Báo cáo khoa học: Solution structure of 2¢,5¢ d(G4C4) Relevance to topological restrictions and nature’s choice of phosphodiester links docx

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Báo cáo khoa học: Solution structure of 2¢,5¢ d(G4C4) Relevance to topological restrictions and nature’s choice of phosphodiester links docx

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Solution structure of 2¢,5¢ d(G 4 C 4 ) Relevance to topological restrictions and nature’s choice of phosphodiester links Bernard J. Premraj 1 , Swaminathan Raja 1 , Neel S. Bhavesh 2 , Ke Shi 3 , Ramakrishna V. Hosur 2 , Muttaiya Sundaralingam 3 and Narayanarao Yathindra 1 1 Department of Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai, India; 2 Department of Chemical Sciences, TIFR, Colaba, Mumbai, India; 3 Department of Chemistry, The Ohio State University, Columbus, OH, USA The N MR structure o f 2¢,5¢ d(GGGGCCCC) was deter- mined to gain insights into the structural differences between 2¢,5¢-and3¢,5¢-linked DNA duplexes that may be relevant in elucidating nature’s choice of sugar-phosphate links to encode genetic information. The oligomer assumes a duplex with extended nucl eotide repeats formed out of mostly N-type sugar puckers. With the e xception of the 5 ¢-terminal guanine that assumes the syn glycosyl conformation, all other bases prefer the anti glycosyl conformation. Base pairs in the duplex exhibit slide ()1.96 A ˚ ) and intermediate values for X-displacement ()3.23 A ˚ ), as in ADNA, while their inclination to the helical axis is not prominent. Major and minor grooves display features intermediate to A and BDNA. The duplex structure of iso d(GGGGCCCC) may therefore be best characterized as a hybrid of A and BDNA. Importantly, the results confirm that even 3 ¢ deo xy 2¢,5¢ DNA supports duplex formation only i n the presence of distinct slide (‡ )1.6 A ˚ ) a nd X-displacement (‡ )2.5 A ˚ )for base pairs, and hence does not favor an ideal BDNA topology characterized by their near-zero values. Such restrictions on base pair movements in 2¢,5¢ DNA, w hich are clearly absent in 3¢,5¢ DNA, are expected to impose con- straints on its ability for deformability of the kind ob served in DNA during its co mpaction and interaction with proteins. It is therefore c onceivable t hat selection pressure relating to the optimization of t opological features might have been a factor in the rejection of 2¢,5¢ links in preferenc e to 3¢,5¢ link s. Keywords: structure of 2¢,5¢ DNA; evolution of 3¢,5¢ vs. 2¢,5¢ links in nucleic acids; AB hybrid structure ; restrained base pair movements; topological restrictions in 2¢,5¢ DNA. Nature’s selection of 3 ¢,5¢ linkages ( instead of 2¢,5¢ linkages) in nucleic acids, to encode genetic information, is intriguing. Thefactthat2¢,5 ¢ links are formed i n a bundance and serve as a template in nonenzymatic reactions suggest that they might have been the ancestors of the biotic 3¢,5¢ links, which could h ave e volved from a pool of 3¢,5¢ and 2 ¢,5¢ links [1]. Nucleic a cids with 2¢,5¢ links satisfy one of the critical features required for the fidelity of replication, namely that theyassociatetoformWatsonandCrickbase-paired duplex structures [2–5], although with weaker affinity than 3¢,5¢-linked DNA strands. However, detailed knowledge about stereochemistry, polymorphism and topological properties of 2¢,5¢ DNA duplexes, which may provide insights into the factors that determine nature’s choice of sugar-phosphate links from a stereochemical perspective, is sparse [6–9]. In fact, there are only two reports of NMR structure determination – one on a 2 ¢,5¢ DNA fragment [10] and one on a 2¢,5¢ RNA fragment [ 11] – both of w hich suggest an A-type duplex structure with s ome stereochem- ical details that differ from genomic DNA and RNA duplexes. In this context, it is relevant to recognize the results from recent modeling studies on 2¢,5¢ nucleic ac ids, which suggest that 2¢,5¢ DNA cannot form a 10-fold BDNA-like duplex (like 3 ¢,5¢ DNA) without the mandatory slide (‡ )1.6 A ˚ ) and X-displacement (‡ )2.5 A ˚ )[9].Witha view to probe further i nto the structural properties o f 2¢,5¢ DNA, we report here a high-resolution NMR study of the 2¢,5¢ DNA fragment that pos sesses a g uanine tract f ollowed by a cytosine t ract, to d iscern also possible sequence e ffects. The results show that iso d(GGGGCCCC) [d(G 4 C 4 )] assumes a duplex that conforms to neither a canonical BDNA nor an A DNA family, but a duplex characterized by featu res of both A and BDNA. Possible implications of this on the topological restrictions of 2¢,5¢ DNA, and its rejection by nature, are discussed. Materials