x and the pseudorotation angle P of [d(G,T,G,)]*. The values for the averaged structure are designated with filled circles and the extreme values for the five simulation structures are designated by vertical bars. The dashed lines designate values for canonical B-DNA (Saenger. 1984).

640 Keniry et al. [ E l / / : .I. Bioclzem. 233)

tion. all the \tern guanines have backbone torsion angles at or near those characteristic of B-DNA. The 5' end of the loop at G3pT4 and T4pT5 (and GI 3pT14 and T14pT15) has backbone angles similar to B-DNA. There is a sharp turn at T5pT6 (and T15pT16) which is achieved by replacing the B-type backbone torsions angles, c ' - i ~ -u /?-;,' with the torsion angles E --[+-u'-pt- ;fa. There is a further adjustment at the backbone direction at T7pG8 where the normal B-type torsions, <--a--y ' are replaced by i ' -(I ' - 7 1 ' so that the remaining part of the stern backbone can follow a normal B-like twist. The sugar puckers fall mainly around the C2'-endo region, although G2, T4 and T14 are close to the 04'-endo conformation while the T6 and T I 6 sugars fall in the C3'-exo conformation. The combination of strong H1'- H4' NOE cross-peaks with the strong H3'-H4' DQFCOSY cross- peaks for the T4, T14, and G2 residues are all consistent with the 04'-eiido conformation and a correspondingly low value for d (Fig. 8).

DISCUSSION

An unexpected feature that has emerged from the recently determined NMR and X-ray structures of guanine-rich DNA is the variety of folding patterns that can be assumed by this form of DNA. There are many factors that affect the folding pattern, including the identity of the cation and subtle variations in nu- cleotide sequence and composition. Since there is increasing evi- dence for a number of biological roles for guanine-rich DNA (Bock et al., 1992; Blackburn, 1994; Stavenhagen and Zakian, 1994), an understanding of the factors that determine the global fold will contribute to our understanding of chromosome protec- tion and organization. the recognition of guanine-rich DNA by proteins and the role of guanine-rich DNA in enzyme regulation (Zahler et al.. 1991). We have determined a high-resolution structure for a DNA quadruplex that is an example of a DNA fragment which has sequential guanines i n the syn conformation. Two strands of [d(G,T,G,)]2 associate in the presence of N a ' in solution to form a DNA yuadruplex in which each strand loops diagonally across the guanine-rich stein.

Structural features of the thymine loop. The stacking of the thymine bases over the outer G-quartets is presented in Fig. 9. Our model of the quadruplex loop stacks two bases over the stem and has hydrogen bonding between the two stacked bases. T4 stacks over the adjacent guanine base in the outer G-quartet (Fig. 9), as does T I 4 in the other loop. T5 in turn stacks over T4, and T I 5 similarly stacks over Tl4. T6 assumes an unusual conformation, as mentioned above, that is clearly stabilized by hydrogen bonding to T4 and by stacking over a non-sequential base, GI 1 in the outer G-quartet (Fig. 9). This stacking pattern is mirrored i n the other loop, resulting in hydrogen bonding of T16 with T I 4 and stacking over GI . These stacking interactions may be responsible for the somewhat unusual sugar pucker for the T4, T6, T I 4 and T I 6 sugars in the structure determined from molecular-dynamics analysis. The length of the hydrogen bond between the H3 of T6 and the 0 2 of T4 was found to be 0.2 11 +- 0.025 nin with a bond angle of 161 ' 5 4". The equivalent bond between T I 4 and T I 6 has an internuclear distance of 0.205 5 0.022 nm and a bond angle of 161 O C 10". T7 and TI 7 are the most poorly determined of the loop bases because of the sinall number of interresidue NOE to these residues. We see no evidence for any greater mobility of T7 or T17 compared to the other loop residues, as reported in a closely related DNA quadruplex (Smith and Feigon, 1993). The resonance linewidths of T7 and T17 are the same as the other loop protons. T7 and T17 may be held in place by van der Waals' interactions or

Fig. 9. Stereoviews of (top) the loop conformation of the averaged structure of [d(G,T,G,)], illustrating the stacking and hydrogen bonding of the thymine bases and (bottom) stacking of the thymine bases (red) over the outer G-quartet guanine bases (yellow) in the averaged structure of [d(G,T,G,)],.

further refinement of the model with tighter backbone restraints may reveal hydrogen bonding between TS and T7 and between T I 5 and T17.

