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Nucleic Acids Research Pages 2981-2988  

A novel palindromic triple-stranded structure formed by homopyrimidine dodecamer d-CTTCTCCTCTTC and homopurine hexamer d-GAAGAG
Introduction
Materials And Methods
Results And Discussion
   The ordered structure has a palindromic triple-stranded conformation
   The palindromic structure is stabilized by Hoogsteen and Watson-Crick hydrogen bonding
   Nucleotides have `anti' conformation and C2[prime]-endo sugar puckers
   NMR spectrum as a function of pH and temperature shows that the C+.G:C triads remain intact at neutral pH
   Molecular mechanics calculations show that individual strands adopt conformation close to B-DNA
Acknowledgements
References
A novel palindromic triple-stranded structure formed by homopyrimidine dodecamer d-CTTCTCCTCTTC and homopurine hexamer d-GAAGAG

A novel palindromic triple-stranded structure formed by homopyrimidine dodecamer d-CTTCTCCTCTTC and homopurine hexamer d-GAAGAG

Sukesh R. Bhaumik, Kandala V. R. Chary*, Girjesh Govil, Keliang Liu1,+, H. Todd Miles1

Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India and 1National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA

Received February 9, 1998; Revised and Accepted April 22, 1998

ABSTRACT

We have carried out NMR and molecular mechanics studies on a complex formed when a palindromic homopyrimidine dodecamer (d-CTTCTCCTCTTC) and a homopurine hexamer (d-GAAGAG) are mixed in 1:1 molar ratio in aqueous solutions. Such studies unequivocally establish that two strands of each oligomer combine to form a triple-stranded DNA structure with a palindromic symmetry and with six T.A:T and six C+.G:C hydrogen-bonded base triads. The two purine strands are placed head to head, with their 3[prime] ends facing each other in the center of the structure. One-half of each pyrimidine strand contains protonated and the other half contains non-protonated cytosines. The two half segments containing protonated cytosines are hydrogen bonded to each of the two purine hexamers through Hoogsteen T.A and C+.G base pairing. The segments containing non-protonated cytosines are involved in Watson-Crick (A:T and G:C) base pairing. This leads to a palindromic triplex with a C2-dyad symmetry with respect to the center of the structure. The complex is less stable at neutral pH, but the cytosines involved in Hoogsteen base pairing remain protonated even under these conditions. Molecular mechanics calculations using NMR constraints have provided a detailed three-dimensional structure of the complex. The entire stretches of purine, and the pyrimidine nucleotides have a conformation close to B-DNA.

INTRODUCTION

The sequence-specific recognition of DNA duplexes by a third strand (1-12), has implications in gene-regulation and site-specific cleavage of genomic DNA (13-16). Interest in DNA triplexes is growing since homopyrimidine deoxyoligonucleotides may be used as `antisense' DNA in chemotherapy, where the gene expression can be influenced by the formation of a triple-stranded structure (17-20). Also, a third strand may be covalently linked to a DNA cleaving agent such as EDTA-Fe(II) to generate artificial nucleases useful in chromosome mapping (9,21-25).

There have been several studies on the structural characterization of pyrimidine(Y).purine(R): pyrimidine(Y) DNA triplexes (26-34). The second Y strand in such triplexes is parallel to the purine strand and forms Hoogsteen base-pairs with the standard antiparallel Watson-Crick R:Y double-helical DNA (Watson and Crick base pairs are represented in this paper as R:Y, and Hoogsteen base pairs as Y.R). A related class of systems is single-stranded oligonucleotide sequences, which form intra-molecular triple-stranded structures with well defined strand orientations (35-37). In most of these triple-stranded structures, base-base recognition is achieved through specific hydrogen bonds between T and A:T and between protonated cytosines (C+) and G:C leading to T.A:T and C+.G:C base triads (Fig. 1). An essential condition for the formation of C+.G:C triad is the protonation of C at the N3 position (pKa for free cytosine is 4.6 at 25°C) and hence such triplexes are found to be stable at acidic pH.


Figure 1. Schematics for hydrogen-bonded base pairing in (A) C+.G:C and (B) T.A:T triads, usually observed in triple-stranded DNA structures. The bases Y.R are hydrogen-bonded through Hoogsteen base pairing (vertical base pairs), while R:Y are bonded through Watson-Crick base pairing (horizontal base pairs). The orientation of the strands is indicated by `+' and `-' signs.

We have carried out NMR and molecular mechanics studies, on a complex formed when a palindromic homopyrimidine dodecamer (d-CTTCTCCTCTTC) and a homopurine hexamer (d-GAAGAG) are mixed in 1:1 molar ratio. This paper describes structural details of the conformation adopted by such a molecular system and the influence of hydrogen ion concentration and temperature on its stability.

MATERIALS AND METHODS

Oligonucleotides were synthesized with Applied Biosystems Model 380 B DNA synthesizer using a solid-phase cyanoethyl-phosphoramidite method. Oligonucleotides were purified on 20% denaturing polyacrylamide/bis (19:1) gel with 7.6 M urea, 0.09 M Tris-boric acid buffer, pH 8.3, 0.02 M EDTA. Molar extinction coefficients were measured by phosphate analysis, as described previously (38).

The sample for NMR studies was prepared by mixing 1:1 molar ratio of the pyrimidine and the purine strand. The final composition was as follows: ~11 mg of total material dissolved in 0.6 ml of either 99.9% 2H2O (for experiments on non-exchangeable protons), or in a mixed solvent consisting of 90% H2O and 10% 2H2O (for experiments on exchangeable protons). The final concentration was ~5 mM of each strand, with 0.05 M sodium acetate buffer and 0.1M NaCl. The pH was varied from 4.3 to 7.2. A temperature of 35°C was used in most experiments, though some studies were carried out in an extended temperature range of 10-60°C.