and methods DNA synthesis and NMR sample preparation The 2¢,5¢-linked 3 ¢ deoxy (GGGGCCCC) (iso DNA), was synthesized at 1 lmol scale on an in-house Applied Biosystem 391 automatic DNA synthesizer using solid-state phosphoramidite chemistry [12]. The universal support (purchased from BioGene) was used as the solid support for the synthesis. The standard concentration of phospho- ramidite was d iluted with a n equal volume of acetonitrile. The products were cleaved off the column w ith 5 mL of 37% ammonium hydroxide containing 5% LiCl. The Correspondence to N. Yathindra, Department of Crystallography and Biophysics, U niversity of M adras, Guindy C ampus, Chennai-600 025, India. Fax: + 9 1 4 4 2230 0122, 2 Tel.: + 91 44 2235 1367, E-mail: ny@vsnl.com Abbreviations:d(G 4 C 4 ), d(GGGGCCCC); LALS, linked atom least squares; RDC, residual dipolar couplings. (Received 4 March 2004, revised 30 A pril 2004, ac cepted 21 May 2 004) Eur. J. Biochem. 271, 2956–2966 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04225.x solution was incubated in a 55 °C water bath for 16 h and then lyophilized. The pellet from lyophilization was dis- solved in 5% NaHCO 3 and purified by FPLC. The collected peak elution was lyophilized and the sample stored at )20 °C. NMR samples (0.6 m M )ofthe2¢,5¢ DNA fragment were prepared in 20 m M potassium phosphate buffer containing 0.5 m M EDTA and 100 m M KCl. For experi- ments in D 2 O, the s amples were lyophilized and redissolved in D 2 O. UV melting studies show that the T m of iso d(G 4 C 4 ) is  32 °C under identical buffer conditions. NMR data acquisition NMR experiments were carried out on a 600 MHz Varian Unity-plus spectrometer. 1D spectra in H 2 O were r ecorded using the jump-and-return pulse sequence for H 2 O sup- pression at different t emperatures in t he range o f 2–45 °C [13]. 2D NOESY spectra in H 2 O were recorded at 2 °Cwith mixing times of 80 ms and 300 ms. Phase-sensitive N OESY spectra in D 2 O [14] w ere recorded with mixing times of 7 0, 120, 150, 200, 250 and 300 ms; and TOCSY spectra [15] were recorded with mixing times of 30 and 90 ms at 2 °C. The DQF-COSY spectrum [16,17] and 2D J-resolved spectra [18] were recorded in D 2 Ofor 1 H– 1 H coupling constant estimation. For the various experiments, the time domain data c onsisted of 2048 c omplex points i n t2 and 300–400 complex points in t 1 dimension. The relaxation time delay was between 1 and 3 s for the different 2D experiments. Experimental restraints Data processing and analysis were carried out using VNMR and FELIX packages [ 19] on a Silicon graphics work station. Based on the relative intensities and build-up, the cross peaks in the NOESY s pectra (obtained in D 2 O at various mixing times), are classified as strong, medium-strong, medium, and weak, and the interproton distances are restrained, respectively, to the ranges 2–3 A ˚ , 2.5–3.5 A ˚ , 3–4.5 A ˚ , and 3.5–5.5 A ˚ . The narrow bounds are mostly used for strong intranucleotide cross peaks, for which distance ranges are small and known. As the distance ranges for the observable NOEs are not so large, the NOE distance bounds used are c onsidered to be realistic. The interproton distances involving the exchangeable protons in the H 2 O NOESY spectra are restrained to the ranges 2–4 A ˚ and 3.5– 5.5 A ˚ , corresponding to the s trong and w eak cross peaks, respectively. At this level o f NOE inte nsity quantification, spin diffusion is not expected to influence the distance restraints to a significant extent. Even so, the larger the number of distance restraints, the better it is for internal consistency, and the structures derived would be more reliable. A total of 162 NOE restraints were collected, of which 115 were intranucleotide and 47 internucleotide NOEs. Base (H8/H6) s ugar proton NOEs, especially to the H1¢, also enable deriving constraints on the glycosyl torsion angles. The H8/H6–H1¢ distance is very short ( 2.3–2.5 A ˚ ) for a syn conformation and r elatively much l onger ( 3.5– 4.0 A ˚ )forananti conformation. Thus, the H8/H6–H1¢ NOE will be very strong, even at short mixing times (such as 60–70 ms) if the glycosyl torsion angle is in the syn domain, whereas, u nder the sa me conditions, the peak will be nearly absent for an anti conformation. We observe that G1 has a syn conformation, while all others are in the anti domain (spectra presented in Results). The 2D J-resolved spectra provides precise values of the J(H1¢–H2¢) coupling constants (Table 1). The observed coupling constants are very small, indicating that the sugar geometry b elongs largely to the N domain (in the N domain this coupling constant is near 0–2 