In the present model the equivalent nitrogen-oxygen in- ternuclear distances between T5 and T7 and between T15 and TI 7 are 0.238 2 0.029 nm and 0.282 t 0.053 nm, respectively, and the equivalent bond angles are 112O-C 15" and 93'2 17", respectively. Nevertheless, both T5 and T7 are turned in and form a partial hydrophobic pocket such that one side of each aromatic ring is shielded from the solvent. A similar structural element is observed in duplex hairpins and it has been suggested that shielding one side of the aromatic ring from the solvent may contribute to the stability of the hairpin loop (Blommers et al., 1991). Stereoviews of the quadruplex loop from three different perspectives are displayed in Fig. 10. The upper view is taken from directly above the loop and clearly shows the stacking of T5 over T4, the orientation of T4 relative to T6 and the coinpact nature of the loop created by turning each of the bases into the center of the loop. The middle view is a 60" rotation of the upper view and shows the relative orientation of T7 with respect to T5. The lower view is a further 60" rotation of the middle view.

The loop conformation adopts inany features common to hairpin loops observed in duplex DNA. Although the bend of this particular loop is shallower than that of a duplex DNA hair- pin, there are significant base-stacking and hydrogen-bonding interactions. In common with other DNA loops, the backbone at the 5' end of the loop follows the same general direction as the stein backbone and the first loop base stacks over the stem (Or- bons et al., 1987; Pieters et al., 1990; Blommers et al., 1991). A 180" turn at T5pT6 is achieved by using a ~-- i+-a ' -P'- j '+ tor- sion angle combination which has similarities to the c -( ' -ai -

P'-;i' combination used to achieve the turn i n d(ATCCTATTTA- TAGGAT) (Blommers et al., 1991) and the E ' - ~ ~ - ( x '-/Y-;i+ coinbi- nation in the cyclic dinucleotide d (pApA) (Blommers et al., 1988). The turn occurs between the second and third residue of the loop in the quadruplex but occurs between the third and the fourth residue of the loop in several duplex hairpins (Orbons et

Keniry et al. (ELM J. Biockem. 2.33) 641

T6 T4 T5

Fig. 10. Stereoviews of the loop conformation of the averaged struc- ture of [d(G,T,G,)], from three different perspectives. The middle and bottom views are rotated 60" and 120" respectively about the *-axis compared to the top view.

al., 1987; Blommers et al., 1988; Pieters et al., 1990). Although both loops stack the first loop base over the stem, the quadruplex loop stacks the third loop residue over the stem whereas the duplex hairpins stack the fourth loop residue over the stem (Or- bons et al., 1987; Blommers et al., 1988; Pieters et al., 1990). In both cases there is hydrogen bonding between the two stacked bases.

Another interesting similarity is at the 3' terminus of the loop. The duplex hairpins have a 1'' torsion angle at the fourth loop residue which stacks over the stem (Orbons et al., 1987; Blommers et al., 1988; Pieters et al., 1990) whereas the quadru- plex has a 7' torsion angle at the first stem residue, which is in essence the first stacked residue at the 3' end of the loop. It is interesting to note that the edge-looped form of [d(G4T,G,)l2, determined by X-ray analysis (Kang et al., 1992), has a loop structure closely resembling the hairpin loop in duplex DNA, where the first and the fourth bases are stacked over the stein and the sharp turn occurs between the third and the fourth resi- due in the loop. A major difference between this edge-looped quadruplex and the diagonal-looped form described in this study is the distance between the phosphorus atoms at the 3' and 5' termini of the loop, being approximately 1.75 nm in the edge- looped form and approximately 2 nm in the diagonal-looped form. This distance influences loop conformation through the resulting steric constraint for loop closure (Haasnoot et al.. 1986). Different loop diameters, measured at the base of the loop, and the resulting altered steric constraints may be the ma- jor reason for the edge-looped form stacking the fourth loop base over the stem and the diagonal-looped form stacking the third loop base over the stem.