Figure 2. UV melting behavior of the complex formed by 1:1 molar mixture of d-GAAGAG and d-CTTCTCCTCTTC as a function of pH (1) 5.0, (2) 5.5, (3) 6.5 and (4) 7.4.

NMR experiments were carried out on a Varian Unity+ 600 MHz FT-NMR spectrometer. The NMR spectra in 90% H2O and 10% 2H2O include one-dimensional (1D) 1H spectra recorded with P11 pulse sequence (39) at different pH and temperatures, and two-dimensional (2D) nuclear Overhauser enhancement spectroscopy (NOESY) (40) with a 11 detection pulse and a mixing time of 200 ms. The NMR experiments in 2H2O include 2D clean total correlation spectroscopy (clean TOCSY) (41), with a mixing time of 80 ms and 2D NOESY with a mixing time of 200 ms. The chemical shifts have been measured with respect to TSP.

Junction minimization of nucleic acids (JUMNA) algorithm (42,43), which models nucleic acids using a combination of helicoidal and internal variables, has been used for the energy calculations. Bond lengths are kept fixed and the junction between successive nucleotides and the ring closure are ensured by quadratic constraints on the C4[prime]-O4[prime] and O5[prime]-C5[prime] distances.

The independent variables of each nucleotide are consistently three translations and three rotations, which position the nucleotide with respect to the helical axis system, the glycosidic dihedral angle, three valence angles and two dihedral angles within the sugar moiety and two backbone dihedrals [epsis] (C4[prime]-C3[prime]-O3[prime]-P) and [xi] (C3[prime]-O3[prime]-P-O5[prime]). Other sugar and backbone variables are dependent and are determined by the closure conditions that involve the C4[prime]-O1[prime] bond length within the sugar ring, the internucleotide O5[prime]-C5[prime] bond and the valence angles P-O5[prime]-C5[prime] and O5[prime]-C5[prime]-C4[prime]. These constraints are imposed via harmonic energy penalty terms. The corresponding force constants were adjusted to satisfy closure distances to within 0.02 Å and closure angles to within 1°. Energy minimizations were carried out using FLEX force field (42-46), included in JUMNA. The effect of solvent and counterions were simulated using a sigmoidal distance-dependent dielectric function (42-47), with a slope of 0.356 and a plateau value of 78, and by damping of net phosphate charges to -0.5e. Helical analysis was performed using the CURVES algorithm (48).

RESULTS AND DISCUSSION

In an earlier study, the UV melting curves at various pH for a 1:1 mixture of the two oligomers were studied (G.Raghunathan, K.Liu, H.T.Miles and V.Sasisekharan, unpublished). The UV absorbance at some selected pH values (5.0, 5.5, 6.5 and 7.4) is shown in Figure 2 for comparison with the NMR results. These results show that the system undergoes sharp monophasic melting over the entire range of pH's used, and the molecular system adopts an ordered secondary structure at lower temperatures. The melting temperature (Tm) decreases with increase in pH, suggesting that either the secondary structure formed at acidic pH undergoes a conformational transition as the pH is raised, or that the stability of the structure present under acidic conditions decreases at higher pH.

The ordered structure has a palindromic triple-stranded conformation

There are a total of six cytosines and six thymines in the Y strand, and three guanosines and three adenosines in the R strand. Some of the possible structures for the complex formed at acidic pH, when the purine and pyrimidine strands are mixed in 1:1 stoichiometry are: (i) an antiparallel DNA duplex with Watson-Crick base-pairing (Fig. 3A); (ii) a parallel-stranded DNA duplex with Hoogsteen base-pairing (Fig. 3B); (iii) a DNA duplex (formed from one pyrimidine and two purine strands) which consists of both parallel- and antiparallel-stranded domains (Fig. 3C). Such a duplex would coexist with a free homopyrimidine oligomer; (iv) a palindromic triple-stranded structure formed from two strands each of the purine and the pyrimidine oligomers, with six C+.G:C and six T.A:T base triads (Fig. 3D); (v) a non-palindromic triple-stranded structure with five C+.G:C, five T.A:T and two mismatch base triads C+-A-C and T-G-T (Fig. 3E). One can think of other possible structures too. However, under the experimental conditions used here, the NMR results conclusively prove the existence of structure 3D, as discussed later.

For DNA structures stabilized by hydrogen-bonded base pairing, information on the hydrogen bond schematics can be derived from exchangeable imino and amino proton resonances, and their NOE correlations with other intra- and inter-strand base protons. Each base pair in an ordered DNA structure has one or two imino protons, which are involved in hydrogen bonding. In addition, amino protons belonging to A, G, C and C+ may be involved in hydrogen bonding. Thus, the number of imino and amino proton signals and their positions in a 1H NMR spectrum depends on the type of hydrogen-bonded base pairs present, and on the nature of conformational equilibrium when more than one structure is present.