Hz, whereas it varies between 9 and 10 Hz in the S domain). A common practice is to consider the sugar geometry as an equilib- rium mixture of N and S types, and the coupling constants as weighted averages. However, there are also reports in the literature [18] where the sugar ring is believed t o be rigid, and is primarily of a single type, at least in the interior of the duplex. In the present case, we observe that the terminal residues, for example, C8 and G2, where one would have expected greater dynamism, exhibit very small values ( 1.5Hz) for J(H1¢–H2¢). If one considers an equilibrium model, for a 10% contribution of the S domain, the contribution to t he coupling c onstant would be around 1 Hz. Moreover, it is c lear from the steepness of the curve displaying the dependence of coupling constants on pseudorotation phase angle P (Fig. 1 ), that the P range i n the N domain is not going to be very different regardless of whether the S contribu- tion is explicitly considered. Thus, from t he small values of the coupling constants for the terminal residues, it is evident t hat the sugar geometries are dominantly in the N domain only. This will be also true for the internal residues. Now, in the N domain, especially in the P range 30–80°, the dependence of H1¢–H2¢ coupling on P is very steep and this significantly narrows the range of permis- sible P-values for a given coupling constant value [18,20]. Taking these factors into consideration, sugar puckers were restrained to the P ranges indicated in Table 1 a nd these were then converted to respective dihedral angle ranges in the sugar rings. The hydrogen bond restraints were given as two distances per hydrogen bond (a total of 36 restraints) for the central hexamer (see below). Based on the observation that the peak count for t he duplex is the same as expected from a single strand in the various spectral data (indicating that the duplex is highly symmetric and the two strands are Table 1. J(H1¢–H2¢) coupling and the c orresponding ranges of phase ang le of pse udorotation (P°). G1 G2 G3 G4 C5 C6 C7 C8 J(H1¢–H2¢) Hz 2.9 1.7 3.5 2.9 3.8 1.8 5.1 1.5 Range of phase angle of pseudorotation (P°) 49–58 35–47 55–64 49–58 57–66 37–48 68–77 33–45 Ó FEBS 2004 2¢,5¢ DNA with hybrid features of A and BDNA 1 (Eur. J. Biochem. 271) 2957 equivalent), NCS restraints were imposed to ob tain sym- metry between the two strands forming the duplex. Structure calculation Structure calculation of the iso d(GGGGCCCC) was carried out using X - PLOR 3.8.5 [21]. The topology and parameter files w ere appropriately modified t o handle 2¢,5¢ linkages to obtain optimum geometry at the 2 ¢,5¢ phospho- diester linkage. Ideal A- and B-type duplex models for iso DNA (possessing helical parameters identical to those of the canonical ADNA and B DNA), obtained p reviously [9] using the linked atom least squares (LALS) refinement approach [22], were used as the starting models for structure calculation. This is justified considering that the NMR spectra in water clearly estab lish Watson and Crick b ase pair formation between antiparallel strands. The model iso ADNA duplex is characterized by the s ame value of slide, X-displacement and the helical parameters, as ADNA. On the other hand, the iso BDNA model, while possessing the same h elical parameters as BDNA, is distinguished by a nonzero slide ( ‡ )1.7 A ˚ ) and X-displacemen t (‡ )2.5 A ˚ ), in sharp c ontrast to the ideal BDNA that is characterized b y zero values for them. Nonzero slide and X-displacement are found to b e mandatory to generate a 10-fold 2¢,5¢ duplex, even with 3¢ deoxy sugars [9]. Thus, the iso BDNA and iso ADNA models are very different from each other, and choosing these two as initial models removes any starting model bias i n the results of c alculation. Such a strategy a lso saves computational efforts compared to starting the calculation from a completely extend ed structure. In the latter case, m uch effort i s expended for the formation of the base pair itself . Syn conformation was imposed for the 5¢ end guanine (see below). The initial model was subjected to restrained energy minimization using the conjugate gradient algorithm and was guided by the experimental NOE distance restraints as well as dihedral restraints. A conform ational search w as performed on the octamer duplex using the Ôsimulated annealingÕ protocol [23], f ollowed by s tructure refinement using the Ôgentle refineÕ protocol of X - PLOR 3.8.5. A distance- dependent dielectric constant was used throughout the structure calculation to mimic the presence of high dielectric solvent, typically for simulating water (when explicit water