Thermal stability and energetics of 15-mer DNA duplex interstrand crosslinked by trans-diamminedichloroplatinum(II)

The effect of the location of the interstrand cross-link formed by trans-diamminedichloroplatinum(II) (transplatin) on the thermal stability and energetics of 15-mer DNA duplex has been investigated. The duplex containing single, site-specific cross-link, thermodynamically equivalent model structures (hairpins) and nonmodified duplexes were characterized by differential scanning calorimetry, temperature-dependent uv absorption, and circular dichroism. The results demonstrate that the formation of the interstrand cross-link of transplatin does not affect pronouncedly thermodynamic stability of DNA: the cross-link induces no marked changes not only in enthalpy, but also in "reduced" (concentration independent) monomolecular transition entropy. These results are consistent with the previous observations that interstrand cross-links of transplatin structurally perturb DNA only to a relatively small extent. On the other hand, constraining the duplex with the interstrand cross-link of transplatin results in a significant increase in thermal stability that is primarily due to entropic effects: the cross-link reduces the molecularity of the oligomer system from bimolecular to monomolecular. Importantly, the position of the interstrand cross-link within the duplex modulates cooperativity of the melting transition of the duplex and consequently its thermal stability.     

Influence of an exocyclic guanine adduct on the thermal stability, conformation, and melting thermodynamics of a DNA duplex.

As part of an overall program to characterize the impact of mutagenic lesions on the physiochemical properties of DNA, we report here the results of a comparative spectroscopic study on pairs of DNA duplexes both with and without an exocyclic guanine lesion. Specifically, we have studied a family of four 13-mer duplexes of the form d(CGCATGYGTACGC).d(GCGTACZCATGCG) in which Y is either the normal deoxyguanosine residue (G) or the exocyclic guanine adduct 1,N2-propanodeoxyguanosine (X), while Z is either deoxycytosine (C) or deoxyadenosine (A). Thus, the four duplexes studied, which can be designated by the identity of their central Y.Z base pair, are a Watson-Crick duplex (GC), a duplex with a central mismatch (GA), and two duplexes with exocyclic guanine lesions (X), that differ only by the base opposite the lesion (XC and XA). The data derived from our spectroscopic measurements on these four duplexes have allowed us to evaluate the influence of the exocyclic guanine lesion, as well as the base opposite the lesion, on the conformation, thermal stability, and melting energetics of the host DNA duplex. To be specific, our circular dichroism (CD) spectra show that the exocyclic guanine lesion induces alterations in the duplex structure, while our temperature-dependent optical measurements reveal that these lesion-induced structural alterations reduce the thermal stability, the transition enthalpy, and the transition free energy of the duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and thermodynamic features of spiroiminodihydantoin damaged DNA duplexes

Oxidation of guanine or 8-oxo-7,8-dihydroguanine can produce spiroiminodihydantoin (Sp) R and S stereoisomers. Both in vitro and in vivo experiments have shown that the Sp stereoisomers are highly mutagenic, causing G --> C and G --> T transversion mutations. Therefore, they are of interest as potential endogenous cancer causing lesions. However, their structural properties in DNA duplexes remain to be elucidated. We have employed computational methods to study the Sp lesions in 11-mer DNA duplexes with A, C, G, and T partners. Molecular dynamics simulations have been carried out to obtain ensembles of structures, and the trajectories were employed to analyze the structures and compute free energies. The structural and thermodynamic analyses reveal that the Sp stereoisomers energetically favor positioning in the B-DNA major groove, with minor groove conformers also low energy in some cases, depending on the partner base. The R and S stereoisomers adopt opposite orientations with respect to the 5' to 3' direction of the modified strand. Both syn and anti glycosidic bond conformations are energetically feasible, with partner base and stereochemistry determining the preference. The lesions adversely impact base stacking and Watson-Crick hydrogen bonding interactions in the duplex, and cause groove widening. The chemical nature of the partner base determines specific hydrogen bonding and stacking properties of the damaged duplexes. The structural characteristics may relate to observed mutagenic properties of the Sp stereoisomers, including possible stereoisomer-dependent differences.     

