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)     

The insertion of two 8-methyl-2'-deoxyguanosine residues in tetramolecular quadruplex structures: trying to orientate the strands

In this article, we report a structural study, based on NMR and CD spectroscopies, and molecular modelling of all possible d(TG(3)T) and d(TG(4)T) analogues containing two 8-methyl-2'-deoxyguanosine residues (M). Particularly, the potential ability of these modified residues to orientate the strands and then to affect the folding topology of tetramolecular quadruplex structures has been investigated. Oligodeoxynucleotides (ODNs) TMMGT (T12) and TMMGGT (F12) form parallel tetramolecular quadruplexes, characterized by an all-syn M-tetrad at the 5'-side stacked to all-anti M- and G-tetrads. ODNs TMGMT (T13) and TMGGMT (F14) form parallel tetramolecular quadruplexes, in which an all-anti G core is sandwiched between two all-syn M-tetrads at the 5'- and the 3'-side. Notably, the quadruplex formed by T13 corresponds to an unprecedented structure in which the syn residues exceed in number the anti ones. Conversely, ODN TGMGMT (F24) adopts a parallel arrangement in which all-anti G-tetrads alternate with all-syn M-tetrads. Most importantly, all data strongly suggest that ODN TMGMGT (F13) forms an unprecedented anti-parallel tetramolecular quadruplex in which G and M residues adopt anti and syn glycosidic conformations, respectively. This article opens up new understandings and perspectives about the intricate relationship between the quadruplex strands orientation and the glycosidic conformation of the residues.     

Comparison of the B- and Z-form hairpin loop structures formed by d(CG)5T4(CG)5

The partially self-complementary synthetic DNA oligonucleotide d(CG)5T4(CG)5 has been studied by using 1H and 31P NMR and circular dichroism. Results show that, under low-salt conditions (120 mM NaCl buffer), an intramolecular hairpin loop exists in which the double-helical stem region is B-form and the thymidine loop residues have predominantly southern (C2'-endo) sugar conformations. The thymidine glycosidic torsion angles are intermediate between syn and anti or exist as an equilibrium mixture of residues in the two extremes. NOESY data indicate that the structure of the loop region is very similar to that found for d(CG)2T4(CG)2 [Hare, D. R., & Reid, B. R. (1986) Biochemistry 25, 5341-5350]. Under high-salt conditions (6 M NaClO4 buffer), the dominant form (approximately equal to 85%) is an intramolecular hairpin structure in which the stem region forms a Z-form double helix. As in the B-form, the loop thymidine residues are intermediate between the syn and anti conformations or exist as an equilibrium mixture of the two, but the thymidine sugar conformations differ in that they are biased toward northern (C3'-endo) conformations.     

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite deoxyguanosine in a DNA duplex. Epsilon dA(syn).dG(anti) pairing at the lesion site

Proton NMR studies are reported on the complementary d(C-A-T-G-G-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dG 9-mer duplex), which contains exocyclic adduct 1,N6-ethenodeoxyadenosine positioned opposite deoxyguanosine in the center of the helix. The present study focuses on the alignment of dG5 and epsilon dA14 at the lesion site in the epsilon dA.dG 9-mer duplex at neutral pH. This alignment has been characterized by monitoring the NOEs originating from the NH1 proton of dG5 and the H2, H5, and H7/H8 protons of epsilon dA14 in the central d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment of the epsilon dA.dG 9-mer duplex. These NOE patterns establish that epsilon dA14 adopts a syn glycosidic torsion angle that positions the exocyclic ring toward the major groove edge while all the other bases including dG5 adopt anti glycosidic torsion angles. We detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment which establish formation of right-handed helical conformations on both strands and stacking of the dG5(anti).epsilon dA14(syn) pair between stable dG4.dC15 and dG6.dC13 pairs. The energy-minimized conformation of the central d(G4-G5-G6).d(C13-epsilon A14-C15) segment establishes that the dG5(anti).epsilon dA14(syn) alignment is stabilized by two hydrogen bonds from the NH1 and NH2-2 of dG5(anti) to N9 and N1 of epsilon dA14(syn), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution structure of a DNA duplex containing 8-hydroxy-2'-deoxyguanosine opposite deoxyguanosine