is not used). The starting s tructure was heated t o 1000 K , and sets of 100 structures tha t are s ignifican tly different from one another were extracted during high-temperature dynamics. Each of the structures was subjected to 18 ps of high-temperature dynamics followed by s low cooling to 100 K , at steps of 50 K. During each cooling step the structures were subjected to 500 fs of molecular dynamics. Finally, the structures were energy minimized using the conjugate gradient algorithm. This was followed by a refinement using the Ôgentle refineÕ protocol, where each of the structures was subjected to 20 ps of m olecular dynamics at 300 K. Average coordinates over the last 10 ps of molecular dynamics simulation were computed and then refined by conjugate gradient minimization. The NOE distance restraints, hydrogen bond restraints (given as two distances per hydrogen bond), and dihedral restraints on the sugar conformation were applied throughout the entire calculation with force constants of 50 kcalÆmol )1 ÆA ˚ )2 , 100 k calÆmol )1 ÆA ˚ )2 and 300 kcalÆmol )1 ÆA ˚ )2 , respectively. NCS restraints w ith a force constant of 300 kcalÆmol )1 ÆA ˚ )2 were imposed t o obtain symmetry between the t wo strand s of the duplex. Results 1D and 2D proton spectra The 1D 1 H NMR spectrum (Fig. 2A) of the octamer iso d(GGGGCCCC) displays three peaks corresponding to the imino protons at 13.25 (G4), 12.80 (G2) and 12.70 (G3) p.p.m., expected from Watson and C rick base pairs i n an antiparallel duplex. Sequence-specific assignments for the exchangeable and nonexchangeable protons were made from the NOESY and TOCSY spectra following the procedures developed f or 3¢,5¢ duplexes [24]. T he observa- tion of NOE changes from G imino to C amino protons of nonterminal base pairs in the NOESY water spectra (Fig. 2 B) further substantiates the formation of Watson and Crick base pairing between G and C. The uninterrupted self and sequential connectivity from H8/H6 to H1¢ (Fig.3A),aswellasH8/H6toH2¢ (Fig. 3B) in the N OESY spectra suggest a right-handed helical structure. These sequential connectivities are consistent throughout the various regions of the spectra. From the temperature dependence of the G imino resonances in 1D spectra in H 2 O (data not shown), the melting temperature of the duplex was seen to be  30 °C. Tables 2 a nd 3 3 show the chemical shift values for a ll the assigned sugar and base protons. The stereospecific assign- ments involving the 3¢ and 3¢¢ protons were based on the 2¢)3¢ and 2 ¢)3¢¢ NOE intensities in the 70 m s NOESY spectrum. As the H2¢–H3¢ proton separation is shorter t han the H2¢–H3¢¢ separation, irrespective o f the sugar confor- mation, the H2¢–H3¢ NOE intensity should be stronger at shorter mixing times. The relative intensities of the cross- peaks of the interproton base to sugar NOEs in the NOESY spectrum (Fig. 3C), at mixing times varying from 70 to 300 m s, indicate that the 5¢-terminal guanine exists in the Fig. 1. Plots showing the dependence of the 3 -bond coupling constants (J) on the phase a ngle of ps eudorotation (P). 2958 B. J. Premraj et al. (Eur. J. Biochem. 271) Ó FEBS 2004 syn conformation, while other bases favor t he anti confor- mation. This is a recurring feature found in 2¢,5¢-linked dimers [25–27] and oligomers [10,11]. In the crystal struc- tures o f 2 ¢,5¢-linked dinucleoside monophosphates, the syn conformation is stabilized by an intramolecular hydrogen Fig. 2. NMR spectra and the NOESY spectrum. (A) 1D H 2 O exchangeable NMR spectra of iso d(GGGGCC CC) in 100 m M KCl, pH 7.0, and at 2 °C, showing the imino and amino proton signals. (B) Selected region of the NOESY spectrum (mixing time 300 m s) in H 2 O solution showing N OE correlation sfromGiminotoCaminoprotons. CNH 2(i) and CNH 2(e) refer to the internal (H-bon ded) and external (free) amino p roton s of the cytosine base. Fig. 3. (H8/H6)–H1¢ cross-peak region of a 300 ms 2D NOESY spectrum o f iso d(GGGGCCCC) in D 2 Osolutionat2°C, showing the uninterrupted sequential connectivities from (A) (H8/H6) to H1¢ protons (B) (H8/H6) to H2¢ proto ns (C). Stacked plot of the H8/H6 to H1¢ region showing a high intensity for the H8–H1¢ cross-peak of G1, suggesting syn glycosyl conformation for the terminal G1 residue . Ó FEBS 2004 2¢,5¢ DNA with hybrid features of A and BDNA 1 (Eur. J. Biochem. 