The occurence of the syn-C3' endo conformation and the distorted backbone conformations for C4'-C5' and P-O5' in oligo and polynucleotides.

The nucleoside constituents of nucleic acids prefer the anti conformation (1). When the sugar pucker is taken into account the nucleosides prefer the C2'endo-anti conformation. Of the nearly 300 nucleosides known, about 250 are in the anti conformation and 50 are in the syn-conformation, i.e., anti to syn conformation is 5:1. The nucleotide building blocks of nucleic acids show the same trend as nucleosides. Both the deoxy-guanosine and riboguanosine residues in nucleosides and nucleotides prefer the syn-C2'endo conformation with an intra-molecular hydrogen bond (for nucleosides) between the O5'-H and the N3 of the base and, a few syn-C3'endo conformations are also observed. Evidence is presented for the occurrence of the C3'endo-syn conformation for guanines in mis-paired double helical right-handed structures with the distorted sugar phosphate C4'-C5' and P-O5' bonds respectively, from g+ (gg) and g- to trans. Evidence is also provided for guanosine nucleotides in left-handed double-helical (Z-DNA) oligo and polynucleotides which has the same syn-C3'endo conformation and the distorted backbone sugar-phosphate bonds (C4'-C5' and P-O5') as in the earlier right-handed case.     

The occurrence of the syn-C3′ endo conformation and the distorted backbone conformations for C4′-C5′ and P-O5′ in oligo and polynucleotides

Abstract

The nucleoside constituents of nucleic acids prefer the anti conformation (1). When the sugar pucker is taken into account the nucleosides prefer the C2′endo-anti conformation. Of the nearly 300 nucleosides known, about 250 are in the anti conformation and 50 are in the syn-conformation, i.e., anti to syn conformation is 5:1. The nucleotide building blocks of nucleic acids show the same trend as nucleosides. Both the deoxy-guanosine and ribo- guanosine residues in nucleosides and nucleotides prefer the syn-C2′endo conformation with an intra-molecular hydrogen bond (for nucleosides) between the O5′- H and the N3 of the base and, a few syn-C3′endo conformations are also observed. Evidence is presented for the occurrence of the C3′endo-syn conformation for guanines in mis-paired double helical right-handed structures with the distorted sugar phosphate C4′-C5′ and P-O5′ bonds respectively, from g+ (gg) and g- to trans. Evidence is also provided for guanosine nucleotides in left-handed double-helical (Z-DNA) oligo and polynucleotides which has the same syn-C3′endo conformation and the distorted backbone sugar-phosphate bonds (C4′-C5′ and P- O5′) as in the earlier right-handed case.     

Conformational analysis of arabinonucleosides and nucleotides. A comparison with the ribonucleosides and nucleotides.

Conformations of arabino nucleosides and nucleotides have been analyzed by semiempirical energy calculations. It is found that the change in the configuration of the O(2')-hydroxyl group in arabinoses compared to riboses exerts significant influence on the conformational priorities of the glycosyl and the exocyclic C(4')-C(5') bond torsions. While the anti conformations for the bases are preferred, the anti in equilibrium or formed from syn interconversion is considerably hampered compared to ribosides due to large energy barrier. Further the preferred anti glycosyl torsions are shifted to higher values for C(3')-endo puckers and in ribosides. While the gauche+ conformation around the C(4')-C(5') bond is favored for C(3')-endo arabinosides, it is strongly stabilized for C(2')-endo arabinosides only in the presence of the intrasugar hydrogen bond O(2')-H ... O(5'). The net attractive electrostatic interactions between the phosphate and the base stabilizes the preferred conformations of 5'-arabinonucleotides also.     

Atomic coordinates and molecular conformation of yeast phenylalanyl tRNA. An independent investigation.