Deoxyguanosine residues are hydroxylated by reactive oxygen species at the C-8 position to form 8-hydroxy-2'-deoxyguanosine (8-OG), one of the most important mutagenic lesions in DNA. Though the spontaneous G:C to C:G transversions are rare events, the pathways leading to this mutation are not established. An 8-OG:G mispair, if not corrected by DNA repair enzymes, could lead to G:C to C:G transversions. NMR spectroscopy and restrained molecular dynamics calculations are used to refine the solution structure of the base mismatch formed by the 8-OG:G pair on a self complementary DNA dodecamer duplex d(CGCGAATT(8-O)GGCG)(2). The results reveal that the 8-OG base is inserted into the helix and forms Hoogsteen base-pairing with the G on the opposite strand. The 8-OG:G base-pairs are seen to be stabilized by two hydrogen bonding interactions, one between the H7 of the 8-OG and the O6 of the G, and a three-center hydrogen bonding between the O8 of the 8-OG and the imino and amino protons of the G. The 8-OG:G base-pairs are very well stacked between the Watson-Crick base-paired flanking bases. Both strands of the DNA duplex adopt right-handed conformations. All of the unmodified bases, including the G at the lesion site, adopt anti glycosidic torsion angles and form Watson-Crick base-pairs. At the lesion site, the 8-OG residues adopt syn conformations. The structural studies demonstrate that 8-OG(syn):G(anti) forms a stable pair in the interior of the duplex, providing a basis for the in vivo incorporation of G opposite 8-OG. Calculated helical parameters and backbone torsional angles, and the observed 31P chemical shifts, indicate that the structure of the duplex is perturbed near lesion sites, with the local unwinding of the double helix. The melting temperature of the 8-OG:G containing duplex is only 2.6 deg. C less than the t(m) of the unmodified duplex.     

Binding modes of inhibitors to ribonuclease T1 as elucidated by the analysis of two-dimensional NMR

Aromatic proton and high field shifted methyl proton resonances of RNase T1 complexed with Guo, 2'GMP, 3'GMP or 5'GMP were assigned to specific amino acid residues by 2D-NMR spectra in comparison with the crystal structure of RNase T1-2'GMP complex. The spatial proximities of amino acid residues as elucidated by NOESY spectra were found to be quite similar among free RNase T1 and the inhibitor complexes, showing that large conformational changes did not occur upon complex formation. However, small but appreciable conformational changes were induced which were reflected by the systematic chemical shift changes of some amino acid residues in the active site. Furthermore, we confirmed that RNase T1 contains two specific binding sites, one for the guanine base and the other for the phosphate moiety. The inhibitors are forced to adapt their conformations to fit the guanine base and the phosphate moiety to each binding site on the enzyme. This is consistent with our previous studies that 2'GMP and 3'GMP take syn form as a bound conformation, while 5'GMP takes anti conformation around glycosidic bonds.     

NMR structural studies of the ionizing radiation adduct 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) opposite deoxyadenosine in a DNA duplex. 8-Oxo-7H-dG(syn).dA(anti) alignment at lesion site