271) 2959 bond between the purine N3 and O5¢H of the sugar residue, besides sugar O4¢–base (syn) base interaction [9,25–27]. The (H1¢–H2¢) coupling constants derived from 2D J-resolved spectra clearly indicate that all of the 3¢ deoxy sugars belong to the N type, except for C7 which has a slightly higher coupling constant (5.1 Hz). Structural features of 2¢,5¢ d(GGGGCCCC) The 3D structure of 2¢,5¢ d(GGGGCCCC) was obtained by simulated annealing molecular dynam ics using X - PLOR 3.8.5 [21]. Experimental restraints and structure convergence parameters are listed in Table 4. The convergent structures are clustered into families: BFI (Fig. 4A) with 39 structures, and BFII (Fig. 4B) with 20 structures when the starting model was ideally iso BDNA; and AFI (Fig. 4C) with 85 structures and AFII (Fig. 4D) with 15 s tructures when the starting model was iso ADNA. Structures represented by BFI and AFI families (FI) differ considerably in their overall topologies from the structures represented by BFII and AFII families (FII). The root mean square devi- ation (rmsd) b etween FI and FII is greater t han 3 A ˚ , while it is less than 1 A ˚ for structures within FI or FII. The structures were selected using standard criteria on the basis of proper c ovalent geometry, the least number of distance and dihedral violations, symmetry and low energy. The duplex model AFI (Fig. 5A), c losely resembles BFI (Fig. 5 B). T he rmsd between the average structure of AFI (Fig. 5 A) and BFI (Fig. 5 B) is 0.8 A ˚ . Thus, in spite of the large rmsd (> 4 A ˚ ) in the starting structures, the final structures fall into similar families, indicating that the structures are not biased by the c hoice of the initial m odel. This also indicates that the experimental restraints are sufficient and consistent to define good convergent struc- tures. In view of this, it is b elieved th at there is no need for any further refinements using residual dipolar couplings (RDCs), as often performed in longer DNA stretches [28–30]. Likewise, we also did not f eel the need f or any relaxation matrix refinement, w hich takes into account spin diffusion e xplicitly, which may be r equired if t he NOE data set is very small. At the same time, relaxation matrix refinement puts a greater demand on the accuracy of NOE quantification. In the final structures, the terminal GC pairs are not well defined owing to insufficient NOEs. Hence, structural features manifested in the cen tral hexame r of iso d(G 4 C 4 ), corresponding to the GGGCCC du plex in the f amily AFI, which has the highest population of converged structures and also has very good convergence, are considered for detailed discussion. Calculated values of X-displacement and slide for the base pairs in AFI are given in Table 5 . Average values of X-displacement and slide of GC base p airs at the GG step (Fig. 5 A) are )3.25 A ˚ and )1.62 A ˚ (Table 5), r espectively. On the other hand, slide for the GC pair at the GC step that links the G stretch with the C stretch is rather high ()3.3 2 A ˚ ). The nature of the base stacking interaction in the iso d(GGGGCCCC) duplex, as seen in AFI, is shown in Fig. 6A. Stacking at the G 2 G 3 and G 3 G 4 steps involves overlap of t he six-membered ring of one gu anine with the imidazole r ing o f the adjacent guanine, while there i s only Table 3 . Chemical s hifts (p .p.m ) f or iso d(GGGGCCCC) 2 exchange- able proto ns. Base H 1 H 22 /H 42 (e) H 21 /H 41 (i) G1 – – – G2 12.76 – – G3 12.71 – – G4 13.25 – – C5 – 6.94 8.7 C6 – 6.93 8.58 C7 – 7.12 8.51 C8 – – – Table 2. Chemical shifts (p.p.m) for iso d(GGGGCCCC) 2 non- exchangeable protons. Residue H6/H8 H1¢ H2¢ H3¢ H3¢¢ H4¢ H5¢/H5¢¢ H5 G1 7.97 5.98 5.18 2.53 2.37 4.58 3.88,3.69 – G2 7.78 5.83 4.7 2.45 2.31 4.61 4.18,4.08 – G3 7.62 5.96 4.91 2.56 2.48 4.78 4.51,4.14 – G4 7.58 5.99 4.61 2.48 2.38 4.77 – – C5 7.5 6.08 4.62 2.44 – 4.76 4.39,4.07 5.10 C6 7.88 5.94 4.5 2.31 – 4.71 4.10 5.48 C7 7.73 6.03 4.66 2.49 2.31 4.04 4.27 5.52 C8 7.84 5.66 4.26 1.84 1.82 4.56 4.39,4.0 5.61 Table 4 . NMR restraints for iso d(GGGGCCCC) 2 . NOE distance restraints (per strand) Non-exchangeable NOE restraints 140 Exchangeable NOE restraints 22 Total restraints 162 Intra-residue 115 Inter-residue 47 Sugar dihedral restraints (per strand) 40 Hydrogen bond restraints 36 BFI (model obtained when iso BDNA is used as the starting duplex) Number of convergent structures 39 rmsd from the average structure 0.5 A ˚ )1.0 A ˚ NOE violation > 0.2 A ˚ 1 Dihedral angle violation > 5° Nil BFII (modelobtained when iso BDNA is used as the starting duplex) Number of convergent structures 20 rmsd from the average structure 0.3 A ˚ )1.0 A ˚ NOE violation > 0.2 A ˚ 1 Dihedral angle violation > 5° Nil AFI (model obtained when ADNA is used as the starting duplex) Number of convergent structures 85 rmsd from the average structure 0.1 A ˚ )0.6 A ˚ NOE violation > 0.2 A ˚ 1 Dihedral angle violation > 5° Nil AFII (model obtained when ADNA is used as the starting duplex) Number of convergent structures 15 rmsd from the average structure 0.1 A ˚ )0.5 A ˚ NOE violation > 0.2 A ˚ 1 Dihedral angle violation > 5° Nil 2960 B. J. Premraj et al. (Eur. J. Biochem. 