Structural characterization of separated H DNA conformers

Polypyrimidine/polypurine DNA sequences in plasmids can adopt protonated triplex-containing structures (H DNA) in response to negative superhelical stress and low pH. A d(TC)17-d(GA)17 insert adopts two isomeric protonated structures, which differ in degree of helical unwinding. The variant forms of individual topoisomers were separated by agarose gel electrophoresis and their reactivities to permanganate and acid-induced depurination were compared. Depurination patterns of the individual conformers indicate that in the more mobile form (H-y5) the 5'-half of the d(GA)n strand participates in a triplex while in the other (H-y3) the 3'-half forms the triplex. The H-y5 form is more stable than the H-y3 form at low negative superhelix densities. Because of the difference in helical unwinding, the H-y5 form becomes relatively less stable as the superhelix density increases. Topological models of the two forms show that providing there is no linkage at the tips of the triple helical segments one more positive twist is localized in the H-y5 form than in the H-y3 form. The foldback in the pyrimidine strand of the H-y5 form is less accessible to solvent than that of the H-y3 form as assessed by its lower reactivity to permanganate. Consideration of a pyrimidine loop model (Harvey, S. C., Luo, J., & Lavery, R. (1988) Nucleic Acids Res. 16, 11795-11809) suggests that the unique stability of the H-y5 form results from Watson-Crick base pairs between residues of the d(TC)n loop and the d(GA)n strand as it exits the triplex.     

NMR studies of triple-strand formation from the homopurine-homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4

The complexes formed by the homopurine and homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4 have been investigated by one- and two-dimensional 1H NMR. Under appropriate conditions [low pH, excess d(TC)4 strand] the oligonucleotides form a triplex containing one d(GA)4 and two d(TC)4 strands. The homopurine and one of the homopyrimidine strands are Watson-Crick base paired, and the second homopyrimidine strand is Hoogsteen base paired in the major groove to the d(GA)4 strand. Hoogsteen base pairing in GC base pairs requires hemiprotonation of C; we report direct observation of the C+ imino proton in these base pairs. Both homopyrimidine strands have C3'-endo sugar conformations, but the purine strand does not. The major triplex formed appears to have four TAT and three CGC+ triplets formed by binding of the second d(TC)4 strand parallel to the d(GA)4 strand with a 3' dangling end. In addition to the triplexes formed, at least one other heterocomplex is observed under some conditions.     

The DNA sequence at echinomycin binding sites determines the structural changes induced by drug binding: NMR studies of echinomycin binding to [d(ACGTACGT)]2 and [d(TCGATCGA)]2

The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers [d(ACGTACGT)]2 and [d(TCGATCGA)]2 have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic and chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer [d(ACGTACGT)]2 is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to [d(TCGATCGA)]2 is not cooperative. At low temperatures, both the internal and terminal A.T base pairs adjacent to the binding site in the [d(ACGTACGT)]2-2 echinomycin complex are Hoogsteen base paired (Gilbert et al., 1989) as observed in related crystal structures. However, as the temperature is raised, the internal A.T Hoogsteen base pairs are destabilized and are observed to be exchanging between the Hoogsteen base-paired and an open (or Watson-Crick base-paired) state. In contrast, in the [d(TCGATCGA)]2-2 echinomycin complex, no A.T Hoogsteen base pairs are observed, the internal A.T base pairs appear to be stabilized by drug binding, and the structure of the complex does not change significantly from 0 to 45 degrees C. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While we conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.     

Nuclear magnetic resonance study of hydrogen-bonded ring protons in oligonucleotide helices involving classical and nonclassical base pairs.

A study of the exchangeable ring nitrogen protons in aqueous solutions of oligonucleotide complexes involving Watson-Crick base pairs as well as Hoogsteen pairs and other nonclassical hydrogen bonding schemes shows that resolvable resonances in the low-field (-10 to -16 ppm from sodium 4,4-dimethyl-4-silapentanesulfonate) region can be detected in a variety of structures other than double stranded helices. Ring nitrogen proton resonances arising from the following hydrogen-bonding situations are reported: (1) AT and GC Watson-Crick base pairs in a self-complementary octanucleotide, dApApApGpCpTpTpT; (2) U-A-U base triples in complexes between oligo-U15 and AMP; (3) C-G-C+ base triples in complexes between oligo-C17 and GMP at acid pH; (4) s4U-A-s4U base triples in complexes between oligo-s4U15 and AMP, all of which involve both Watson-Crick and Hoogsteen base pairing to form triplexes; (5) C-C+ base pairing between protonated and unprotonated C residues in oligo-C17 at acid pH; and (6) I4 base quadruples in the four strand association among oligo-I at high salt. The behavior of the dA3G-CT3 helix is consistent with both fraying of the terminal base pairs and presence of intermediate states as the helix opens. In the monomer-oligomer complexes, under the conditions used here, the exchange appears to be governed by the dissociation rate of monomer from the complex. These findings suggest that those tertiary structure hydrogen bonds in tRNA involving ring nitrogen protons should have representative resonances in the low-field (11-16 ppm) proton NMR region in H2O.     