The atomic coordinates of yeast tRNA(Phe) in the monoclinic crystal form have been determined by an independent analysis from a model built into a 3 A MIR map. The overall molecular structure is found to be in agreement with those reported for the same crystal form by Ladner et al. (1975) and for the orthorhombic form by Quigley et al. (1975) and Kim et al. (1975). However, significant differences between any two of the four models are found in certain local regions of the molecule. The structure is analyzed in terms of the nucleotide stereochemistry and internucleotide phosphodiesters. A striking observation is that the majority of the nucleotide moieties occur in the conformation preferred by the constituent mononucleotides themselves. The internucleotide P-O bonds afford the primary source of flexibility for the folding of the polynucleotide backbone while the sugar pucker and C(4')-C(5') torsions provide the secondary source of flexibility.     

Stereochemical effects of non-ionic methylphosphonates on nucleotide conformations.

The preferred conformations of deoxyribo and ribonucleoside 3'-methylphosphonates are analysed by minimizing the conformational energy as a function of all the major parameters including the sugar ring for both the S- and R-isomers. The results show that neither the substitution nor the nature of the diastereomer affects significantly the preferred conformations compared to the naturally occurring nucleoside 3'-phosphates. The preferred range of C3'-O3' bond torsions or the phase angles of pseudorotation (P) of the sugar are unaffected. The chiral substitution on the phosphate always adopts a conformation distal to the secondary C3' carbon atom in the minimum energy conformational state. Further, it introduces certain restrictions on the preferred range of P-O3' torsions depending on the methylphosphonate configuration. Methylphosphonate, especially the S-isomer, renders the normal gauche- range of P-O3' bond torsions responsible for the stacked helical duplexes to be energetically unfavourable besides introducing a high energy barrier between trans and gauche conformations. Therefore it is suggested that duplexes with S-methylphosphonate may favour extended phosphodiester conformations. These factors explain the observed lower melting temperature as well as the downfield shifts in the 31P signals in duplexes containing the S-isomer.     

Polynucleotide folding in yeast tRNAPhe: elucidation of short-, medium-, and long-range interactions of sugar-phosphate-sugar backbone and base using a "blocked" nucleotide probe

It has recently been proposed that the repeating backbone nucleotide may be regarded as consisting of two blocks of equal magnitude representable by two virtual bonds. Implicit consideration of the nucleotide (ψ,ψ) and internucleotide (ω′,ω) geometry that generate variety in polynucleotide conformations, and of the constancy of the repeating structural moieties (P-C4′ and C4′-P) independent of the above rotations, has enabled us to utilize this scheme in the study of ordered structures such as di-, oligonucleotides and, most significantly, tRNA. The polynucleotide folding dictated by short-, intermediate-, and long-range interactions in the monoclinic and orthorhombic forms is described and compared through circular plots depicting the virtual bond torsions and distance plots constructed independently for backbone as well as bases. The torsions and the bond angles associated with the virtual bonds afford a clear distinction between ordered helical segments from loops and bends of tRNA. Lower virtual bond torsions (?60° to 60°) concomitant with higher values of virtual bond angles characterize various bend regions, while torsions around 160°–210° typify ordered helical strands. The distance plot elucidates the type of interaction associated with various sub-structures (helix–helix, helix–loop, and loop–loop) that form the constituents of different structural domains. Several other features such as the manifestation of the P₁₀ loop and the approximate twofold symmetry in the tRNA molecule are conspicuous on the distance plot.     

Conformational studies of (2''-5'') polynucleotides: theoretical computations of energy, base morphology, helical structure, and duplex formation.

A detailed theoretical analysis has been carried out to probe the conformational characteristics of (2'-5') polynucleotide chains. Semi-empirical energy calculations are used to estimate the preferred torsional combinations of the monomeric repeating unit. The resulting morphology of adjacent bases and the tendency to form regular single-stranded structures are determined by standard computational procedures. The torsional preferences are in agreement with available nmr measurements on model compounds. The tendencies to adopt base stacked and intercalative geometries are markedly depressed compared to those in (3'-5') chains. Very limited families of regular monomerically repeating single-stranded (2'-5') helices are found. Base stacking, however, can be enhanced (but helix formation is at the same time depressed) in mixed puckered chains. Constrained (2'-5') duplex structures have been constructed from a search of all intervening glycosyl and sugar conformations that form geometrically feasible phosphodiester linkages. Both A- and B-type base stacking are found to generate non-standard backbone torsions and mixed glycosyl/sugar combinations. The 2'- and 5'-residues are locked in totally different arrangements and are thereby prevented from generating long helical structures.     