271) Ó FEBS 2004 minimal stacking between cytosines. Likewise, stacking at the GC s tep, which links the G stretch w ith the C stretch, i s minimal o wing to a larger s lide ()3.32 A ˚ ). Superposition of thebasepairsofthe(GGGCCC) 2 fragment of the iso d(G 4 C 4 ) duplex with the ideal iso BDNA (Fig. 7), demon- strates a strong resemblance in the stacking patterns. An estimate of the dimensions of major and minor grooves is obtained by generating a 12mer duplex using t he central hexamer of the average structure (AFI) as the repeat using the program FREEHELIX [31]. The groove topologies of AFI show significantly different features f rom the ideal duplex models. T he major groove is wide (17 A ˚ ), while its minor groove is narrow (10.3 A ˚ ). The 3¢ deoxy sugars i n iso d(G 4 C 4 ) favor N-type pucker, corresponding to the C4¢ exo conformational domain ( P ¼ 38–64°), except in the residue C7, which favors C4 ¢ exo/O4¢ endo pucker, corresponding to P ¼ 54–90° (Table 1) in AFI. In any case, none of the sugars shows a tendency for S-type sugar conformation. Base pairs in A FI are slightly overwound, an d the duplex has 9 bp per t urn, with an average helical twist o f 38.4° and a rise of 3.76 A ˚ (Table 5). The average helical twist at the GG and CC steps is  41 °, w hile it is 28° at the GC step. Slight underwinding at this step is accompanied by a higher slide of )3.32 A ˚ . Base p airs are nearly perpendicular t o the helix axis (inclination angle 3 °).Thetwocentralbasepairs of the duplex are p ractically planar and they do not exhibit significant propeller twist (Table 6), while the base pairs flanking them possess a larger value of )22°. Phosphodi- ester conformations at all the GG steps, as well as at the GC step, conform to t he (g – ,g – ) domain, while they correspond to the (t,g – ) at the CC steps (Table 7 ). Discussion It is now well established that nucleic acids, even with 2 ¢,5¢ linkages, associate to form Watson and Crick paired duplexes [2–5,10,11,32–37]. They also selectively associate with DNA and RNA with a v arying degree of stability. Interestingly, it has b een shown recently that 2¢,5¢ RNA fragments form even hairpins with a stability comparable to RNA hairpins [38]. In an effort to obtain a comprehensive understanding of the stereochemistry that govern the structures of 2 ¢,5¢ nucleic acids, we recently reported the Fig. 5. Stereo plot of the average structure of is o d(G 4 C 4 ). (A) AFI an d (B) BFI. Fig. 4. Stereo plot of th e families of conv erged structures of is o d(GGGGCCCC) 2 . (A) B FI (39 structures), (B) BFII (20 structures), (C) AFI (85 structures), a nd (D) AFII ( 15 structures). Table 5. Base-step parameters i n the average structure (AFI) of the iso d(GGGGCCCC) duplex. Base step Slide (A ˚ ) X-disp (A ˚ ) Twist (°) Rise (A ˚ ) G2-G3 )1.53 )3.36 42.3 3.68 G3-G4 )1.71 )3.13 39.67 3.61 G4-C5 )3.32 )3.19 28.02 4.23 C5-C6 )1.71 )3.13 39.63 3.61 C6-C7 )1.53 )3.23 42.37 3.68 Average )1.96 )3.20 38.39 3.76 Ó FEBS 2004 2¢,5¢ DNA with hybrid features of A and BDNA 1 (Eur. J. Biochem. 271) 2961 NMR structure of a 2¢,5¢ RNAfragment[11]thatexhibited interesting features which supported our predictions from modeling studies [8,9]. We re port here the results o f high- resolution NMR structure of a 2¢,5¢-linked D NA fragment d(GGGGCCCC). The structural model, AFI, that emerged from N OE and other NMR data, exhibit slide ()1.96 A ˚ )and intermediate X-displacement ()3.32 A ˚ )forthebasepairs, a feature normally seen only in ADNA duplexes. However, the magnitude of X-displacement observed here is lower () 4.7 A ˚ ) than that found in ADNA. Interest- ingly, the slide ()3.32 A ˚ ) at the lone GC step, linking the G stretch with the C stretch, is found to be nearly twice that found at the GG steps ()1.62 A ˚ ), indicating possible sequence effects. A comparison of the stacking pattern observed at the GG steps of the present structure with those in ideal ADNA, iso ADNA and iso BDNA duplexes (Fig. 6B) brings out a strong similarity. It is interesting t hat the similarity in stacking p ersists, notwith- standing different values f or X displacement that c harac- terizes these duplexes (Table 5). However, it should be noted that all of them possess nearly the same slide ()1.7 A ˚ ). Thus, the base stacking pattern in iso d(G 4 C 4 )is like that in ADNA, except at the GC step where a large slide causes adjacent b ases to move aw ay, resu lting in minimal overlap between them. Another unusual feature is the predominance of N-type pucker i n nearly all the 3 ¢ deoxy s ugars in 2¢,5¢ d(G 4 C 4 ). This is in sharp contrast to the S -type puckers preferred by 2¢ deoxy sugars in DNA duplexes. This has been Fig. 6. Base stacking at different steps in the AFI dup lex of is o (G 4 C 4 ) and the GG steps of is o BDNA: iso ADNA and ADN A . Note the identic al base stacking at the GG s teps of AFI a nd ideal duplex es. Figures were d rawn using 3 DNA v1.5 [ 47]. 