Proton NMR studies are reported on the complementary d(C1-C2-A3-C4-T5-A6-oxo-G7-T8-C9-A10-C11-C12).d(G13-G14-T15- G16-A17-A18-T19- A20-G21-T22-G23-G24) dodecanucleotide duplex (designated 8-oxo-7H-dG.dA 12-mer), which contains a centrally located 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) residue, a group commonly found in DNA that has been exposed to ionizing radiation or oxidizing free radicals. From the NMR spectra it can be deduced that this moiety exists as two tautomers, or gives rise to two DNA conformations, that are in equilibrium and that exchange slowly. The present study focuses on the major component of the equilibrium that originates in the 6,8-dioxo tautomer of 8-oxo-7H-dG. We have assigned the exchangeable NH1, NH7, and NH2-2 base protons located on the Watson-Crick and Hoogsteen edges of 8-oxo-7H-dG7 in the 8-oxo-7H-dG.dA 12-mer duplex, using an analysis of one- and two-dimensional nuclear Overhauser enhancement (NOE) data in H2O solution. The observed NOEs derived from the NH7 proton of 8-oxo-7H-dG7 to the H2 and NH2-6 protons of dA18 establish an 8-oxo-7H-dG7(syn).dA 18(anti) alignment at the lesion site in the 8-oxo-7H-dG.dA 12-mer duplex in solution. This alignment, which places the 8-oxo group in the minor groove, was further characterized by an analysis of the NOESY spectrum of the 8-oxo-7H-dG.dA 12-mer duplex in D2O solution. We were able to detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(A6-oxo-G7-T8).d(A17-A18-T19) trinucleotide segment centered about the lesion site that establishes stacking of the oxo-dG7(syn).dA(anti) pair between stable Watson-Crick dA6.dT19 and dT8.dA17 base pairs with minimal perturbation of the helix. Thus, both strands of the 8-oxo-7H-dG.dA 12-mer duplex adopt right-handed conformations at and adjacent to the lesion site, the unmodified bases adopt anti glycosidic torsion angles, and the bases are stacked into the helix. The energy-minimized conformation of the central d(A6-oxo-G7-T8).d(A17-A18-T19) segment requires that the 8-oxo-7H-dG7(syn).dA18(anti) alignment be stabilized by two hydrogen bonds from NH7 and O6 of 8-oxo-7H-dG7(syn) to N1 and NH2-6 of dA18(anti), respectively, at the lesion site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformational feasibility of a hairpin with two purines in the loop. 5'-d-GGTACIAGTACC-3'

Structural feasibility and conformational requirements for the sequence 5'-d-GGTACIAGTACC-3' to adopt a hairpin loop with I6 and A7 in the loop are studied. It is shown that a hairpin loop containing only two nucleotides can readily be formed without any unusual torsional angles. Stacking is continued on the 5'-side of the loop, with the I6 stacked upon C5. The base A7, on the 3'-side of the loop, can either be partially stacked with I6 or stick outside without stacking. Loop closure can be achieved for both syn and anti conformations of the glycosidic torsions for G8 while maintaining the normal Watson-Crick base pairing with the opposite C5. All torsional angles in the stem fall within the standard B-family of DNA helical structures. The phosphodiesters of the loop have trans,trans conformations. Loop formation might require the torsion about the C4'-C5' bond of G8 to be trans as opposed to the gauche+ observed in B-DNA. These results are discussed in relation to melting temperature studies [Howard et al. (1991) Biochemistry (preceding paper in this issue)] that suggest the formation of very stable hairpin structures for this sequence.     

Alternating d(GA)n DNA sequences form antiparallel stranded homoduplexes stabilized by the formation of G.A base pairs.

Alternating d(GA)n DNA sequences form antiparallel stranded homoduplexes which are stabilized by the formation of G.A pairs. Three base pairings are known to occur between adenine and guanine: AH+ (anti).G(syn), A(anti).G(anti) and A(syn).G(anti). Protonation of the adenine residues is not involved in the stabilization of this structure, since it is observed at any pH value from 8.3 to 4.5; at pH < or = 4.0 antiparallel stranded d(GA.GA) DNA is destabilized. The results reported in this paper strongly suggest that antiparallel stranded d(GA.GA) homoduplexes are stabilized by the formation of alternating A(anti).G(anti) and G(anti).A(syn) pairs. In this structure, all guanine residues are in the anti conformation with their N7 position freely accessible to DMS methylation. On the other hand, adenines in one strand adopt the anti conformation, with their N7 position also free for reaction, while those of the opposite strand are in the syn conformation, with their N7 position hydrogen bonded to the guanine N1 group of the opposite strand. A regular right-handed helix can be generated using alternating G(anti).A(syn) and A(anti).G(anti) pairs.     