Although the quadruplex structure has been determined using NOE and coupling constant data, the final structure should account for the chemical shift data. There are several chemical shift values for deoxyribose protons in the loop that differ con- siderably from values observed in right-handed duplex DNA. The H2' of T4 is located directly above the plane of the T.5 aromatic ring, rationalizing the upfield shift of this proton com- pared to the usual H2' shift range. Similarly, the H2' of T6 is

located below the plane of the T7 ring, the H4' of T5 is located above the plane of the T6 ring and the H4' of T7 is located directly above the plane of the aromatic rings of G8 explaining the upfield 4hifts of each of these resonance5. The downfield shift of the H2' of G8 is explained by the unusual y'torsion angle at G8 pulling this proton more in plane with the G8 aromatic ring.

Why guanines do not follow strict syn-anti alternation. Sev- eral folded DNA quadruplexes have now been studied by NMR and by X-ray crystallography (Aboul-ela et al., 1992; Kang et al., 1992; Wang and Patel, 1993a,b; Laughlan et al., 1994; Schultze et al., 1994a,b; Wang et al., 1994). All of these quad- ruplexes follow strict syn-anti alternation of the guanines in the quadruplex stem. Syz-anti alternation of DNA bases has been observed before in Z-DNA (Wang et al., 1979; Drew et al., 1980) but sequential bases in the syn conformation have never previously been observed, so it is initially surprising that the quadruplex formed by a dimer of [d(G,T,G,)], does not follow strict syn-ccnti alternation. Closer scrutiny of quadruplexes that may form with odd numbers of G-quartets and with loops at opposite ends of the stem, however, reveals that it is not possible to maintain strict syn-anti alternation without introducing an ele- ment of asymmetry in the sequential conformation of two other- wise identical strands. For example, to maintain strict syn-anti alternation of the bases and form the full complement of G- quartets, the base at the 5' end of one strand must be in the syn conformation while the base at the 5' end of the other strand must be in the anti conformation. An asymmetric conforma- tional element does occur in the two strands of [d(G,T4G,)l2 but this element occurs at the central base of each GGG sequence.

In all but one of the folded quadruplex structures determined, including the close analog to this quadruplex, [d(G,T,G,)],, the guanine at the 5' end of the diagonal loop is always in the anti conformation and the guanine at the 3' end of the diagonal loop is always in the syn conformation, while the guanine at the 5' end of the strand is always in the syn conformation and the guanine at the 3' end of the strand is always in the anti confor- mation. So why does this particular quadruplex deviate from strict syn-anti alternation? Why are the relative conformations of the guanine bases in the outer G-quartets of [d(G,T,G,)], and [d(G,T,G,)], identical ? The answer lies in the contributions to the stability of the complete structure, including the relative in- stability of the G,-G, stack (Strahan et al., 1994) along with effects from the loop thymines. The thymine residues fold into a relatively rigid structure. Inspection of the refined molecular models suggests that there are favorable energetic contributions from the stacking of the thymines over the guanines in the outer G-quartet, from hydrogen bonding between the stacked thy- mines and from van der Waals' interactions between the thymine bases. It is worthwhile to consider the effect on loop conforma- tion if, for example, the guanine at the 5' end of the loop is in the syrz conformation. For a sequential thymine to stack effi- ciently over a syn guanine, the thymine itself would have to be in the s y conformation which, for pyrimidines, is sterically unfavorable. Steric considerations would also force T6 to as- sume another position, resulting in a loop conformation that is radically different to that which is observed.

One might then ask why, in our structure, the stacking at the 5' end of the loop is so important. We suggest that the answer to this question lies in the folding principle for DNA hairpin loops proposed by Hilbers and coworkers (Haasnoot et al., 1986, 1987). Several hairpin loops in DNA fold such that the stacking of the DNA stem continues in the 3' direction into the loop as far as two to three bases and is then followed by a sharp turn (Orbons et al., 1987; Blommers et al., 1988; Pieters et al., 1990).