2962 B. J. Premraj et al. (Eur. J. Biochem. 271) Ó FEBS 2004 anticipated in view of certain stereochemical arguments [8,9]. Exclusive preference for the N-type sugar puckers has, in fact, been indicated by the early NMR studies on 2¢,5¢-AAA [39] and crystal structures of 3¢ deoxynucleo- sides [25–27]. Such preference for N-type pucker has also been confirmed by recent 1 H N MR analysis on a number of 3¢ d eoxynucleosides and stereo-electronic arguments [40,41]. Unconstrained molecular dynamics simulations of a2¢,5¢ DNA duplex, l asting a few nanoseconds, have also demonstrated the retention of N-type pucker for the sugar [42]. It should be recognized at this juncture that the consequence of N-type sugar pucker is to render the preferred nucleotide conformation to b e extended in 2¢,5¢ DNA a nd compact in 3 ¢,5¢ DNA [8,9]. It is well known that the extended nucleotide repeats lead to an extended BDNA, and the compact nucleotide repeat leads to a compact ADNA type of duplexes (Fig. 8). The 2 ¢,5¢ DNA fragment d (G 4 C 4 ) is thus composed of extended nucleo- tide repeats that are normally part of BDNA but with a distinct X-displacement, slide and base stacking like in ADNA. Thus, the duplex model AFI of 2¢,5¢ d(G 4 C 4 ), possesses composite features of both A and BDNA. In view of these, it is perhaps appropriate to regard the structure of iso d(G 4 C 4 ) as a hybrid structure of A and B forms. It is grati fying that the more populated AFI family of iso d(G 4 C 4 ) resembles the ideal iso BDNA-like duplex [9], which is also characterized by similar values of slide, intermediate displacement, base stacking p attern and extended nucleotide repeat formed out of N-type sugar puckers (Table 5). Furthermore, the overall groove topol- ogies of iso d(G 4 C 4 ) resemble BDNA, with the widths of the major groove and the minor groove having values of 17 A ˚ and 10.3 A ˚ , respectively (Table 8 ). It has been demonstrated from modeling investigations that 2¢,5¢ isomers, even with 3¢ deoxyriboses, cannot form duplexes without base pair displacements [9]. Results of CD and FTIR investigations on iso DNA fragments comprising a variety of base sequences also seem to converge to suggest that they favo r A-type r ather than B -type duplexes (S. Raja & N. Y athindra, unpublished observation). F urthermore, it has been found [43] that iso d(CGCGCG) does not associate to form left-handed ZDNA. T his has been attributed to the inaccessibility [42] to form t he well-known water-mediated hydrogen bond stabilization i nteraction between the amino group of the syn guanine and the anion oxygen of the phosphate group [44]. These clearly point to the constraint on the r ange of duplex helical structures possible for nucleic acids with 2¢,5¢ linkages. The lateral slide of the sugar-phosphate chain from the periphery (as in 3¢,5¢ links) towards the helix interior in Fig. 7. Superposition of the G 3 C 3 fragment of AFI with ideal iso BDNA. Root mean square deviation with respect to base pairs is 0.6 A ˚ . Table 6. Propeller twist (°) of base pairs in th e average structure (AFI) of the iso d(GGGGCCCC) duplex. Base pair Propeller twist (°) G2–C15 )22.4 G3–C14 )19.6 G4–C13 1.0 C5–G12 1.0 C6–G11 )19.4 C7–G10 )22.4 Average )14.3 Table 7. Conformation angles (°) in the av erage structure (AFI) of th e iso d(GGGGCCCC) 2 duplex. Residue a (P-O5¢) b (O5¢-C5) c (C4¢-C5¢) n (C2¢-O2¢) f (P-O2¢) v (C1¢-N) P G1 – – 29 )115 )86 86.2 49 G2 )50 167 46 )78 )122 )137 37.6 G3 )31 138 36 )69 )131 )137 56.4 G4 )45 143 32 )87 )89 )142 49.5 C5 )68 )177 27 )76 )161 )138 62.7 C6 )25 128 35 )60 )133 )151 37.4 C7 )28 131 34 )90 )144 )140 70.7 C8 )44 158 39 – – )151 34.1 Ó FEBS 2004 2¢,5¢ DNA with hybrid features of A and BDNA 1 (Eur. J. Biochem. 