Figure 3. Some of the possible solution conformations for the 1:1 molar homopurine-homopyrimidine complex composed of an hexamer d-GAAGAG and a dodecamer d-CTTCTCCTCTTC: (A) antiparallel Watson-Crick duplex with three A:T and three G:C base pairs; (B) parallel duplex with three T.A and three C+.G Hoogsteen base pairs; (C) DNA duplex formed from one pyrimidine and two purine strands, which consists of both parallel- and antiparallel-stranded domains. The antiparallel-stranded domain is stabilized by three G:C and three A:T Watson-Crick base pairs while the parallel-stranded domain is stabilized by three C+.G and three T.A Hoogsteen base pairs; (D) palindromic triple-stranded structure formed from two strands each of the purine and the pyrimidine oligomers, with six T.A:T and six C+.G:C triads. In this structure the two purine strands are aligned with their 3[prime] ends facing head to head; (E) non-palindromic DNA triplex with five T.A:T, five C+.G:C and two mismatch C+-A-C and T-G-T triads (shown in boxes). In this structure the two purine strands are aligned head to tail such that the 3[prime] end of the first, faces the 5[prime] end of the second.

NMR studies have been carried out on a mixture of purine and pyrimidine strands mixed in 1:1 molar ratio at pH 4.3, 5.3 and 7.2. In each case, a study of the effect of temperature was also made. The melting behavior of the 1D 1H NMR spectrum is similar to the one observed from the UV melting curves (Figs 2 and 4). For detailed 2D NMR studies, a pH of 5.3 and a temperature of 35°C was found optimum. Under these conditions, the exchange behavior of the amino and imino protons is favorable and the resonance lines are relatively sharp. The melting temperature of the complex as observed by UV studies (Fig. 2) is such that one can work at the ambient temperature of the NMR probe.

Figure 4A shows the imino proton region of the 1D 1H NMR spectrum recorded in a mixed solvent of 90% H2O and 10% 2H2O, as a function of pH. The hydrogen-bonded imino protons of G, T and protonated cytosine (C+) can be easily identified from their characteristic resonance frequencies which lie in the 12-16 p.p.m. region. Two important points emerge from this Figure: (i) the system acquires a highly ordered structure over the entire range of conditions used here, with imino protons of several purine and pyrimidine bases locked in hydrogen-bonded base pairs; and (ii) the structure shows only minor variations under the experimental conditions used.

Figures 5 and 6 show selected regions of the 2D NOESY spectrum of the sample recorded in 90% H2O and 10% 2H2O at 35°C and pH 5.3. The strategies for assignment of individual exchangeable and non-exchangeable protons in ordered DNA structures based on the chemical shifts, J-coupling correlations and intra- and inter-strand NOEs, are well documented (49,50). Only salient features which have a bearing on the structure of the present system will be discussed. It may be pointed out that the sequence-specific resonance assignments in NMR of large molecules and their secondary structure determinations go hand in hand. For better understanding, we have drawn the structure of the complex finally derived from NMR studies, and the resonance assignments at the top of each Figure, even though such information was available to us only after going through the entire sequential assignment procedure.

As a first step, we have identified two sets of cross peaks belonging to amino protons of a protonated cytosine which usually resonate in the 8-10 p.p.m. region, and two sets of cross peaks for a non-protonated cytosine (usual chemical shift range 6-8.5 p.p.m.) (Fig. 5A). In each case, the hydrogen-bonded (HB) and the solvent-exposed (X) amino protons (belonging to the 4-NH2 group in cytosines), could be separately identified. A unique feature of the NOESY spectrum is a network of intra- and inter-base cross peaks involving HB and X amino protons between themselves and with the H5 and H6 protons of the two cystosines. Such peaks in a NOESY spectrum can arise from dipolar interactions between protons of the two cytosines provided these are in close proximity in space. In the pyrimidine strands, there are no sequentially adjacent cytosines which can lead to intra-strand C+->C NOEs. Therefore, such a pattern is indicative of the formation of a hydrogen-bonded C+:C base pair or a C+.G:C triad involving cytosines from two different pyrimidine strands. An alternative possibility that the cross peaks are due to the two cytosines undergoing chemical exchange between the C and the C+ forms has been ruled out from the complete sequence specific resonance assignment. Among the proposed structures, only 3D and 3E satisfy the observation of inter-strand C->C+ NOE cross peaks, as a consequence of the presence of C+.G:C base triads in these structures.


Figure 4. The effect of temperature (T) and pH on the 1D 1H NMR spectrum of the triple-stranded structure formed by 1:1 molar ratio of d-GAAGAG and d-CTTCTCCTCTTC in a mixed solvent of 90% H2O and 10% 2H2O. The 12-16 p.p.m. region shown here, is due to the hydrogen-bonded imino proton resonances of T(3NH), G(1NH) and C+(3NH). The peaks are labeled based on the complete sequential assignments of the imino protons: (A) spectra at three different pH, below the corresponding melting temperatures (Tm) as determined from the UV melting curves and NMR experiments. (B) Spectra as a function of temperature at pH 7.2; the broadening is indicative of weakening and breaking of hydrogen bonds involving imino protons for individual nucleotides. Beyond the melting temperature the resonances broaden out completely in a highly cooperative fashion. (C) Similar spectra at pH 5.3, where the melting temperature is much higher.

Both structures 3D and 3E involve two purine hexamers sandwiched between two palindromic pyrimidine dodecamers and are stabilized by Y.R:Y types of hydrogen-bonded triads. The total number of observed resonances is half of that expected from a non-symmetrical Y.R:Y triple-stranded structure such as the one in Figure 3E. Structure 3D has a C2 dyad symmetry, with the two short purine strands facing head to head in the center, and the protonated and non-protonated cytosines in the two pyrimidine strands lying on opposite ends. Thus, this structure is consistent with the observation of fewer NMR signals than expected from an unsymmetrical structure. After the completion of sequential assignment, it became clear that the cytosines giving rise to the pattern in Figure 5A are protonated C16 and non-protonated C9 arising from the triple-stranded structure in Figure 3D, which are spatially close due to the formation of C16+.G4:C9 triad.