642 Keniry et al. (Eur: J . Bioc-hem. 233)

Exactly the same principle is seen in the quadruplex. The stack- ing pattern of the stem is continued through T4 and T5 followed by a sharp turn at TSpT6. At this point the quadruplex loop structure deviates from the duplex hairpin structure by seeking tn enhance stacking and hydrogen-bond interactions imposed by the differing geometry of the C-quartet. If the loop conformation is an important determinant or the quadruplex structure, then variations in the composition and length of the loop may induce radical changes in the sequential conformation and structure of the quadruplex stem residues. For example, the presence of an adenine in the loop of the human telomeric quadruplex, d[AG,- (T2AG,),] may explain the occurrence of strict sprz-unti alterna- tion of the bases in this quadruplex even though only three G- quartets are formed (Wang and Patel, 1993a). Furthermore, there is some evidence which suggests that d(G,T,AG,) forms a folded dimeric quadruplex in which the two loops are at the same end of the quadruplex (Balagurumoorthy and Brah- machari, 1994). Sequence-dependent structural variations in DNA hairpins have also been observed (Williamson and Boxer, 1989). The effect of loop composition and sequence on quadru- plex structure is presently under investigation in our laboratory.

Other quadruplexes belonging to the [d(G,T,G,)], family. After considering the experimental evidence presented above for the structure of the [d(G,T,C,)12 folded quadruplex and previous studies of the [d(C,T,C,)], folded quadruplex, it is possible to speculate on the conformation of other dimeric hairpin quad- ruplexes. All the folded quadruplexes formed from d(G,,T,G,,) where n is even [as well as d(G,T,G,) for which some NMR data is available (Wang et al., 1991)] should have strictly al- ternating syn-anti guanines because these structures can enhance the base stacking and hydrogen-bonding interactions without the need to deviate from strict syn-anti alternation of the guanines. When n is even, the two guanines that are involved in base stacking with the loop thymines, the 5’ terminal guanine and the guanine at the 5’ end of the loop can be syn and anti, respective- ly, without disrupting strict syri-anti alternation. All folded quad- ruplexes formed from [d(C,,T,C,)], where n is odd must have at least one sequential syn-syn segment in the guanine stem of each strand if the stacking arrangement of the loop and stein observed in [d(G,T,G,)], is to be maintained. If the 5’ terminal base is syn then the base at the 5‘ end of the loop cannot be anti unless strict syn-anti alternation of the guanine bases is abandoned. On the assumption that the number of sequential syz-anti guanines is maximized and that the disruption in syn-anti alternation oc- curs at one end of the quadruplex stem, one can hypothesize that all folded dimer quadruplexes of this type form from two strands which have the following conformation: d[G,(G,G,],,,-,,,2

for n = 3,7,11 ,IS... In summary, the oligonucleotide d(G,T,G,) folds into a di-

meric, diagonal-looped hairpin quadruplex containing three gua- nine quartets. The final folded conformation is determined by hydrogen bonding, base stacking and steric constraints in much the same way that these factors determine the conformation of hairpin loops in duplex DNA. Much work has focused on the structure of the G-quartet, but this and other work prove that the length and the base content of the linking sequences between the guanine-rich regions are also important factors. Organism- specific telomere base sequences may specify protein-recogni- tion features of the final folded structure.

TTTT(CsG.~),,,-,,/,G,) and d[(GsG,),,,-,,/,G,~Gs(GsG,),,,-i,/,I

This research was supported by the National Institutes of Health through grant GM 51650 awarded by the National Institute of General Medical Sciences, US Department of Health and Human Services, and benefited from the use of NMR facilities at the Australian National Uni-

versity. We also acknowledge the Computer Graphics Laboratory, Uni- versity of California, San Francisco, supported by grant RRO1081 from the Division of Research Resources, National Institutes of Health. US Department of Health and Human Services. The authors are grateful to Professor Tom James, University of California, San Francisco for a copy of the programs, CORMA and MARDICRAS and to Professor Richard Lavery, Instirut de Biologir Ph?siro-Clzirniclue, Paris for a copy of the program, CURVES 4. I .

REFERENCES Aboul-ela, F., Murchie, A. I. & Lilley, D. M. J. (1992) NMR study

of parallel-stranded tetraplex formation by the hexadeoxynucleotide d(TG,T), Nature 360, 280-282.