Covalent carcinogenic O6-methylguanosine lesions in DNA. Structural studies of the O6 meG X A and O6meG X G interactions in dodecanucleotide duplexes

High-resolution proton and phosphorus nuclear magnetic resonance studies are reported on the self-complementary d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6meG X A 12-mer when N3 = A3 and O6meG X G 12-mer when N3 = G3), which contain symmetry-related A3 X O6meG10 and G3 X O6meG10 interactions in the interior of the helices. We observe inter-base-pair nuclear Overhauser effects (NOE) between the base protons at the N3 X O6meG10 modification site and protons of flanking G2 X C11 and G4 X C9 base-pairs, indicative of the stacking of N3 and O6meG10 bases in both O6meG X A 12-mer and O6meG X G 12-mer duplexes. We have assigned all the base and a majority of the sugar protons from two-dimensional proton-correlated and nuclear Overhauser effect experiments on the O6meG X A 12-mer duplex and O6meG X G 12-mer duplex in solution. The observed NOEs establish that the A3 and O6meG10 at the modification site and all other residues adopt the anti configuration about the glycosidic bond, and that the O6meG X A 12-mer forms a right-handed duplex. The interaction between the bulky purine A3 and O6meG10 residues in the anti orientation results in large proton chemical shift perturbations at the (G2-A3-G4) X (C9-O6meG10-C11) segments of the helix. By contrast, we demonstrate that the O6meG10 residue adopts a syn configuration, while all other bases adopt an anti configuration about the glycosidic bond in the right-handed O6meG X G 12-mer duplex. This results in altered NOE patterns between the base protons of O6meG10 and the base and sugar protons of flanking C9 and C11 residues in the O6meG X G 12-mer duplex. The phosphorus backbone is perturbed at the modification site in both duplexes, since the phosphorus resonances are dispersed over 2 parts per million in the O6meG X A 12-mer and over 1 part per million in the O6meG X G 12-mer compared to a 0.5 part per million dispersion for an unperturbed DNA helix. We propose tentative pairing schemes for the A3 X O6meG10 and G3 X O6meG10 interactions in the above dodecanucleotide duplexes.     

Two-dimensional proton nuclear magnetic resonance investigation of the synthetic deoxyribonucleic acid decamer d(ATATCGATAT)2

In this study two-dimensional NMR techniques (COSY and NOESY) have been used in conjunction with one-dimensional NMR results to complete the assignment of the proton NMR spectrum of the double-stranded DNA decamer, d(ATATCGATAT)2, and to obtain qualitative information about numerous interproton distances in this molecule and some limited information about conformational dynamics. COSY and NOESY measurements have been combined to systematically assign many of the resonances from the H1' and H2',2" sugar protons to specific nucleotides in the double helix. This method relies on the fact that sugar protons within a specific nucleotide are scalar coupled and that base protons (AH8, GH8, TH6, and CH6) in right-handed helices can interact simultaneously with their own H2',2" sugar protons and those of the adjacent (5'-3') nucleotide attached to its 5' side (i.e., XpA not ApX). A COSY experiment is used to identify sugar resonances within a residue whereas the NOESY experiment allows the neighboring sugar to be connected (linked). The CH5 and CH6 resonances in the spectrum can immediately be identified by the COSY experiment. The methyl protons of thymine residues exhibit strong through-space interbase interactions both with their own TH6 proton and with AH8 proton on the adjacent (5'-3') adenine residue. These interactions are used both to make assignments of the spectra and to establish that the thymine methyl groups are in close proximity to the AH8 protons of adjacent adenine residues [Feigon, J., Wright, J. M., Leupin, W., Denny, W. A., & Kearns, D. R. (1982) J. Am. Chem. Soc. 104, 5540].(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular mechanics studies on poly(purine).poly(pyrimidine) sequences in DNA: polymorphism and local variability