NMR structural studies of intramolecular (Y+)n.(R+)n(Y-)nDNA triplexes in solution: imino and amino proton and nitrogen markers of G.TA base triple formation.

We reported previously on NMR studies of (Y+)n.(R+)n(Y-)n DNA triple helices containing one oligopurine strand (R)n and two oligopyrimidine strands (Y)n stabilized by T.AT and C+.GC base triples [de los Santos, C., Rosen, M., & Patel, D. J. (1989) Biochemistry 28, 7282-7289]. Recently, it has been established that guanosine can recognize a thymidine.adenosine base pair to form a G.TA triple in an otherwise (Y+)n.(R+)n(Y-)n triple-helix motif. [Griffin, L. C., & Dervan, P. B. (1989) Science 245, 967-971]. The present study extends the NMR research to the characterization of structural features of a 31-mer deoxyoligonucleotide that folds intramolecularly into a 7-mer (Y+)n.(R+)n(Y-)n triplex with the strands linked through two T5 loops and that contains a central G.TA triple flanked by T.AT triples. The G.TA triplex exhibits an unusually well resolved and narrow imino and amino exchangeable proton and nonexchangeable proton spectrum in H2O solution, pH 4.85, at 5 degrees C. We have assigned the imino protons of thymidine and amino protons of adenosine involved in Watson-Crick and Hoogsteen pairing in T.AT triples, as well as the guanosine imino and cytidine amino protons involved in Watson-Crick pairing and the protonated cytidine imino and amino protons involved in Hoogsteen pairing in C+.GC triples in the NOESY spectrum of the G.TA triplex. The NMR data are consistent with the proposed pairing alignment for the G.TA triple where the guanosine in an anti orientation pairs through a single hydrogen bond from one of its 2-amino protons to the 4-carbonyl group of thymidine in the Watson-Crick TA pair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct identification of NH...N hydrogen bonds in non-canonical base pairs of RNA by NMR spectroscopy.

It is shown that the recently developed quantitative J(NN)HNN-COSY experiment can be used for the direct identification of hydrogen bonds in non-canonical base pairs in RNA. Scalar(2h)J(NN)couplings across NH.N hydrogen bonds are observed in imino hydrogen bonded GA base pairs of the hpGA RNA molecule, which contains a tandem GA mismatch, and in the reverse Hoogsteen AU base pairs of the E-loop of Escherichia coli 5S rRNA. These scalar couplings correlate the imino donor(15)N nucleus of guanine or uridine with the acceptor N1 or N7 nucleus of adenine. The values of the corresponding(2h)J(NN)coupling constants are similar in size to those observed in Watson-Crick base pairs. The reverse Hoogsteen base pairs could be directly detected for the E-loop of E.coli 5S rRNA both in the free form and in a complex with the ribosomal protein L25. This supports the notion that the E-loop is a pre-folded RNA recognition site that is not subject to significant induced conformational changes. Since Watson-Crick GC and AU base pairs are also readily detected the HNN-COSY experiment provides a useful and sensitive tool for the rapid identification of RNA secondary structure elements.     

NMR investigation of Hoogsteen base pairing in quinoxaline antibiotic--DNA complexes: comparison of 2:1 echinomycin, triostin A and [N-MeCys3,N-MeCys7] TANDEM complexes with DNA oligonucleotides.