Balagurunioorthy, P. & Brahmachari. S. K. (1994) Structure and stability of human telomeric sequence. J . Riol. Cheni. 26Y. 21 858-21 869.

Bax, A. & Lerner, L. (1988) Measurement of ‘H-’H coupling constants in DNA fragments by 2D NMR, J. Mrrgr7. Reson. 79, 429-438.

Blackburn, E. H. (1990) Telomeres: Structure and synthesis. J. Biol. Chenz. 265, 5919-5921.

Blackburn. E. H. ( I 991) Structure and function of telomeres. Nutwe 350, 569-573.

Blackburn, E. H. (1994) Telomeres: No elid in sight, Cell 77, 621-623. Blommers, M. J. J.. Haasnoot, C. A. G., Walters. J. A. L. I . , Van der

Marel, G. A,, Van Boom, J. H. & Hilbers, C. W. (1988) Solution structure of the 3’-5’ cyclic dinucleotide d (pApA) . A combined NMR, UV Melting, and molecular mechanics study, Biochemi.str). 27, 8361 -8369.

Blommers, M. J. J., van de Ven, F. J.. Van der Marel, G. A,, Van Boom, J . H. & Hilbers, C. W. (1991) The three-dimensional structure of a DNA hairpin in solution: Two-dimensional NMR studies and struc- tural analysis of d(ATCCTATTTATAGGAT), Eur: J . Biockenr. 201, 33-51.

Bock, L. C., Griffin, L. C., Latham. J. A,. Vermaas, E. H. & Took, J. J. (1992) Selection of single-stranded DNA molecules that bind and inhibit thrombin, Nut~ire 355, 564-566.

Borgias, B. A. & James, T. L. (1988) COMATOSE - a method for constrained refinement of macromolecular structure based on two- dimensional nuclear Overhauser effect spectra, J. M a p . Reson. 79,

Borgias, B. A. & James, T. L. (1990) MARDIGRAS - a procedure for matrix analysis of relaxation for discerning geometry of an aqueous structure, J. M U ~ J I . Reso~i. 87, 475-487.

Briinger, A. (1992) XPLOR 3.1 A sysretn ,fbr X-ray crjstallograp/iy and NMR, Yale University Press, New Haven.

Davis, A. L. & Wimperis, S. (1989) A solvent suppression technique giving NMR spectra with minimal amplitude and phase distortion, I. Magti. Re~on. 84, 620-626.

Drew, H., Takano, T., Tanaka, S., Itakura, K. & Dickerson, R. E. (1980) High-salt d(CpGpCpG), A left-handed Z’ DNA double helix, Nature 286, 561-573.

Evans, T., Schon, E., Gora-Maslak, G.. Patterson, J. & Efstratiadis, A. (1984) S I -hypersensitive sites in eukaryotic promotor regions, Nit- cleic Acids Res. 12, 8043-8058.

Ferrin, T. E., Huang, C. C., Jarvis, L. E. & Langridge, R. (1988) The MIDAS display system, J. Mol. Graphics 6, 13-27.

Funabiki, H., Hagan, I., Uzawa, S. & Yanagida, M. (1993) Cell cycle- dependent specific positioning and clustering of centromeres and te- lomeres in fission yeast, J . Cell B i d . 121. 961 -976.

Gray, B. N., Owen, E. A. & Keniry, M. A. (1994) The solution confor- mation of a trisdeconucleotide containing the consensus binding site of the dnaA initiation protein, ELK J. Bioc-hem. 226, 115-124.

Gupta, G., Garcia, A. E., Guo, Q., Lu, M. & Kallenbach, N. R. (1993) Structure of a parallel-strandcd tetramer of the Oxyfrichn telomeric DNA sequence dT,G,, Biochemi.str?: 32. 7098-7103.

Haasnoot, C. A. G., Hilbers, C. W.. Van der Marel, G. A., Van Boom, J. H.. Singh, U. C.. Pattabiraman, N. & Kollman, l? A. (1986) On loop folding in nucleic acid hairpin-type structures, J. Bioniol. Struct.