Energy minimization has been carried out on three poly(purine).poly(pyrimidine) sequences--d(G)10.d(C)10, d(A)10.d(T)10, and d(AG)5.d(CT)5--using the molecular mechanics program AMBER (Assisted Model Building and Energy Refinement). In order to extensively scan the conformational space available, five different helical models were studied, three of them being right-handed helices while the other two were left helical. For all three sequences the right-handed A- and B-type helices are energetically slightly preferred over the left helices, but the energy difference between the various right-handed helices is only marginal. A detailed analysis has been carried out to characterize the local structural variability in the refined structures, both in terms of torsion angles as well as other parameters such as base-pair tilt, wedge roll, and wedge tilt, etc. All three sequences exhibit similar structural features for a particular form, but both the forms A and B show significant deviations from fiber models. In particular, the A-form structures have higher unit rise (2.7 A), and lower unit twist (31 degrees) and base-pair tilt (12 degrees), compared to the fiber model, which has corresponding values of 2.56 A, 32.7 degrees, and 20 degrees, respectively. All these changes indicate that the refined models are closer to the A-form structure observed in crystals of oligonucleotides. In the refined B-for models, the helical parameters are close to the fiber B-form, although the torsion angles show considerable variations. None of the three sequences examined, including the d(A)n.d(T)n sequence, show any pronounced curvature for the B-form structure.     

Molecular mechanics studies on the conformations of 2',3'-dideoxy-2',3'-didehydroguanine nucleoside, D4G.

Conformational energy calculations have been presented on guanine nucleoside in which the furanose ring is replaced by 2',3'-dideoxy-2',3'-didehydrofuran using molecular mechanics and conformational analysis. Conformational energies have been evaluated using the MM2 and AMBER94 force field parameters at two different dielectric constants. The results are presented in terms of isoenergy contours in the conformational space of the glycosidic (chi) and C4'-C5' (gamma) bonds torsions. In general, the chi-gamma interrelationships differ from the corresponding plots for unmodified nucleosides and nucleotides, reported previously. Consistency of the calculated preferred conformations with the x-ray data is sensitive to the force field employed.     

Index to Subjects,Vol. 27

Abstract

A constrained model building procedure is used to generate nucleic acid structures of the familiar A-, B-, and Z-DNA duplexes. Attention is focused upon the multiple structural solutions associated with the arrangements of nucleic acid base pairs rather than the optimum sugar-phosphate structure. The glycosyl (χ) and sugar torsions (both the ring puckering and the exocyclic C5′-C4′ (ψ) torsion) are treated as independent variables and the resulting O3′…O5′ distances are used as closure determinants. When such distances conform to the known geometry of phosphate chemical bonding, an intervening phosphorus atom with correct C-O-P valence angles can be located. Four sequential torsion angles- φ,ω,ω,ω and φ about the C3′-O3′-P-O5′-C5′ bonds are then obtained as dependent variables. The resulting structures are categorized in terms of conformation, ranked in potential energy, and analyzed for torsional correlations. The numerical results are quite interesting with implications regarding nucleic acid models constructed to fit less than ideal experimental data. The multiple solutions to the problem are useful for comprehending the conformational complexities of thelocal sugar-phosphate backbone and for understanding the transitions between different helical forms. According to these studies, unique characterization of a nucleic acid duplex involves more than the determination of its base pair morphology, its sugar puckering preferences, or its groove binding features.     

Multinuclear nuclear magnetic resonance studies of Na cation-stabilized complex formed by d(G-G-T-T-T-T-C-G-G) in solution. Implications for G-tetrad structures.