NMR and computational characterization of the N-(deoxyguanosin-8-yl)aminofluorene adduct [(AF)G] opposite adenosine in DNA: (AF)G[syn].A[anti] pair formation and its pH dependence

This paper reports on a combined two-dimensional NMR and energy minimization computational characterization of the conformation of the N-(deoxyguanosyl-8-yl)aminofluorene adduct [(AF)G] positioned across adenosine in a DNA oligomer duplex as a function of pH in aqueous solution. This study was undertaken on the d[C1-C2-A3-T4-C5-(AF)G6-C7-T8-A9-C10-C11].[G12-G13-T14 -A15-G16-A17-G18- A19-T20-G21-G22] complementary undecamer [(AF)G 11-mer duplex]. The modification of the single G6 on the pyrimidine-rich strand was accomplished by reaction of the oligonucleotide with N-acetoxy-2-(acetylamino)fluorene and subsequent deacetylation under alkaline conditions. The HPLC-purified modified strand was annealed with the unmodified purine-rich strand to generate the (AF)G 11-mer duplex. The exchangeable and nonexchangeable protons are well resolved and narrow in the NMR spectra of the (AF)G 11-mer duplex so that the base and the majority of sugar nucleic acid protons, as well as several aminofluorene ring protons, have been assigned following analysis of two-dimensional NOESY and COSY data sets at pH 6.9, 30 degrees C in H2O and D2O solution. The NOE distance constraints establish that the glycosidic torsion angle is syn at (AF)G6 and anti at A17, which results in the aminofluorene ring being positioned in the minor groove. A very large downfield shift is detected at the H2' sugar proton of (AF)G6 associated with the (AF)G6[syn].A17[anti] alignment in the (AF)G 11-mer duplex. The NMR parameters demonstrate formation of Watson-Crick C5.G18 and C7.G16 base pairs on either side of the (AF)G6[syn].A17[anti] modification site with the imino proton of G18 more stable to exchange than the imino proton of G16. Several nonexchangeable aminofluorene protons undergo large downfield shifts as do the imino and H8 protons of G16 on lowering of the pH from neutrality to acidic values for the (AF)G 11-mer duplex. Both the neutral and acidic pH conformations have been defined by assigning the NOE constraints in the [C5-(AF)G6-C7].[G16-A17-G18] segment centered about the modification site and incorporating them in distance constrained minimized potential energy calculations in torsion angle space with the DUPLEX program. A series of NOEs between the aminofluorene protons and the DNA sugar protons in the neutral pH conformation establish that the aminofluorene ring spans the minor groove and is directed toward the G16-A17-G18 sugar-phosphate backbone on the partner strand.(ABSTRACT TRUNCATED AT 400 WORDS)     

The solution structure of d(G(4)T(4)G(3))(2): a bimolecular G-quadruplex with a novel fold