Haasnoot, C. A. G., Blommers, M. J. J . & Hilbers, C. W. (1987) Confor- mational aspects of hairpin loops in DNA oligonucleotides, Springer Sex Biophys. I , 212-216.

493 -51 2.

Dyn. 3, 843-857.

Keniry et al. (ELM J . Biochem. 233) 643

Hare, D. R., Wemmer. D. E., Chou, S.-H., Drobny, G. & Reid, B. R. ( I 983) Assignment of the non-exchangeable proton resonances ot d(C-G-C-G-A-A-T-T-C-G-C-G) using two-dimensional nuclear mag- netic resonance methods. J. Mol. Biol. 171, 319-336.

Kang, C., Zhang, X., Ratliff. R., Moyzis, R. & Rich, A. (1992) Crystal structure of four-stranded Oxyfrichatelomeric DNA, Naturc, 356,

Kessler, H., Griesinger, C., Kerssebauni, R.. Wagner, K. & Ernst, R. R. (1 9 87) Separation of cross-relaxation and J cross-peaks in 2D rotat- ing-frame NMR spectroscopy, J. Am. Clzem. Soc. 109, 607-609.

Kilpatrick, M. W., Torri, A,, Kang, D. S., Engler, J. A. & Wells, R. D. (1986) Unusual DNA structures in the adenovirus genome, J . B id .

Kim, S.-G., Lin, L.-J. & Reid, B. R. (1992) Determination of nucleic acid backbone conformation by proton NMR, Biochemisfty 31,

Laughlan, G., Murchie, A. I. H., Norman, D. G., Moore, M. H., Moody, P. C. E., Lilley, D. M. J. & Luisi, B. (1994) The high-resolution crystal structure of a parallel-stranded guanine tetraplex, Science

Lavery, R. & Sklenar, H. (1992) CURVES 4.1 helical analysis ofirregu- lar nucleic acids, Laboratoire de Biochimie Theorique CNRS. Paris.

Liu, H., Thomas, P. D. & James, T. L. (1992) Averaging of cross-relax- ation rates and distances for methyl, methylene and aromatic ring protons due to motion or overlap. Extraction of accurate distances iteratively via relaxation matrix analysis of 2D NOE spectra, J . M a p . K i ~ o n . 98, 163-175.

Liu, Z., Frantz, J. D., Gilbert, W. & Tye, B.-K. (1993) Identification and characterization of a nuclease activity specific for G4 tetrastranded DNA, Proc. Nafl Acml. Sci. USA 90, 3157-3161.

Orbons, L. P. M., van Beuzekom, A. A. & Altona, C. (1987) Conforma- tional and model-building studies of the hairpin form of the mis- matched DNA octamer d(mSC-G-m'C-G-T-G-mSC-G), 1. Biomol. Struct. & Dyn. 4, 965-987.

Pearson, G. A. (1977) A general baseline-recognition and baseline-flat- tening algorithm, J. Mugn. Reson. 27, 265-272.

Pieters, J. M. L., de Vroom, E., Van der Marel, G. A,, Van Boom, J. H., Koning, T. M. G., Kaptein, R. & Altona, C. (1990) Hairpin structures of DNA containing arabinofuranosylcytosine. A combination of nuclear magnetic resonance and molecular dynamics, Biochemktry

Saenger, W. (1984) Principles o j nucleic acid structure, pp. 230-232, Springer-Verlag, New York.

Scaria, P. V., Shire, S. J. & Shafer, R. H. (1992) Quadruplex structure of d(G,T,G,) stabilized by K' or Na ' is an asymmetric hairpin di- mer, Proc. Naf l Acad. Sci. USA 89, 10336-10340.

Schultze, P., Macaya, R. F. & Feigon, J. (19944 Three-dimensional so- lution structure of the thrombin-binding DNA aptamer

Schultze, P., Smith, F. W. & Feigon, J. (1994b) Refined solution struc- ture of the dimeric quadruplex formed from the Oxytricha telomeric oligonucleotide d(GGGGTTTTGGGG), Strucfiire 2, 221 -233.

Smith, F. W. & Feigon, J. (1992) Quadruplex structure of 0,rytricha telomeric DNA oligonucleotides, Nuture 356, 164- 168.