Crystallization of a DNA duplex 15-mer containing unpaired bases: d(CGCGAAATTTACGCG)

Crystals of an almost self-complementary DNA 15-mer d(CGCGAAATTTACGCG) have been grown by the vapor diffusion technique at 4 degrees C. The space group is I222 with a = 37.3 A, b = 54.6 A and c = 104.8 A. Solution studies showed that the 15-mer forms a duplex with the extra adenine residue unpaired: (sequence; see text) Crystals are stable at 4 degrees C and are suitable for medium-resolution structural studies.     

NMR with 13C, 15N-doubly-labeled DNA: The shape Antennapedia homeodomain complex with a 14-mer DNA duplex

Nearly complete ¹H, ¹³C and¹⁵ N NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the free state and complexed with the uniformly ¹⁵N-labeled Antennapedia homeodomain. The DNA was either fully ¹³C,¹⁵N-labeled or contained uniformly ¹³C, ¹⁵N-labeled nucleotides only at those positions which form the protein–DNA interface in the previously determined NMR solution structure of the Antennapedia homeodomain–DNA complex. The resonance assignments were obtained in three steps: (i) identification of the deoxyribose spin systems via scalar couplings using 2D and 3D HCCH-COSY and soft-relayed HCCH-COSY; (ii) sequential assignment of the nucleotides via¹ H–¹H NOEs observed in 3D¹³ C-resolved NOESY; and (iii) assignment of the imino and amino groups via ¹H–¹H NOEs and¹⁵ N–¹H correlation spectroscopy. The assignment of the duplex in the 17 kDa protein–DNA complex was greatly facilitated by the fact that ¹H signals of the protein were filtered out in ¹³C-resolved spectroscopy and by the excellent carbon chemical shift dispersion of the DNA duplex. Comparison of corresponding ¹³C chemical shifts of the free and the protein-bound DNA indicates conformational changes in the DNA upon complex formation.     

Effects of monofunctional platinum binding on the thermal stability and conformation of a self-complementary 22-mer

We investigated the effect of various monofunctional platinum complexes on the thermal stability and conformation of a self-complementary 22-mer duplex oligonucleotide by means of CD and UV melting profiles. We studied several families of triamine complexes of the general formula PtA2AmCl where A2=(NH3)2 and ethylenediamine and where Am=N1-4-methyl-pyridine, N7-guanosine, and 9-ethyl-guanine. Platination by the N1-4-methyl-pyridine and 9-ethyl-guanine complexes led to a decrease in the Tm of the oligonucleotide by 2-11.5 degrees C while platination with the N7-guanosine complexes led to a rise in the melting temperature of the oligonucleotides by 4.5 degrees C. A similar inverse correlation between the two groups of platinum compounds was found in the CD spectra. In all cases, the cis isomer had a more pronounced effect on both the melting curve and the CD spectrum. The cis isomer was found to have a more destabilizing effect than its trans counterpart. This indicates that the cis geometry in fact forces a greater structural constraint on the backbone of the double helix. We have also found that the sugar of the guanosine has a significant influence on both the Tm and CD spectra; the sugar moiety contributes to the stability of the double helix, probably through the formation of hydrogen bonds.     

The effect of a single boranophosphate substitution with defined configuration on the thermal stability and conformation of a DNA duplex

Substitution of one non-bridging oxygen in a natural phosphodiester internucleotide linkage with a borano (-BH3) group results in a chiral phosphorus center in boranophosphate. UV thermal melting profiles were recorded for DNA duplexes formed between a DNA 9-mer with either an Sp or a Rp boranophosphate linkage in the middle and the complementary DNA 9-mer, as well as for their unmodified parent duplex. The thermal stability of the DNA duplexes was in the order of normal > Sp borano > Rp borano. CD spectra of all three duplexes exhibited typical B-DNA profile, which closely resembled each other.     