The G-rich 11-mer oligonucleotide d(G(4)T(4)G(3)) forms a bimolecular G-quadruplex in the presence of sodium ions with a topology that is distinct from the folds of the closely related and well-characterized sequences d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)). The solution structure of d(G(4)T(4)G(3))(2) has been determined using a combination of NMR spectroscopy and restrained molecular dynamics calculations. d(G(4)T(4)G(3))(2) forms an asymmetric dimeric fold-back structure consisting of three stacked G-quartets. The two T(4) loops that span diagonally across the outer faces of the G-quartets assume different conformations. The glycosidic torsion angle conformations of the guanine bases are 5'-syn-anti-syn-anti-(T(4) loop)-anti-syn-anti in one strand and 5'-syn-anti-syn-anti-(T(4) loop)-syn-anti-syn in the other strand. The guanine bases of the two outer G-quartets exhibit a clockwise donor-acceptor hydrogen-bonding directionality, while those of the middle G-quartet exhibit the anti-clockwise directionality. The topology of this G-quadruplex, like other bimolecular fold-back structures with diagonal loops, places each strand of the G-quartet region next to a neighboring parallel and an anti-parallel strand. The two guanine residues not involved in G-quartet formation, G4 and G12 (i.e. the fourth guanine base of one strand and the first guanine base of the other strand), adopt distinct conformations. G4 is stacked on top of an adjacent G-quartet, and this base-stacking continues along with the bases of the loop residues T5 and T6. G12 is orientated away from the core of G-quartets; stacked on the T7 base and apparently involved in hydrogen-bonding interactions with the phosphodiester group of this same residue. The cation-dependent folding of the d(G(4)T(4)G(3))(2) quadruplex structure is distinct from that observed for similar sequences. While both d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)) form bimolecular, diagonally looped G-quadruplex structures in the presence of Na(+), K(+) and NH(4)(+), we have observed this folding to be favored for d(G(4)T(4)G(3)) in the presence of Na(+), but not in the presence of K(+) or NH(4)(+). The structure of d(G(4)T(4)G(3))(2) exhibits a "slipped-loop" element that is similar to what has been proposed for structural intermediates in the folding pathway of some G-quadruplexes, and therefore provides support for the feasibility of these proposed transient structures in G-quadruplex formation.     

NMR studies of abasic sites in DNA duplexes: deoxyadenosine stacks into the helix opposite the cyclic analogue of 2-deoxyribose

Proton and phosphorus NMR studies are reported for the complementary d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-F-C-A-T-G) nonanucleotide duplex (designated APF 9-mer duplex) which contains a stable abasic site analogue, F, in the center of the helix. This oligodeoxynucleotide contains a modified tetrahydrofuran moiety, isosteric with 2-deoxyribofuranose, which serves as a structural analogue of a natural apurinic/apyrimidinic site [Takeshita, M., Chang, C.N., Johnson, F., Will, S., & Grollman, A.P. (1987) J. Biol. Chem. 262, 10171-10179]. Exchangeable and nonexchangeable base and sugar protons, including those located at the abasic site, have been assigned in the complementary APF 9-mer duplex by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H2O and D2O solution at low temperature (0 degrees C). These studies indicate that A5 inserts into the helix opposite the abasic site F14 and stacks with flanking G4.C15 and G6.C13 Watson-Crick base pairs. Base-sugar proton NOE connectivities were measured through G4-A5-G6 on the unmodified strand and between the base protons of C15 and the sugar protons of the 5'-flanking residue F14 on the modified strand. These studies establish that all glycosidic torsion angles are anti and that the helix is right-handed at and adjacent to the abasic site in the APF 9-mer duplex. Two of the 16 phosphodiester groups exhibit phosphorus resonances outside the normal spectral dispersion indicative of altered torsion angles at two of the phosphate groups in the backbone of the APF 9-mer duplex.     

Yeast tRNAAsp: codon and wobble codon-anticodon interactions. A transferred nuclear Overhauser enhancement study

The conformations of the ribotrinucleoside bisphosphates GpApC and GpApU, the codon and wobble codon for aspartic acid respectively, bound to yeast tRNAAsp in solution, have been examined by means of time-dependent transferred nuclear Overhauser enhancement measurements to determine distances between bound ligand protons. The conformations of the two bound ribotrinucleoside bisphosphates are shown to be very similar with an overall root-mean-square difference in interproton distances of 0.03 nm. The ribose conformations of all the residues are 3'-endo; the glycosidic bond torsion angles of the A and C residues of GpApC and of the A and U residues of GpApU are in the low anti range. These features are typical of an A-RNA type structure. In contrast, the G residue of both GpApC and GpApU exists as a mixture of syn and anti conformations. The overall conformation of the two bound ribotrinucleoside bisphosphates is also similar to A-RNA and the stability of the complexes is enhanced by extensive base-base stacking interactions. In addition, it is shown that the binding of the codon GpApC to tRNAAsp induces self-association into a multicomplex system consisting of four GpApC-tRNAAsp complexes, whereas the wobble codon GpApU fails to induce any observable self-association.     