Figure 5. Selected regions of pure-absorption NOESY spectrum of 1:1 molar mixture of d-GAAGAG and d-CTTCTCCTCTTC recorded in a mixed solvent of 90% H2O and 10% 2H2O at 35°C and pH 5.3. Experimental parameters were as follows: t1max = 22.5 ms, t2max = 205.0 ms, recycle delay = 1 s, 96 scans/t1 increment, time-domain data points were 512 and 2048 along t1 and t2 dimensions, respectively. The 1H-carrier frequency was kept on the water resonance. The data were multiplied with sine bell window functions shifted by [pi]/4 and [pi]/8 along t1 and t2 axes, respectively and zero-filled to 1024 data points along t1 dimension prior to stripped 2D-FT. The digital resolution along F2 and F1 axes, corresponds to 3.9 and 1.85 Hz/pt, respectively. The chemical shift positions of the various imino and amino protons obtained after complete sequential assignments are marked along the F1 and F2 axes. On the right hand, we have indicated the intra- and inter-strand connectivities involving protons belonging to various nucleotides in the triple-stranded complex. (A) NOE connectivities from C(H5)/C(H6)/C(4NH2)/C+(4NH2) protons (F1 axis) to the C+(4NH2) protons (F2 axis). The suffix `x' to the nucleotide number identifies the exposed amino proton (X) of the cytosine amino group. The hydrogen-bonded (HB) and the non-hydrogen-bonded (X) protons are marked separately in the Figure. (B) Inter-imino proton NOE connectivities. The intra-nucleotide cross peaks are shown with the corresponding nucleotide number along the sequence. The inter-nucleotide cross peaks are shown with the corresponding numbers of the nucleotide units to which the two protons belong [for example, 10-11 identifies an NOE cross peak between the T11(3NH) (F1 axis) and the T10(3NH) (F2 axis)].


Figure 6. Selected region of pure-absorption NOESY spectrum of 1:1 molar mixture of d-GAAGAG and d-CTTCTCCTCTTC. Experimental conditions and the NMR parameters are the same as those shown in Figure 5. This region shows NOE cross peaks between H2/H6/H8/NH2 protons and the imino protons [C+(3NH), T(3NH) and G(1NH)]. The NOE cross-peaks (a-g) in each column (identified by the imino proton) are assigned as follows (hydrogen-bonded and exposed amino protons abbreviated as HB and X, respectively). In the overlapped regions, the two columns of peaks are distinguished by single letter a-f with and without primes. [C+16(3NH) -> A3(H8) (a), G4(H8) (b), A5(H8) (c), C+16(4NH2) (X, d), C+16(4NH2)(HB, e)]. [T10(3NH) -> C9(4NH2) (X, a), A3(H2) (b), A2(H2) (c), C9(4NH2) (HB, d), C+16(4NH2) (X, e), C+16(4NH2) (HB, f)]. [T8(3NH) -> C9(4NH2) (X, a), A5(H2) (b), A5(6NH2) (c), C9(4NH2) (HB, d), C+16, C+18 (4NH2) (X, e), C+18(4NH2) (HB, f), C+16(4NH2) (HB, g)]. [T11(3NH) -> A3(H2) (a), A2(H2) (b)]. [T14(3NH) -> A3(H8) (a[prime]), A2(H8) (b[prime])]. [T17(3NH) -> G6(H8) (a), G4(H8) (b), A5(H8) (c), C9(4NH2) (HB, d), C+16(4NH2) (X, e), C+16(4NH2) (HB, f)]. [T15(3NH) -> A3(H8) (a), A2(H8) (b), A3(6NH2) (c), C9(4NH2) (HB, d), C+16(4NH2) (X, e), C+16(4NH2) (HB, f)]. [G6(1NH) -> C7(4NH2) (X, a), A5(H2) (b), C7(4NH2) (HB, c), C+18(4NH2) (X, d), C+18(4NH2) (HB, e)]. [G4(1NH) -> C9(4NH2) (X, a), A3(H2) (b), A5(H2) (c), A3(6NH2) (d), C9(4NH2) (HB, e), C+16(4NH2) (X, f); C+16(4NH2) (HB, g)].


The palindromic structure is stabilized by Hoogsteen and Watson-Crick hydrogen bonding