271) 2963 2¢,5¢ nucleic acids causes t he base pairs t o s lide, resulting i n the intrinsic requirement of slide, and hence X-displace- ment, that manifest in all 2¢,5¢ nucleic acid duplexes. This limits the access to a lower range of values of both s lide (< )1.5 A ˚ ) and X-displacement (< )2.5 A ˚ )in2¢,5 ¢ nucleic acids. In contrast, nucleic acids with 3¢,5¢ links have a w ider range of access for both slide (0–2.5 A ˚ ) and X-displacement (0–4.7 A ˚ ) that includes ranges forbidden for the 2¢,5¢ isome r. This enables 3¢,5¢-linked nucleic acids to assume a variety of duplexes with distinct topological features and also afford other capabilities, such as b ending, kinking and curvature, which form the basis for nucleic acid compaction and specificity of interaction with proteins. It is therefore anticipated that the restricting factors in 2¢,5¢ n ucleic ac ids, Fig. 8. Shape and dimension (adjacent P–P separations) of the repeating nucleotide units in 2¢,5¢- and 3¢,5¢-linked nucleic acids. An equatorial (e) link renders the adjacent phosphates to be proximal, leading to a compact nucleotide (P–P  5.9 A ˚ ), while an axial (a) link renders them to be distal, leading to an e xtended nucleotide (P–P  7.0 A ˚ ). Table 8. Comparison of structural features of the iso d(GGGGCCCC) 2 duplex (AFI) with the ideal A and B types of duplexes formed by 3¢,5¢ and 2¢,5¢ links. Features/parameters BDNA ADNA iso BDNA iso ADNA AFI X-disp (A ˚ ) )0.1 )4.7 )2.5 )4.7 )3.2 Slide (A ˚ ) 0.4 )1.6 )1.7 )1.67 )1.96 Twist (°) 36 32.7 36 32.7 38.4 Rise (A ˚ ) 3.4 2.56 3.4 2.56 3.76 No: res./turn 10 11 10 11  9.4 Inclination (°) 3.4 20.0 0 19.3 3 P–P separation (A ˚ ) 7 5.9 7.5 5.9 7.4 Sugar pucker S type N type N type S type N type C2¢endo C3¢endo C3¢endo C2¢endo C4¢exo Major groove (A ˚ ) 17 8.2 19.8 10.7 17 Minor groove (A ˚ ) 11.7 16.9 10.5 14.8 10.3 2964 B. J. Premraj et al. (Eur. J. Biochem. 271) Ó FEBS 2004 which are mentioned above, probably impose additional constraints limiting these capabilities. Also, it has been shown f rom modeling c onsideration t hat the lateral slide of the sugar-phosphate chain leads to overwinding of the 2¢,5¢-linked single-stranded helix to enhance the adjacent base–base or s ugar–base stabilizing i nteractions [9,42,45]. Tighter winding of the 2¢,5¢ single-stranded DNA helix, compared with 3¢,5¢ DNA, probably offers restrictions to the folding abilities of even single-stranded 2 ¢,5¢ DNA. Hence, it may be argued that topological restrictions inherent to the 2¢,5¢-linked helical duplexes might have a lso contributed towards their rejection. It is worth mention ing that the inherent low thermal stability of 2¢,5¢ links might have been another factor involved in nature’s selection of the 3¢,5¢ links. T hus, optimization o f the topology o f duplex helix, besides the optimization o f b ase p air s tability [46], must have been important in the chemical etiology of nucleic acid structures. Conclusions Systematic investigations of 2¢,5¢ nucleic acids h ave provided new p erspectives on the stereochemical d etails pertaining to their ability, or lack of it, to form dup lex structures akin to their naturally occurring 3 ¢,5¢ isomers. In parallel t o our finding [8,9] of the critical features that distinguish the shapes and dimensions of the r epeating nucleotides of 3¢,5¢ and 2 ¢,5¢ isomers, we have provided structural details of an iso RNA [11] and an iso DNA duplex fragment ( present work) from NMR studies. T ogether, these should p rovide a structural basis for understanding much of the experimental data from solution stu dies concerning the associations of 2¢,5¢ nucleic acids a nd also with DNA and R NA. C ompar- ison of the structure deduced for iso d(GGGGCCCC), from the current study, and that of iso d(CGGCGCCG) [10] suggestthateven2¢,5¢ DNAs are pr one t o s equence e ffects, as evidenced by some differences seen in structures of the two sequences. The former sequence a ssumes a hybrid structure of A and BDNA duplexes, while the latter assumes an ADNA-like duplex with mixed C 2¢ endo and C3¢ endo sugar puckers for t he central hexamer. The fact that both these sequences, s tudied by NMR, point to a non-BDNA duplex structure, suggest a constrained nature o f base p air movements i n 2 ¢,5¢ nucleic acids vis-a ` -vis their 3¢,5¢ isomers. This is in complete conformity with the modeling studies [8,9] which indicate that slide and X-displaceme nt of base pairs lower than )1.7 A ˚ and )2.5 A ˚ , respectively, are inaccessible owing to the inherent chemistry o f the 2 ¢,5¢- linked sugar-phosphate backbone. It seems, then, that a need for greater topological flexibility of DNA helices might have had a bearing on the selection of 3¢,5¢ links over 2¢,5¢ links during the course of evolution. Acknowledgements NMR a nd c omputational facilities, provided by the National F acility for High R esolution NMR at the Tata Institute of Fundamental Research, Mumbai, are gratefully a cknowledged. N.Y. a nd B.J.P. thank DST and CSIR f or a research g rant and senior f ellowship, respectively. 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