Binding modes of inhibitors of ribonuclease T1 as elucidated by analysis of two-dimensional NMR

Aromatic proton and high field shifted methyl proton resonances of RNase T1 complexed with Guo, 2'GMP, 3'GMP, or 5'GMP were assigned to specific amino acid residues by analyses of the two-dimensional NMR spectra in comparison with the crystal structure of the RNase T1-2'GMP complex. These assignments were subsequently correlated to those of free RNase T1 [Hoffmann & Rüterjans (1988) Eur. J. Biochem. 177, 539-560]. The spatial proximities of amino acid residues as elucidated by NOESY spectra were found to be quite similar among free RNase T1 and the inhibitor complexes, showing that large conformational changes did not occur upon complex formation. However, small but appreciable conformational changes were induced, which were reflected by the systematic chemical shift changes of some amino acid residues in the active site. Furthermore, we confirmed that RNase T1 contains two specific binding sites, one for the guanine base and the other for the phosphate moiety. The inhibitors are forced to adapt their conformations to fit the guanine base and the phosphate moiety to each binding site on the enzyme. This is consistent with our previous studies that 2'GMP and 3'GMP take the syn form as a bound conformation, while 5'GMP takes the anti conformation around glycosidic bonds [Inagaki et al. (1985) Biochemistry 24, 1013-1020]. The slow-exchange process between free and bound forms involving Tyr42 and Tyr45 was found to be specific to the recognition of the guanine base.     

A double chain reversal loop and two diagonal loops define the architecture of a unimolecular DNA quadruplex containing a pair of stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads flanked by a G-(T-T) Triad and a T-T-T triple.

The architecture of G-G-G-G tetrad-aligned DNA quadruplexes in monovalent cation solution is dependent on the directionality of the four strands, which in turn are defined by loop connectivities and the guanine syn/anti distribution along individual strands and within individual G-G-G-G tetrads. The smallest unimolecular G-quadruplex belongs to the d(G2NnG2NnG2NnG2) family, which has the potential to form two stacked G-tetrads linked by Nn loop connectivities. Previous studies have focused on the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), where Nn was T2 for the first and third connecting loops and TGT for the middle connecting loop. This DNA aptamer in K(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(anti)-G(syn)-G(anti) tetrads, adjacent strands which are antiparallel to each other and edge-wise connecting T2, TGT and T2 loops. We now report on the NMR-based solution structure of the d(G2T4G2CAG2GT4G2T) sequence, which differs from the thrombin-binding DNA aptamer sequence in having longer first (T4) and third (GT4) loops and a shorter (CA) middle loop. This d(G2T4G2CAG2GT4G2T) sequence in Na(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads, adjacent strands which have one parallel and one antiparallel neighbors and distinct non-edge-wise loop connectivities. Specifically, the longer first (T4) and third (GT4) loops are of the diagonal type while the shorter middle loop is of the double chain reversal type. In addition, the pair of stacked G-G-G-G tetrads are flanked on one side by a G-(T-T) triad and on the other side by a T-T-T triple. The distinct differences in strand directionalities, loop connectivities and syn/anti distribution within G-G-G-G tetrads between the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2) quadruplex reported previously, and the d(G2T4G2CAG2GT4G2T) quadruplex reported here, reinforces the polymorphic nature of higher-order DNA architectures. Further, these two small unimolecular G-quadruplexes, which are distinct from each other and from parallel-stranded G-quadruplexes, provide novel targets for ligand recognition. Our results demonstrate that the double chain reversal loop connectivity identified previously by our laboratory within the Tetrahymena telomere d(T2G4)4 quadruplex, is a robust folding topology, since it has now also been observed within the d(G2T4G2CAG2GT4G2T) quadruplex. The identification of a G-(T-T) triad and a T-T-T triple, expands on the available recognition alignments for base triads and triples.     