The NOE cross peaks between the imino protons (Fig. 5B), confirm the formation of a palindromic DNA triplex with six C+.G:C and six T.A:T triples. We identify six imino proton resonances of thymines (two of which are nearly degenerate) from the observation of intra-nucleotide T(CH3)-T(3NH) NOEs (not shown). These are located at 13.20, 13.45, 13.52, 13.68, 14.12 and 14.52 p.p.m. From the chemical shifts of these protons, it is clear that all six thymines are hydrogen bonded. We have also identified two hydrogen-bonded imino proton resonances of guanosines at 12.82 and 12.92 p.p.m. (Fig. 5B). Both these G(1NH) protons show NOEs to the T(3NH) proton at 14.12 p.p.m.. This suggests that the G4-A5-G6 segment of the purine strand is in close spatial proximity to the thymine responsible for the imino proton signal at 14.12, and that this thymine is hydrogen-bonded to A5, thereby leading to inter-strand NOE between its imino proton and those of G4 and G6. In terms of structure 3D, the signal at 14.12 p.p.m. is thus assigned to T8(3NH) of the A5:T8 base pair, while those at 12.82 and 12.92 p.p.m. belong to G4(1NH) and G6(1NH) in the G4:C9 and G6:C7 base pairs. The G(1NH) proton at 12.82 p.p.m. shows a strong NOE to the T(3NH) signal at 14.52 p.p.m.; these resonances therefore belong to G4(1NH) and T10(3NH), respectively. The resonance at 12.92 p.p.m. can thus be assigned to G6(1NH). The assignments of the imino protons for G4, G6, T8 and T10 serve as starting points for sequential assignments of the remaining imino proton resonances. Sequential connectivities have been observed between the base-paired imino protons along the following pathway: G6(1NH) (belonging to G6:C7 base pair) -> T8(3NH) (A5:T8) -> G4(1NH) (G4:C9) -> T10(3NH) (A3:T10) -> T11(3NH) (A2:T11) (Fig. 5B). The type of NOE pattern observed, where cross peaks are present between G(1NH) of one base pair, and T(3NH) of the adjacent base pair, is indicative of Watson-Crick hydrogen bonding between the segments G1-A2-A3-G4-A5-G6 and C12-T11-T10-C9-T8-C7, aligned antiparallel to one another. Cytosines C7, C9 and C12, do not show imino proton signals, and are therefore uncharged. We do not observe a NOE cross peak between G1(1NH) and T11(3NH), but this is expected, since G1:C12 is a terminal base-pair and is expected to show base-pair fraying effects due to segmental motions of the three strands at the ends of the triple-stranded structure (49,50).

The imino protons of T8 and T10 show NOE cross peaks with the T(3NH) signals located at 13.45 and 13.20 p.p.m., respectively. These interactions are possibly due to the spatial proximity of T8 and T10 in the strand C7-T8-C9-T10-T11-C12 (which has uncharged cytosines), with the thymines in the section of the pyrimidine strand containing the C+ residue observed in Figure 5A. In terms of structure 3D, such NOEs are a result of T8->T17, and T10->T15 short contacts. Using T17(3NH) and T15(3NH) signals thus assigned, the following intra-strand sequential connectivities can be established: T17(3NH) (belonging to A5.T17 base pair) -> C16+(3NH) (G4.C16+) -> T15 (3NH) (A3.T15) -> T14(3NH) (T14.A2). No NOEs were observed from G4(1NH) with either T15(3NH) or T17(3NH), as expected from Hoogsteen base-pairing between the segments G1-A2-A3-G4-A5-G6 and C13+-T14-T15-C16+-T17-C18+. Again, a sequential NOE was not observed between T14 and C13+, which can be ascribed to terminal base pair fraying (49,50). We also do not observe an NOE between imino protons of T17 and C18+.

After the sequential assignments discussed above, we are left with three unassigned broad resonances at 12.75, 14.82 and 15.12 p.p.m.. The first of these, is in the region of hydrogen-bonded G(1NH) imino protons and can thus be assigned to G1. The other two are in the range where hydrogen-bonded C+(3NH) resonances are expected and can therefore be attributed to C18+(3NH) and C13+(3NH). Both these peaks are fairly broad at acidic pH, as seen from the 1D NMR spectrum.

Figure 6 shows the NOE cross peaks between the hydrogen-bonded imino protons on one hand, and base protons such as H2, H6, H8 and NH2 (HB and X) on the other. The assignment of the various cross peaks has been carried out using the usual procedures (49,50) and are indicated in the legend of Figure 6. This section of the NOESY spectrum shows a highly intricate pattern which is consistent with structure 3D. The hydrogen-bonding schemes for the C+.G:C and T.A:T triads involving Hoogsteen and Watson-Crick base pairing (Fig. 1) are confirmed by the NOE cross peaks. For example, in the T.A:T triads, a strong NOE is expected between the T(3NH) and the A(H2) protons for the Watson-Crick A:T base-pair, while for the Hoogsteen T.A base pair, a strong NOE is expected between T(3NH) and A(H8). Similarly, for the Watson-Crick G:C base pair in a C+.G:C triad, a strong G(1NH)->C(4NH2,HB) and a medium intensity G(1NH)->C(4NH2,X) NOE cross peak are expected. For the Hoogsteen C+.G base pair, a strong G(H8)->C+(3NH) cross peak is expected. NOEs, diagonally across the strands between adjacent G:C and A:T Watson-Crick base pairs, are also expected. All these features are observed in Figure 6. The intricate networks of inter- and intra-strand NOESY cross peaks discussed above, unequivocally establish the hydrogen-bonded base-paired network and thus the conformation of the palindromic triplex as shown in Figure 3D.

Nucleotides have `anti' conformation and C2[prime]-endo sugar puckers

Even though the imino proton region of the triple-stranded structure is well resolved, the non-exchangeable proton region of the NOESY spectrum recorded in 2H2O shows considerable overlap of peaks. Only some of the non-exchangeable sugar protons of the purine nucleotides which are better resolved, have been assigned.

Wherever the H2[prime], H2[prime][prime] protons were clearly identified, an attempt was made to extract J-coupling information from the DQF-COSY spectrum (not shown here). The three bond coupling constants belonging to the sugar protons indicate that such nucleotides assume sugar conformation in the S domain of the pseudorotational map. Furthermore, from the relative intensities of the resolved NOEs between the base and the sugar protons, we conclude that the glycosidic bond angle is in an `anti' conformation. In view of the serious spectral overlaps, very few of the sequential NOEs between the base protons and the sugar protons of the neighboring nucleotides, could be assigned.