Impact of the third-strand orientation on the thermodynamic stability of the four-way DNA junction

The physical properties of a triple-helical DNA four-way junction J(T2T4) have been characterized by means of UV spectroscopy, CD spectroscopy, and differential scanning calorimetry (DSC). J(T2T4) is another four-way junction that was designed in addition to J(T1T3) (N. Makube and H. H. Klump (2000) Arch. Biochem. Biophys. 377, 31-42) to study the effects of third strands on the stability of the four-way junction with triple-helical arms. The pH titration curves illustrate the sequential folding of single strands to double-helical four-way junctions and finally the binding of third strands to their respective W-C duplexes. CD measurements confirm triplex formation under appropriate pH and ionic strength conditions. The CD spectra also suggest different melting patterns for the triple-helical arms of J(T2T4). The melting temperature as a function of pH or ionic strength characterizes the effect of the third strands on the structural stability. Increased sodium concentration and low pH conditions enhances and stabilizes the overall structure of the junction. The results also indicate that all triplexes in J(T2T4) are formed in the absence of salt and at low pH; however, the junction may, under these conditions, assume a conformation different from the one assumed in the presence of salt. Through the deconvolution of DSC data, the calorimetric enthalpies associated with melting of arms of the junctions were determined. The loops are designed to have the same enthalpic effect on the different arms. The stabilizing effect of the loops is more pronounced when those loops are shifted from arms 1 and 3 in J(T1T3) to arms 2 and 4 in J(T2T4) without changing any of the sequences. Overall, J(T2T4) is slightly more stable than J(T1T3). The differences can be attributed to sequence effects rather than structural effects. All the results illustrate that binding of the third strand in either of the two orientations 5'5'3' (J(T2T4)) or 5'3'3' (J(T1T3)) stabilizes the underlying double-helical four-way junction and its triple-helical arms.     

Effect of guanine nucleotides on the conformation and stability of chloroplast elongation factor Tu

The effect of guanine nucleotides and kirromycin on the conformation and stability of the chloroplast elongation factor Tu (EF-Tuchl) from Euglena gracilis has been investigated. Free EF-Tuchl is quite thermolabile but the protein is greatly stabilized by guanine nucleotides. The temperature dependence of the thermal inactivation of EF-Tuchl was used to calculate the amount of stabilization energy conferred by the guanine nucleotides. GDP increases the activation energy for the denaturation process by 77 kcal/mol while GTP increases the activation energy by 51 kcal/mol. The difference in heat stability of free EF-Tuchl and the EF-Tuchl.GDP complex was used to determine a dissociation constant of 1.3 x 10(-7) M at 37 degrees C. The temperature dependence of the dissociation constant allowed the calculation of a delta H degree obsd of -55 kcal/mol and a delta S degree obsd of -146 cal/(mol degree) for GDP binding to EF-Tuchl.EF-Tuchl was found to have a trypsin-sensitive region similar to that observed for Escherichia coli EF-Tu. This loop region was protected by GTP and kirromycin but not by GDP.     

Transition characteristics and thermodynamic analysis of DNA duplex formation: a quantitative consideration for the extent of duplex association

Transition characteristics and thermodynamic properties of the single-stranded self-transition and the double-stranded association were investigated and analyzed for 9-, 15- and 21-bp non-self-complementary DNA sequences. The multiple transition processes for the single-stranded self-transition and the double-stranded association were further put forth. The experimental results confirmed that the double-stranded association transition was generally imperfect and the thermodynamic properties of the single-stranded self-transition would exert an influence on a duplex formation. Combining ultraviolet melting experiments in various molar ratios, the extent of duplex association was estimated for three double-stranded DNAs. In our experimental range, the extent of duplex association decreases with increasing the number of base pairs in DNA sequences, which suggest that the short oligonucleotides may proceed in a two-state transition while the long oligonucleotides may not. When the extent of duplex association was considered, the true transition enthalpies of a duplex formation derived from UV and differential scanning calorimetry measurements were in good agreement.     