There has been much recent interest in the self-association of short deoxyguanosine-rich motifs within single-stranded DNAs to generate monovalent cation modulated four-stranded helical segments called G-quadruplexes stabilized by hydrogen-bonded G-tetrad alignments. We have addressed structural aspects of this novel alignment and report on multinuclear 1H, 31P and 13C nuclear magnetic resonance studies on the d(G2T4CG2) deoxynonanucleotide with Na cation as counterion in aqueous solution at low temperature. This sequence forms stable structures even though it cannot align by Watson-Crick hydrogen bond formation (see the paper on d(G2T5G2) describing optical and calorimetric measurements by Jin, R., Breslauer, K. J., Jones, R. A. & Gaffney, B. L. (1990), Science, 250, 543-546). The four narrow exchangeable protons detected between 11.5 and 12.0 parts per million (p.p.m.), which are common to the d(G2T4CG2) deoxynonanucleotide and the d(G2TCG2) deoxyhexanucleotide sequences, are assigned to deoxyguanosine imino protons hydrogen-bonded to carbonyl acceptor groups. These narrow imino protons are not detected for d(IGN5IG) and d(I2N5G2), where two deoxyguanosine residues are replaced by two deoxyinosine residues in the deoxynonanucleotide sequences. This implies that the 2-amino protons of deoxyguanosine must also participate in hydrogen bond formation and stabilize the structured conformation of d(G2T4CG2) in Na cation-containing solution. We have completely assigned the base and sugar H1', H2',2', H3', and H4' protons of the d(G2T4CG2) oligomer following analysis of two-dimensional nuclear Overhauser enhancement spectroscopy and two-dimensional correlated spectroscopy data sets in 0.1 M-NaCl, 10 mM-sodium phosphate, 2H2O solution at 0 degree C. The relative magnitude of the nuclear Overhauser enhancements (NOEs) between the base H8 and its own sugar H1' protons of individual deoxyguanosine residues establishes that G1 and G8 adopt syn orientations while G2 and G9 adopt anti orientations about the glycosidic bond in the d(G1-G2-T3-T4-T5-T6-C7-G8-G9) sequence in both Na and K cation-containing aqueous solution. Consequently, any structure proposed for the tetramolecular complex of d(G2T4CG2) must exhibit alternating G(syn) and G(anti) glycosidic torsion angles within each strand. The directionality and magnitude of the observed NOEs are consistent with the G(syn)-G(anti) steps adopting right-handed helical conformations in solution. We also note that the H8 protons of G1 and G8 (7.35 to 7.45 p.p.m.) in a syn alignment are shifted significantly upfield from the H8 protons of G2 and G9 (8.0 to 8.3 p.p.m.) in an anti alignment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformational Studies on Nucleosides with Furanose Ring Modifications. 1.

Abstract

Conformational energy calculations have been presented on adenine and thymine nucleosides in which the furanose ring is replaced by 2′,3′-dideoxy-2′,3′-didehydrofuran using molecular mechanics and conformational analysis. Conformational energies have been evaluated using the MM2 and AMBER94 force field parameters at two different dielectric constants. The results are presented in terms of isoenergy contours in the conformational space of the glycosidic (χ) and C4′-C5′ (γ) bonds torsions. In general, the χ-γ interrelationships exhibit similarities with the corresponding plots for unmodified nucleosides and nucleotides, reported previously. Consistency of the calculated preferred conformations with the X-ray data is sensitive to the force field employed.     

The identification of the A-type RNA helices in a 55mer RNA by selective incorporation of deuterium-labelled nucleotide residues (Uppsala NMR-window concept)