NMR spectrum as a function of pH and temperature shows that the C+.G:C triads remain intact at neutral pH

An essential condition for the formation of a triple-stranded structure with C+.G:C base triads is protonation of cytosine at the N3 position. The pKa for free cytosine is 4.6 at 25°C. Hence, triple-stranded structures involving C+.G:C triads are generally stable only at acidic pH. At higher pH, such structures may melt into randomly coiled strands or result in the formation of other ordered structures such as duplexes (51). In the present case, the triple-stranded structure is stable even at pH 7.2 and the cytosines involved in Hoogsteen base pairing remain protonated. However, the stability and thus the melting temperature Tm of the triplex decreases with increasing pH, as shown both by UV and NMR melting curves (Figs 2 and 4).


Figure 7. A stereoview of the energy-minimized conformation of the triple-stranded structure involving two strands each of d-GAAGAG and d-CTCTCCTCTTC. Both the purine (R) strands are shown in yellow, while two pyrimidine strands are shown in pink and green. As discussed in the text, this triplex structure has C2-dyad symmetry with respect to the center of the structure. In the top half of the triplex, yellow (R) and green (Y) strands are involved in Hoogsteen base-pairing, while yellow (R) and pink (Y) strands are involved in Watson-Crick base pairing. In the lower half of the complex, yellow (R) and green (Y) strands are involved in Watson-Crick base-pairing, while yellow (R) and pink (Y) strands are involved in Hoogsteen base pairing.

Scheme 1.
5' C+ T T C+ T C+ 3'
5' G A A G A G 3'
3' C T T C T C 5'

Scheme 2.
  24                   13  
5' C+ T T C+ T C+ C T C T T C 3'
  1                   12  
5' G A A G A G//G A G A A G 5'
  36                   25  
3' C T T C T C C+ T C+ T T C+ 5'

The melting behavior of the triple-stranded palindromic structure has been monitored as a function of temperature by 1D NMR at pH 7.2 and 5.3 (Fig. 4B and C). Using the assignments of the imino and amino protons, one can monitor melting of the structure at the level of individual nucleotides. The assignment of all the 3NH protons belonging to C+13, C+16 and C+18 at pH 5.2 and 7.2 suggests that in this triple-stranded structure all the cytosines are protonated even at pH 7.2. It is also observed that rapidly exchanging C13+(3NH) resonance (which is broad even well below the melting temperature), broaden out faster. This is followed by the broadening of other imino protons such as C+18(3NH) and G1(3NH), at the end of the triple-stranded structure. However, even up to 50°C at pH 5.3, and up to 20°C at pH 7.2, hydrogen-bonded imino protons for all the six thymines, G4 and G6, and even C+16, can be observed as relatively sharp peaks. The middle section, where the two purine strands are present head to head, also shows a high degree of stability as reflected in sharp resonances from the imino protons of G6 right up to the melting point. Beyond the above two temperatures, there is a sudden disappearance of all the exchangeable imino protons, suggesting a highly cooperative melting. The chemical shifts of the various NMR signals show that the melting does not go through any other conformational structure. Similar monophasic triplex->single-strand transitions have been observed in some triplexes (30,31,37). However, in other cases a biphasic transition has been reported, and it has been suggested that a triple-stranded structure first melts into a Watson-Crick duplex and a single strand, which then melts into three single strands at the higher melting temperature.

Molecular mechanics calculations show that individual strands adopt conformation close to B-DNA

In molecular modeling of the structure, first a Y.R:Y triple helix with three C+.G:C and three T.A:T base triads was generated using Insight II (Biosym) and JUMNA as shown in scheme 1. The 3[prime] end of the hexameric purine strand was capped with OH.

This was followed by converting it into a full triple-stranded structure which satisfied the palindromic symmetry, with the required sequence and base paired triads. This resulted in leaving the 3[prime] ends of the purine strands facing each other. The palindromic triple-stranded structure thus generated is shown in scheme 2.

The structure in scheme 2 thus generated, was subjected to energy minimization by JUMNA algorithm, using the information about various hydrogen bonds and NOE distance constraints, derived from NOESY data (81 in total). During the minimization process, no effort was made to maintain the dyad symmetry. However, the torsion angles of the final structure are fairly close to what is expected from palindromic symmetry. The minimum energy structure was critically examined for proper hydrogen-bond lengths and angles in the Watson-Crick and Hoogsteen base pairs, stereo chemical feasibility of the various torsional angles and any sterically hindered non-bonded inter-atomic distances. The structure satisfied all these criteria.

A stereo view of the final structure is shown in Figure 7. The backbone torsional angles, glycosidic bond torsional angles and the pseudorotational phase angles (P), are listed in Table 1. The numbering of the nucleotides corresponds to scheme 2.

The energy-minimized structure shows that the sugar puckers lie in the S domain of the pseudorotational wheel and most of the nucleotides assume a sugar pucker very close to C2[prime]-endo. The sugars of C13 and C24+ have C1[prime]-exo puckers, which are also in the S domain. A slightly different behavior for these two nucleotides can be expected since these are present at the ends of the triplex. The backbone torsional angle [delta] (-O3[prime]-C3[prime]-C4[prime]-C5[prime]-) is dictated by the sugar pucker and also reflects the fact that sugar puckers are in the S domain. Other than this, no major differences in the sugar pucker are seen among the individual nucleotides responsible for Hoogsteen base pairs and those involved in Watson-Crick base pairs.