Structural properties of the [d(G3T4G3)]2 quadruplex: evidence for sequential syn-syn deoxyguanosines.

Two-dimensional 1H NMR studies on the dimeric hairpin quadruplex formed by d(G3T4G3) in the presence of either NaCl or KCl are presented. In the presence of either salt, the quadruplex structure is characterized by half the guanine nucleosides in the syn conformation about the glycosidic bond, the other half in the anti conformation, as reported for other similar sequences. However, 1H NOESY and 1H-31P heteronuclear correlation experiments demonstrate that the deoxyguanosines do not strictly alternate between syn and anti along individual strands. Thus we find the following sequences with regard to glycosidic bond conformation: 5'-G1SG2SG3AT4AT5A-T6AT7AG8SG9AG10A-3' and 5'-G11SG12AG13AT14AT1 5AT16AT17AG18SG19SG20A-3', where S and A denote syn and anti, respectively. This represents the first experimental evidence for a nucleic acid structure containing two sequential nucleosides in the syn conformation. The stacking interactions of the resulting quadruplex quartets and their component bases have been evaluated using unrestrained molecular dynamics calculations and energy component analysis. These calculations suggest that the sequential syn-syn/anti-anti and syn-anti quartet stacks are almost equal in energy, whereas the anti-syn stack, which is not present in our structure, is energetically less favorable by about 4 kcal/mol. Possible reasons for this energy difference and its implications for the stability of quadruplex structures are discussed.     

Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes

We report on proton and phosphorus high resolution NMR investigations of the self-complementary dodecanucleotide d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6 meG.N 12-mers), N = C, T, A and G, which contain N3.O6meG10 interactions in the interior of the helix. These sequences containing a single modified O6meG per strand were prepared by phosphoamidite synthesis and provide an excellent model for probing the structural basis for covalent carcinogenic lesions in DNA. Distance dependent nuclear Overhauser effect (NOE) measurements and line widths of imino protons demonstrate that the N3 and O6meG.10 bases stack into the duplex and are flanked by stable Watson-Crick base pairs at low temperature for all four O6meG.N 12-mer duplexes. The imino proton of T3 in the O6meG.T 12-mer and G3 in the O6meG.N 12-mer helix, which are associated with the modification site, resonate at unusually high field (8.5 to 9.0 ppm) compared to imino protons in Watson-Crick base pairs (12.5 to 14.5 ppm). The nonexchangeable base and sugar protons have been assigned from two dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements on the O6meG.N 12-mer helices. The directionality of the distance dependent NOEs establish all O6meG.N duplexes to be right-handed helices in solution. The glycosidic torsion angles are in the anti range at the N3.O6meG10 modification site except for O6meG10 in the O6meG.G 12-mer duplex which adopts a syn configuration. This results in altered NOEs between the G3 (anti).O6meG10 (syn) pair and flanking G2.C11 and G4.C9 base pairs in the O6meG.G 12-mer duplex. We observe pattern reversal for cross peaks in the COSY spectrum linking the sugar H1' protons with the H2',2" protons at the G2 and O6meG10 residues in the O6meG.N 12-mer duplexes with the effect least pronounced for the O6meG.T 12-mer helix. The proton chemical shift and NOE data have been analyzed to identify regions of conformational perturbations associated with N3.O6meG10 modification sites in the O6meG.N 12-mer duplexes. The proton decoupled phosphorus spectrum of O6meG.T 12-mer duplex exhibits an unperturbed phosphodiester backbone in contrast to the phosphorus spectra of the O6meG.C 12-mer, O6meG.G 12-mer and O6meG.A 12-mer duplexes which exhibit phosphorus resonances dispersed over 2 ppm characteristic of altered phosphodiester backbones at the modification site. Tentative proposals are put forward for N3.O6meG10 pairing models based on the available NMR data and serve as a guide for the design of future experiments.     