The 55-nt long RNA, modelling a three-way junction, with non-uniformly incorporated deuterated nucleotides has been synthesised in a pure form. The NMR-window part in this partially deuterated 55mer RNA consists of natural non-enriched nucleotide blocks at the three-way junction (shown in a square box in Fig. 2), whereas all other nucleotides of the rest of the molecule are partially deuterated (> 97 atom% 2H at C2', C3', C5', C5, and approximately 50 atom% 2H at C4'). The secondary structure of this 55mer RNA was determined by 2D 1H NOESY spectroscopy in D2O or in 10% D2O-H2O mixture. The use of deuterated building blocks in the specific region of the 55mer RNA allowed us to identify two distinct A-type RNA helices in a straightforward manner by observing connectivities of H1' with the basepaired imino and the aromatic H2 of all adenosine nucleotides as the first step for the determination of its tertiary structure in a cost- and time-effective manner without employing any 13C/15N labelling. These two decameric helices involve 40 nucleotides, for which all non-exchangeable H1', H6, H2, H8 and H5 protons (all 40 H1', all 40 H6 or H8 aromatics, all seven H2 of adenine nucleotide and all four non-deuterated H5 of cytosines) as well as all 16 exchangeable imino protons (with the exception of four terminal basepairs) and 16 amino protons of cytosines have been assigned. Since all aromatic-H2', H3' as well as H5'/5' crosspeaks from partially deuterated residues have been eliminated from the NMR spectra, the observation of natural nucleotide residues in the NMR window part has essentially been simplified. It has been found that the crosspeaks from the natural nucleotides located at the three-way junction in the NMR-window part show different degrees of line-broadening, thereby indicating that the various nucleotide residues have very different mobilities with respect to themselves as well as compared to other nucleotides in the helices. The assignment of H2' and H3' in the NMR-window part has been made based on NOESY and DQF-COSY crosspeaks. It is noteworthy that, even in this preliminary study, it has been possible to identify 10 H2' out of total 14 and 9 H3' out of 14. The data show that expanded AU containing a tract of 55mer RNA does not self-organise into a tight third helix, as the two decameric A-type helices, across the three-way junction which is evident from the absence of any additional imino protons, except those that already have been assigned for the two decameric helices.     

Atomic resolution analysis of a 2:1 complex of CpG and acridine orange.

Cytidylyl-3', 5'-guanosine and acridine orange crystallize in a highly-ordered triclinic lattice which diffracts X-rays to 0.85 angstrom resolution. The crystal structure has been solved and refined to a residual factor of 9.5%. The two dinucleoside phosphate molecules form an antiparallel double helix with the acridine orange intercalated between them. The two base pairs of the double helical fragment have a twist angle of 10 degrees and it is found to have a C3' endo-(3', 5')-C2' endo mixed sugar puckering along the nucleotide backbone as has been observed for other simple intercalator complexes. Twenty-five water molecules have been located in the lattice together with a sodium ion. The intercalator double helical fragments form sheets which are held together by van der Waals interactions in one direction and hydrogen bonding interactions in the other. The crystal lattice contains aqueous channels in which sixteen water molecules are hydrogen bonded to the nucleotide, none to the intercalator, five water molecules are coordinated about the sodium ion and four water molecules bind solely to other water molecules. The bases in the base pairs have a dihedral angle of 7 to 8 degrees between them.     

Different conformational forms of Escherichia coli and rat liver 5S rRNA revealed by Pb(II)-induced hydrolysis.

Different stable forms of Escherichia coli and rat liver 5S rRNA have been probed by Pb(II)-induced hydrolysis. In the native A forms of 5S rRNA, Pb2+ reveal single-stranded RNA stretches and regions of increased conformational flexibility or distorted by the presence of bulged nucleotides. Hydrolysis of urea/EDTA-treated E. coli 5S rRNA (B form) shows the presence of two strong helical domains; helix A retained from the A form and a helix composed of RNA regions G33-C42 and G79-C88. Other RNA regions resistant to hydrolysis may be involved in alternative base pairing, causing conformational heterogeneity of that form. Pb(II)-induced hydrolysis distinguishes two different forms of rat liver 5S rRNA; the native A form and the form obtained by renaturation of 5S rRNA in the presence of EDTA. Pb(II)-hydrolysis data suggest that both forms are highly structured. In the latter form, the orientation of the bulged C66 is changed with respect to helix B. At the same time, a new helical segment is possibly formed, composed of nucleotides from helix C and loop c on one side and from helix E and loop d' on the other.