The backbone torsion angles for B-DNA structures are well documented from single crystal X-ray diffraction and NMR studies on DNA duplexes. The torsion angles [alpha] (-O3[prime]- P-O5[prime]-C5[prime]-) and [xi] (-C3[prime]-O3[prime]-P-O5[prime]-) bonds are usually in the g-, g- domain, the [beta] (-O3[prime]-O5[prime]-C5[prime]-C4[prime]-) is invariably trans, while [gamma] (-O5[prime]-C5[prime]-C4[prime]-C3[prime]-) is g+. The torsion angles, [epsis] (C4[prime]-C3[prime]-O3[prime]-P-) are also usually trans. A comparison of these values with those listed in Table 1 show that both the purine and the pyrimidine strands in the triple-stranded structure prefer torsion angles close to those found in B-DNA.

ACKNOWLEDGEMENTS

The facilities provided by the High Field FT NMR National Facility supported by the Department of Science and Technology, India, Department of Biotechnology, India, Council of Scientific and Industrial Research, India and Tata Institute of Fundamental Research, Mumbai, India are gratefully acknowledged. We thank Drs V. Sasisekharan and Sanjay Sanghani for helpful discussions on the symmetry and simulations of the triple-stranded structure.

Table 1. Backbone torsion angles and phase angles (P) of the palindromic DNA triplex obtained by constrained molecular mechanics calculations (the numbering of the nucleotides corresponds with scheme 2)
Residues [alpha] [beta] [gamma] [delta] [epsis] [xi] [chi] P
G1 - - 48 147 -178 -107 66 168
A2 -66 -179 49 137 -173 -102 65 154
A3 -66 -179 50 133 -173 -113 64 145
G4 -66 -175 53 139 -176 -107 64 153
A5 -64 177 52 136 -174 -115 63 150
G6 -69 -171 58 139 - - 59 154
G7 - - 50 140 -175 -108 65 151
A8 -66 -175 52 137 -177 -113 65 145
A9 -67 -179 55 139 -173 -108 64 153
G10 -65 -172 50 137 -178 -106 65 154
A11 -68 -179 53 133 -173 -113 62 152
G12 -64 -178 54 136 - - 61 156
C13 -69 -178 54 127 - - 48 138
T14 -68 180 56 135 -175 -102 63 149
T15 -69 -178 56 137 -174 -109 62 153
C16 -70 -178 56 134 -173 -111 56 149
T17 -68 -178 56 139 -174 -110 62 155
C18 -68 -179 56 136 -173 -111 54 151
C+19 -72 -176 56 141 -172 -108 54 163
T20 -71 -179 57 143 -174 -103 65 166
C+21 -74 -176 54 135 -171 -107 52 152
T22 -72 -180 55 143 -173 -105 62 167
T23 -70 -178 54 135 -174 -108 64 152
C+24 - - 51 118 -170 -102 55 125
C+25 - - 53 139 -168 -101 41 158
T26 -71 -177 55 138 -173 -109 63 153
T27 -71 -180 54 144 -173 -103 64 165
C+28 -75 -177 57 129 -171 -109 54 142
T29 -71 -179 54 143 -174 -103 65 165
C+30 -74 -176 56 134 -172 -109 55 150
C31 -68 -179 55 135 -174 -111 62 149
T32 -69 -180 56 138 -174 -108 64 154
C33 -68 -178 55 132 -173 -111 54 145
T34 -71 -178 56 136 -174 -106 63 150
T35 -67 180 54 137 -174 -111 62 154
C36 -73 -173 51 140 -169 - - 161

REFERENCES

1. Felsenfeld. G., Davies, D. R. and Rich, A. (1957) J. Am. Chem. Soc. 79, 2023-2024.

2. Hoogsteen, K., (1959) Acta Cryst. 12, 822-823.

3. Lipsett, M. N. (1963) Biochem. Biophys. Res. Commun. 11, 224-228.

4. Howard, F. B., Frazier, J., Lipsett, M. N. and Miles, H. T. (1964) Biochem. Biophys. Res. Commun. 17, 93-102.

5. Felsenfeld. G. and Miles, H. T. (1967) Annu. Rev. Biochem. 36, 407-448.

6. Morgan, A. R. and Wells, R. D. (1968) J. Mol. Biol. 37, 63-80. MEDLINE Abstract

7. Lee, J. S., Johnson, D. A and Morgan, A. R. (1979) Nucleic Acids Res. 6, 3073-3091. MEDLINE Abstract

8. Arnott, S. and Selsingh, E. (1974) J. Mol. Biol. 88, 509-521. MEDLINE Abstract

9. Moser, H. E. and Dervan, P. B. (1987) Science 238, 645-650. MEDLINE Abstract

10. Mirkin, S. M., Lyamichev, V. I., Drushlyak, K. N., Dobrynin, V. N., Filippov, S. A. and Frank-Kamenetskii, M. D. (1987) Nature 330, 495-497. MEDLINE Abstract

11. Voloshin, O. N., Mirkin, S. M., Lyamichev, V. I., Belotserkovskii, B. P. and Frank-Kamenetskii, M. D. (1988) Nature 333, 475-476. MEDLINE Abstract

12. Hanvey, J. C., Shimizu, M. and Wells, R. D. (1988) Proc. Natl. Acad. Sci. USA 85, 6292-6296. MEDLINE Abstract

13. Wells, R. D., Collier, D. A., Hanvey, J. C., Shimizu, M. and Wohlrab, F. (1988) Fed. Amer. Soc. Exp. Biol. J. 2, 2939-2949.