Hoogsteen base pairs have been demonstrated to occur in base pairs adjacent to the CpG binding sites in complexes of triostin A and echinomycin with a variety of DNA oligonucleotides. To understand the relationship of these unusual base pairs to the sequence specificity of these quinoxaline antibiotics, the conformation of the base pairs flanking the YpR binding sites of the 2:1 drug-DNA complexes of triostin A with [d(ACGTACGT)]2 and of the TpA specific [N-MeCys3, N-MeCys7] TANDEM with [d(ATACGTAT)]2 have been studied by 1H NMR spectroscopy. In both the 2:1 triostin A-DNA complex and the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the terminal A.T base pairs are Hoogsteen base paired with the 5' adenine in the syn conformation. This indicates that both TpA specific and CpG specific quinoxaline antibiotics are capable of inducing Hoogsteen base pairs in DNA. However, in both 2:1 complexes, Hoogsteen base pairing is limited to the terminal base pairs. In the 2:1 triostin A complex, the internal adenines are anti and in the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the internal guanines are anti regardless of pH, which indicates that the central base pairs of both complexes form Watson-Crick base pairs. This indicates that the sequence dependent nature of Hoogsteen base pairing is the same in TpA specific and CpG specific quinoxaline antibiotic-DNA complexes. We have calculated a low resolution three-dimensional structure of the 2triostin A-[d(ACGTACGT)]2 complex and compared it with other CpG specific quinoxaline antibiotic-DNA complexes. The role of stacking in the formation of Hoogsteen base pairs in these complexes is discussed.     

Interactions of Quinoxaline Antibiotic and DNA.: The Molecular Structure of a Triostin A—d(GCGTACGC) Complex

Abstract

The crystal structure of a DNA. octamer d(GCGTA.CGC) complexed to an antitumor antibiotic, triostin A, has been solved and refined to 2.2 Å resolution by x-ray diffraction analysis. The antibiotic molecule acts as a true bis intercalator surrouding the d(CpG) sequence at either end of the unwound right-handed DNA. double helix. A.s previously observed in the structure of triostin A.—d(CGTA.CG) complex (A.H.-J. Wang, et. al., Science, 225,1115–1121 (1984)), the alanine amino acid residues of the drug molecule form sequence-specific hydrogen bonds to guanines in the minor groove. The two central A · T base pairs are in Hoogsteen configuration with adenine in the syn conformation. In addition, the two terminal G · C base pairs flanking the quinoxaline rings are also held together by Hoogsteen base pairing. This is the first observation in an oligonucleotide of. Hoogsteen G · C base pairs where the cytosine is protonated. The principal functional components of a bis-intercalative compound are discussed.     

Interactions of quinoxaline antibiotic and DNA: the molecular structure of a triostin A-d(GCGTACGC) complex

The crystal structure of a DNA octamer d(GCGTACGC) complexed to an antitumor antibiotic, triostin A, has been solved and refined to 2.2 A resolution by x-ray diffraction analysis. The antibiotic molecule acts as a true bis intercalator surrounding the d(CpG) sequence at either end of the unwound right-handed DNA double helix. As previously observed in the structure of triostin A-d(CGTACG) complex (A.H.-J. Wang, et. al., Science, 225, 1115-1121 (1984)), the alanine amino acid residues of the drug molecule form sequence-specific hydrogen bonds to guanines in the minor groove. The two central A.T base pairs are in Hoogsteen configuration with adenine in the syn conformation. In addition, the two terminal G.C base pairs flanking the quinoxaline rings are also held together by Hoogsteen base pairing. This is the first observation in an oligonucleotide of. Hoogsteen G.C base pairs where the cytosine is protonated. The principal functional components of a bis-intercalative compound are discussed.     

Proton exchange and base pair opening in a DNA triple helix

Nuclear magnetic resonance spectroscopy has been used to characterize opening reactions and stabilities of individual base pairs in two related DNA structures. The first is the triplex structure formed by the DNA 31-mer 5'-AGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3'. The structure belongs to the YRY (or parallel) family of triple helices. The second structure is the hairpin double helix formed by the DNA 20-mer 5'-AGAGAGAACCCCTTCTCTCT-3' and corresponds to the duplex part of the YRY triplex. The rates of exchange of imino protons with solvent in the two structures have been measured by magnetization transfer from water and by real-time exchange at 10 degrees C in 100 mM NaCl and 5 mM MgCl2 at pH 5.5 and in the presence of two exchange catalysts. The results indicate that the exchange of imino protons in protonated cytosines is most likely limited by the opening of Hoogsteen C+G base pairs. The base pair opening parameters estimated from imino proton exchange rates suggest that the stability of individual Hoogsteen base pairs in the DNA triplex is comparable to that of Watson-Crick base pairs in double-helical DNA. In the triplex structure, the exchange rates of imino protons in Watson-Crick base pairs are up to 5000-fold lower than those in double-helical DNA. This result suggests that formation of the triplex structure enhances the stability of Watson-Crick base pairs by up to 5 kcal/mol. This stabilization depends on the specific location of each triad in the triplex structure.     

Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes

Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.     

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.     

The thermal stability of DNA fragments with tandem mismatches at a d(CXYG).d(CY'X'G) site.

Temperature-Gradient Gel Electrophoresis (TGGE) was employed to determine the thermal stabilities of 28 DNA fragments, 373 bp long, with two adjacent mismatched base pairs, and eight DNAs with Watson-Crick base pairs at the same positions. Heteroduplex DNAs containing two adjacent mismatches were formed by melting and reannealing pairs of homologous 373 bp DNA fragments differing by two adjacent base pairs. Product DNAs were separated based on their thermal stability by parallel and perpendicular TGGE. The polyacrylamide gel contained 3.36 M urea and 19.2 % formamide to lower the DNA melting temperatures. The order of stability was determined in the sequence context d(CXYG).d(CY'X'G) where X.X' and Y.Y" represent the mismatched or Watson-Crick base pairs. The identity of the mismatched bases and their stacking interactions influence DNA stability. Mobility transition melting temperatures (T u) of the DNAs with adjacent mismatches were 1.0-3.6 degrees C (+/-0.2 degree C) lower than the homoduplex DNA with the d(CCAG).d(CTGG) sequence. Two adjacent G.A pairs, d(CGAG).d(CGAG), created a more stable DNA than DNAs with Watson-Crick A.T pairs at the same sites. The d(GA).d(GA) sequence is estimated to be 0.4 (+/-30%) kcal/mol more stable in free energy than d(AA).d(TT) base pairs. This result confirms the unusual stability of the d(GA).d(GA) sequence previously observed in DNA oligomers. All other DNAs with adjacent mismatched base pairs were less stable than Watson-Crick homoduplex DNAs. Their relative stabilities followed an order expected from previous results on single mismatches. Two homoduplex DNAs with identical nearest neighbor sequences but different next-nearest neighbor sequences had a small but reproducible difference in T u value. This result indicates that sequence dependent next neighbor stacking interactions influence DNA stability.     

Crystal structure of d(gcGXGAgc) with X=G: a mutation at X is possible to occur in a base-intercalated duplex for multiplex formation

DNA fragments with the sequences d(gcGX[Y]n Agc) (n=1, X=A, and Y=A, T, or G)form base-intercalated duplexes, which is a basic unit for formation of multiplexes such as octaplex and hexaplex. To examine the stability of multiplexes, a DNA with X=Y=G and n=1 was crystallized under conditions different from those of the previously determined sequences, and its crystal structure has been determined. The two strands are coupled in an anti-parallel fashion to form a base-intercalated duplex, in which the first and second residues form Watson-Crick type G:C pairs and the third and sixth residues form a sheared G:A pairs at both ends of the duplex. The G4 and G5 bases are stacked alternatively on those of the counter strand to form a long G column of G3-G4-G5*-G5-G4*-G3*, the central four Gs being protruded. In addition, the three duplexes are associated to form a hexaplex around a mixture of calcium and sodium cations on the crystallographic threefold axis. These structural features are similar to those of the previous crystals, though slightly different in detail. The present study indicates that mutation at the 4th position is possible to occur in a base-intercalated duplex for multiplex formations, suggesting that DNA fragments with any sequence sandwiched between the two triplets gcG and Agc can form a multiplex.     

The first example of a Hoogsteen base-paired DNA duplex in dynamic equilibrium with a Watson-Crick base-paired duplex--a structural (NMR), kinetic and thermodynamic study.

A single-point substitution of the O4' oxygen by a CH2 group at the sugar residue of A6 (i.e. 2'-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5'-d(C1G2C3G4A5A6T7T8C9G10C11G12)2(-3), has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A6:T7 Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)<==>(1B): Keq = k1/k(-1) = 0.56+/-0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k1 (298K) = 3.9 0.8 sec(-1); deltaHdegrees++ = 164+/-14 kJ/mol; -TdeltaS degrees++ (298K) = -92 kJ/mol giving a deltaG degrees++ 298 of 72 kJ/mol. Ea (k1) = 167 14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0 0.6 sec-1, deltaH degrees++ = 153 13 kJ/mol; -TdeltaSdegrees++ (298K) = -82 kJ/mol giving a deltaGdegrees++(298) of 71 kJ/mol. Ea (k-1) = 155 13 kJ/mol]. Acomparison of deltaGdegrees++(298) of the forward (k1) and backward (k-1) conversions, (1A)<==>(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A6:T7 Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A6:T7 basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2'-deoxyaristeromycin moiety, A6, as a result of substitution of the endocyclic 4'-oxygen in the natural sugar with a methylene group in A6. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in alpha, sigma and gamma torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.     