Wobble dG X dT pairing in right-handed DNA: solution conformation of the d(C-G-T-G-A-A-T-T-C-G-C-G) duplex deduced from distance geometry analysis of nuclear Overhauser effect spectra

We report below on features of the three-dimensional structure of the d(C-G-T-G-A-A-T-T-C-G-C-G) self-complementary duplex (designated 12-mer GT) containing symmetrical G X T mismatches in the interior of the helix. The majority of the base and sugar protons in the 12-mer GT duplex were assigned by two-dimensional nuclear Overhauser effect (NOESY) spectra in H2O and D2O solution. A set of 92 short (less than 4.5-A) proton-proton distances defined by lower and upper bounds for one symmetrical half of the 12-mer GT duplex were estimated from NOESY data sets recorded as a function of mixing time. These experimental distances combined with nucleotide bond length parameters were embedded into Cartesian space; several trial structures were refined to minimize bond geometry and van der Waals and chirality error. Confidence in this approach is based on the similarity of the refined structures for the solution conformation of the 12-mer GT duplex. The G and T bases pair through two imino-carbonyl hydrogen bonds, and stacking is maintained between the G X T wobble pair and adjacent Watson-Crick G X C pairs. The experimental distance information is restricted to base and sugar protons, and hence structural features such as base pair overlap, glycosidic torsion angles, and sugar pucker are well-defined by this combination of NMR and distance geometry methods. By contrast, we are unable to define the torsion angles about the bonds C3'-O3'-P-O5'-C5'-C4' in the backbone of the nucleic acid.     

A conformational study of alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) by NMR nuclear Overhauser effect spectroscopy (NOESY) and molecular dynamics calculations.

The conformational preference of the disaccharide alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) (1) about the glycosidic torsion angles, phi and psi, was studied by NMR NOESY spectroscopy and molecular mechanics calculations. The NOE data were consistent with either of two distinct conformations close to minima on a calculated phi/psi potential energy surface. Starting from the lowest energy conformation, a 1-ns molecular dynamics (MD) trajectory was computed in vacuo, from which the NOE curves were simulated and compared to the experimentally observed NOESY data.     

NMR structures of r(GCAGGCGUGC)2 and determinants of stability for single guanosine-guanosine base pairs

Nucleotides in RNA that are not Watson-Crick-paired form unique structures for recognition or catalysis, but determinants of these structures and their stabilities are poorly understood. A single noncanonical pair of two guanosines (G) is more stable than other noncanonical pairs and can potentially form pairing structures with two hydrogen bonds in four different ways. Here, the energetics and structure of single GG pairs are investigated in several sequence contexts by optical melting and NMR. The data for r(5'GCAGGCGUGC3')(2), in which G4 and G7 are paired, are consistent with a model in which G4 and G7 alternate syn glycosidic conformations in a two-hydrogen-bond pair. The two distinct structures are derived from nuclear Overhauser effect spectroscopic distance restraints coupled with simulated annealing using the AMBER 95 force field. In each structure, the imino and amino protons of the anti G are hydrogen bonded to the O6 and N7 acceptors of the syn G, respectively. An additional hydrogen-bond connects the syn G amino group to the 5' nonbridging pro-R(p) phosphate oxygen. The GG pair fits well into a Watson-Crick helix. In r(5'GCAGGCGUGC3')(2), the G4(anti), G7(syn) structure is preferred over G4(syn), G7(anti). For single GG pairs in other contexts, exchange processes make interpretation of spectra more difficult but the pairs are also G(syn), G(anti). Thermodynamic data for a variety of duplexes containing pairs of G, inosine, and 7-deazaguanosine flanked by GC pairs are consistent with the structural and energetic interpretations for r(5'GCAGGCGUGC3')(2), suggesting similar GG conformations.