Smith, F. W. & Feigon, J. (1993) Strand orientation in the DNA quadru- plex formed from the Oxylricha telomere repeat oligonucleotide d(G,T,G,) in solution, Biochrmistp 32, 8682-8692.

126- 131.

C h n . 261, 11 350-11 354.

3564- 3574.

265, 520-524.

29, 788-799.

d(GGTTGGTGTGGTTGG), J . Mol. Biol. 235, 1532- 1547.

Smith, F. W., Lau, F. U'. & Feigon, J. (1994) d(G,T,G,) forms an asym- metric diagonally looped dimeric quadruplex with guanosine 5'-sjn- symunfi and S'-.syr~-anti-urzti N-glycosidic conformations, Proc. Nut1

States, D. J., Haberkorn. R. A. & Ruben, D. J. (1982) A two-dimensional nuclear Overhauser experiment with pure absorption phase in four quadrants, J . Mugn. Reson. 48, 286-292.

Stavenhagen, J. B. & Zakian, V. A. (1994) lnternal tracts of telomeric DNA act as silencers in Sacckaromyces cerevisiae, Genes & Dev 8,

Strahan, G. D., Shafer, R. ti. & Keniry, M. A. (1994) Structural proper- ties of the [d(G,T,G,)], quadruplex : Evidence for sequential s.yn-syn deoxyguanosines, Nucleic Acid,r Res. 22, 5447-5455.

Sundquist, W. I. & Heaphy, S. (1993) Evidence for interstrand quadru- plex formation in the diinerization of human immunodeficiency virus genomic RNA, Proc. Nut1 Acad. Sci. USA 90, 3393-3397.

Thomas. P. D., Basus, V. J. & James, T. L. (1991) Protein solution struc- ture determination using distances from two-dimensional nuclear Overhauser effect experiments: Effect of approximations on the ac- curacy of derived structures, Proc. Nut1 Acad. Sci. USA 88, 1237- 1241.

Wang, A. H.-J., Quigley, G. J., Kolpak, F. J., Crawford, J. L., Van Boom, J. H., van der Marel, G. & Rich, A. (1979) Molecular structure of a left-handed double helical DNA fragment at atomic resolution, Na- ture 282, 680-686.

Wang, K. Y., Krawczyk, S. H., Bischofberger, N., Swaminathan, S. & Bolton, P. H. (1993) The tertiary structure of a DNA aptamer which binds to and inhibits thrombin determines activity, Biochemistry 32,

Wang, K. Y., Swaminathan, S . & Bolton, P. H. (1994) Tertiary struc- ture motif of Oxytrichn telomere DNA, Biochemistry 33, 7517 - 7527.

Wang, Y., Jin, R., Gaffney. B., Jones, R. A. & Breslauer, K. J. (1991) Characterization by 'H NMR of glycosidic conformations in the tetramolecular complex formed by d(GGTTTTTGG), Nuc/eic Acids

Wang, Y. & Patel, D. J. (1992) Guanine residues in d(T,AG,) and d(T2G4) form parallel-stranded potassium cation stabilized G-quad- ruplexes with anti glycosidic torsion angles in solution, Biochemisrry

Wang, Y. & Patel, D. J. (1993a) Solution structure of the human telomeric repeat d[AG,(T,AG,),] G-tetraplex, Structure I , 263 - 282.

Wang, Y. & Patel, D. J. (1993b) Solution structure of a parallel-stranded G-quadruplex DNA, J . Mol. B id . 234, 1171-1183.

Williamson, J . R. & Boxer, S. G. (1989) Multinuclear NMR studies of DNA hairpins. 2. Sequence-dependent structural variations, Bio- chemistry 28, 2831 -2836.

Zahler, A. M., Williamson, J . R., Cech, T. R. & Prescott, D. M. (1991) Inhibition of telomerase by G-quartet DNA structures, Nuture 350,

Zakian, V. A. (1989) Structure and function of telomeres, Annu. Rev.

A c u ~ . Sci. USA 91, 10546-10550.

1411- 1422.

11285-11292.

Re.s. 19, 4619-4622.

31, 8112-8119.

71 8-720.

Gene[. 23, 579-604.