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.     

Structure of the DNA Duplex d(ATTAAT)2 with Hoogsteen Hydrogen Bonds

The traditional Watson-Crick base pairs in DNA may occasionally adopt a Hoogsteen conformation, with a different organization of hydrogen bonds. Previous crystal structures have shown that the Hoogsteen conformation is favored in alternating AT sequences of DNA. Here we present new data for a different sequence, d(ATTAAT)₂, which is also found in the Hoogsteen conformation. Thus we demonstrate that other all-AT sequences of DNA with a different sequence may be found in the Hoogsteen conformation. We conclude that any all-AT sequence might acquire this conformation under appropriate conditions. We also compare the detailed features of DNA in either the Hoogsteen or Watson-Crick conformations.     

Hydration regulates the thermodynamic stability of DNA structures under molecular crowding conditions

Although water is an integral part of DNA structures, the effects of water molecules on various DNA structures which are formed by not only Watson-Crick but also Hoogsteen base pairs are still unclear. Here, we studied quantitatively the effects of molecular crowding on the thermodynamics of a parallel G-quadruplex formation of [d(TG 4)2]4 with Hoogsteen base pairs. It was demonstrated that molecular crowding conditions stabilized the parallel G-quadruplex. Moreover, the plot of stability of the parallel G-quadruplex structure versus water activity suggested that water molecules were released through the G-quadruplex formation. The stabilization of the DNA structures consisting of Hoogsteen base pairs under cell-like conditions may lead to a structural polymorphism of various DNA sequences regulated by water molecules.     

Homopurine and homopyrimidine strands complementary in parallel orientation form an antiparallel duplex at neutral pH with A-C,G-T,and T-C mismatched base pairs

DNA sequences d-TGAGGAAAGAAGGT (a 14-mer) and d-CTCCTTTCTTCC (a 12-mer) are complementary in parallel orientation forming either Donahue (reverse Watson-Crick) base pairing at neutral pH or Hoogsteen base pairing at slightly acidic pH. The structure of the complex formed by dissolving the two strands in equimolar ratio in water has been investigated by nmr. At neutral pH, the system forms an ordered antiparallel duplex with five A : T and four G : C Watson-Crick base pairs and three mismatches, namely G-T, A-C, and T-C. The nuclear Overhauser effect cross-peak pattern suggests an overall B-DNA conformation with major structural perturbations near the mismatches. The duplex has a low melting point and dissociates directly into single strands with a broad melting profile. The hydrogen-bonding schemes in the mismatched base pairs have been investigated. It has been shown earlier that in acidic pH, the system prefers a triple-stranded structure with two pyrimidine strands and one purine strand. One of the pyrimidine strands has protonated cytosines, forms Hoogsteen base pairing, and is aligned parallel to the purine strand; the other has nonprotonated cytosines and has base-pairing scheme similar to the one discussed in this paper. The parallel duplex is therefore less stable than either the antiparallel duplex or the triplex, in spite of its perfect complementarity. © 1997 John Wiley & Sons, Inc. Biopoly 41: 773–784, 1997     

Polypurine/polypyrimidine hairpins form a triple helix structure at low pH.

1D and 2D NMR investigations of the 15 residue deoxynucleotide sequence d(TCTCTC-TTT-GAGAGA) show that above pH = 6.5 the molecule adopts a B-form hairpin conformation. As the pH is lowered below 6.5 molecules progressively associate in pairs to form a partially triple helical, partially single stranded structure in which the bases of the oligopyrimidine d(TC)3 tract from one molecule form Hoogsteen pairs with the d(G-A)3 tract of the other. Imino protons of protonated cytosines can be observed at very low field (approximately 15 ppm). The enthalpy of triplex formation was estimated by NMR techniques to be -16 kcal mol-1. Intense H6 to H3' cross peaks from residues in all three strands suggest the presence of N-type sugars at some but not at all possible sites. Surprisingly strong cross peaks between H5' or H5" and non-exchangeable base protons are also observed. These suggest that certain of the O5'-C5'-C4'-C3' phosphate backbone torsion angles (gamma) are unusual.