14. Htun, H. and Dahlberg, J. E. (1988) Science 241, 1791-1796. MEDLINE Abstract

15. Mirkin, S. M. and Frank-Kamenetskii, M. D. (1994) Annu Rev. Biophys. Biomol. Structure 32, 541-576.

16. Frank-Kamenetski, M. D. and Mirkin, S. M. (1995) Annu. Rev. Biochem. 64, 65-95. MEDLINE Abstract

17. Cooney, M., Czernuszewicz, G., Postel, E. H., Flint, S. J. and Hogan, M. E. (1988) Science 241, 456-459. MEDLINE Abstract

18. Duval-Valentin, G., Thuong, N. T. and Helen, C. (1992) Proc. Natl. Acad. Sci. USA 89, 504-508. MEDLINE Abstract

19. Maher, L. J. III, Wold, B. and Dervan, P. B. (1989) Science 245, 725-730. MEDLINE Abstract

20. Maher, L. J. III (1992) BioEssays 14, 807-815. MEDLINE Abstract

21. Helen, C. (1993) Curr. Opin. Biotechnol. 4, 29-36. MEDLINE Abstract

22. Ts'o, P. O. P., Aurelian, L., Cheng, E. and Miller, P. S. (1992) Annu. NY Acad. Sci. 660, 159-177.

23. Strobel, S. A., Doucette-Stamm, L. A., Riba, L., Housman, D. E. and Dervan, P. B. (1991) Science 254, 1639-1642. MEDLINE Abstract

24. Perrouault, L., Asseline, U., Rivalle, C., Thuong, N. T., Bisagni, E., Giovannangi, C., Ledoan, T. and Helen, C. (1990) Nature 344, 358-360. MEDLINE Abstract

25. Helen, C., Thuong, N. T., Siason-Behmoaras, T. and Francoise, J.-C. (1989) Trends Biotechnol. 7, 310-315.

26. Rajagopal, P. and Feigon, J. (1989) Nature (London) 339, 637-640.

27. de los Santos, C., Rosen, M. and Patel, D. (1989) Biochemistry 28, 7282-7289. MEDLINE Abstract

28. Raghunathan, G., Miles, H. T. and Sasisekharan V. (1993) Biochemistry 32, 455-462. MEDLINE Abstract

29. Liu, K., Sasisekharan, V., Miles, H. T. and Raghunathan, G. (1996) Bioplymers 39, 573-589.

30. Kan, L. S., Callahan, D. E., Trapane, T. L. and Miller, P. S. (1991) J. Biomol. Struct. Dynam. 8, 911-933.

31. Bhaumik, S. R., Chary, K. V. R., Govil, G., Liu, K. and Miles, H. T. (1995) Nucleic Acids Res. 23, 4116-4121. MEDLINE Abstract

32. Plum, G. E., Pilch, D. S., Singleton, S. F. and Breslauer, K. J. (1995) Annu. Rev. Biophys. Biomol. Struct. 24, 319-350. MEDLINE Abstract

33. Radhakrishnan, I. and Patel, D. J. (1994) Biochemistry 33, 11405-11416. MEDLINE Abstract

34. Sun, J. S. and Helen, C. (1993) Curr. Opin. Struct. Biol. 3, 345-356.

35. Sklenar, V. and Feigon, J. (1990) Nature 345, 836-838. MEDLINE Abstract

36. Radhakrishnan, I., Santos, C. D. L. and Patel, D. J. (1991) J. Mol. Biol. 221, 1403-1418. MEDLINE Abstract

37. Macaya, R., Wang, E., Schultze, P., Sklenar, V. and Feigon, J. (1992) J. Mol. Biol. 225, 755-773. MEDLINE Abstract

38. Muraoka, M., Miles, H. T. and Howard, F. B. (1980) Biochemistry 19, 2429-2439. MEDLINE Abstract

39. Hore, P. J. (1983) J. Magn. Res. 55, 283-300.

40. Anil Kumar, Wagner, G., Ernst, R. R. and Wuthrich, K. (1980) Biochem. Biophys. Res. Commun. 96, 1156-1163.

41. Griesinger, C., Otting, G., Wuthrich, K. and Ernst, R. R. (1988)J. Am. Chem. Soc. 110, 7870-7872.

42. Lavery, R.(1988) In Olson, W. K., Sarma, R. H., Sarma, M. H. and Sundaralingam, M. (eds), Structure and Expression, Vol. 3 DNA Bending and Curvature. Adenine Press, NY, pp. 191-211.

43. Lavery, R., Zakrzewska, K. and Sklenar, H. (1995) Comp. Phys. Commun. 91, 135-158.

44. White, J. H. and Bauer, W. R. (1986) J. Mol. Biol. 189, 329-341. MEDLINE Abstract

45. Lavery, R., Sklenar, H., Zakrewska, K. and Pullman, B. (1986)J. Biomol. Struct. Dyn. 3, 989-1014. MEDLINE Abstract

46. Lavery, R., Parker, I. and Kendrick, J. (1986) J. Biomol. Struct. Dyn. 4, 443-462. MEDLINE Abstract

47. Hingerty, B., Richie, R. H., Ferrel, T. L. and Turner, J. E. (1985) Biopolymers 24, 427-439.

48. Lavery, R. and Sklenar, H. (1989) J. Biomol. Struct. Dyn. 6, 655-667. MEDLINE Abstract

49. James, T. L. (1995) Methods in Enzymology; Nuclear Magnetic Resonance and Nucleic Acids. Vol. 261, Academic Press, San Diego, Ch. 16-17.

50. Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids, John Wiley and Sons, New York, NY.

51. Bhaumik, S. R., Chary, K. V. R., Govil, G., Liu, K. and Miles, H. T. (1997) Biopolymers 41, 773-784. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +91 22 215 2971; Fax: +91 22 215 2110; Email: chary@tifr.res.in
+Permanent address: Keliang Liu, Institute of Pharmacology and Toxicology, PO Box 130, Beijing 100850, P.R.China

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