Effect of 6-thioguanine on the stability of duplex DNA

The incorporation of 6-thioguanine (S6G) into DNA is a prerequisite for its cytotoxic action, but duplex structure is not significantly perturbed by the presence of the lesion [J. Bohon and C. R. de los Santos (2003) Nucleic Acids Res., 31, 1331–1338]. It is therefore possible that the mechanism of cytotoxicity relies on a loss of stability rather than a pathway involving direct structural recognition. The research described here focuses on the changes in thermodynamic properties of duplex DNA owing to the introduction of S6G as well as the kinetic properties of base pairs involving S6G. Replacement of a guanine in a G•C pair by S6G results in ~1 kcal/mol less favorable Gibbs free energy of duplex formation at 37°C. S6G•T and G•T mismatch-containing duplexes have almost identical Gibbs free energy at 37°C, with values ~3 kcal/mol less favorable than that of the control. Base pair stability is affected by S6G. The lifetime of the normal G•C base pair is ~125 ms, whereas that of the G•T mismatch is below the detection limit. The lifetimes of S6G•C and S6G•T pairs are ~7 and 2 ms, respectively, demonstrating that, although S6G significantly decreases the stability of the pairing with cytosine, it slightly increases that of a mismatch.     

Coexistence of G-quadruplex and duplex domains within the secondary structure of 31-mer DNA thrombin-binding aptamer

A number of thrombin-binding DNA aptamers have been developed during recent years. So far the structure of just a single one, 15-mer thrombin-binding aptamer (15TBA), has been solved as G-quadruplex. Structures of others, showing variable anticoagulation activities, are still not known yet. In this paper, we applied the circular dichroism and UV spectroscopy to characterize the temperature unfolding and conformational features of 31-mer thrombin-binding aptamer (31TBA), whose sequence has a potential to form G-quadruplex and duplex domains. Both structural domains were monitored independently in 31TBA and in several control oligonucleotides unable to form either the duplex region or the G-quadruplex region. The major findings are as follows: (1) both duplex and G-quadruplex domains coexist in intramolecular structure of 31TBA, (2) the formation of duplex domain does not change the fold of G-quadruplex, which is very similar to that of 15TBA, and (3) the whole 31TBA structure disrupts if either of two domains is not formed: the absence of duplex structure in 31TBA abolishes G-quadruplex, and vice versa, the lack of G-quadruplex folding results in disallowing the duplex domain.     

Coexistence of G-quadruplex and duplex domains within the secondary structure of 31-mer DNA thrombin-binding aptamer

A number of thrombin-binding DNA aptamers have been developed during recent years. So far the structure of just a single one, 15-mer thrombin-binding aptamer (15TBA), has been solved as G-quadruplex. Structures of others, showing variable anticoagulation activities, are still not known yet. In this paper, we applied the circular dichroism and UV spectroscopy to characterize the temperature unfolding and conformational features of 31-mer thrombin-binding aptamer (31TBA), whose sequence has a potential to form G-quadruplex and duplex domains. Both structural domains were monitored independently in 31TBA and in several control oligonucleotides unable to form either the duplex region or the G-quadruplex region. The major findings are as follows: (1) both duplex and G-quadruplex domains coexist in intramolecular structure of 31TBA, (2) the formation of duplex domain does not change the fold of G-quadruplex, which is very similar to that of 15TBA, and (3) the whole 31TBA structure disrupts if either of two domains is not formed: the absence of duplex structure in 31TBA abolishes G-quadruplex, and vice versa, the lack of G-quadruplex folding results in disallowing the duplex domain.     

Structure of an 11-mer DNA duplex containing the carbocyclic nucleotide analog: 2'-deoxyaristeromycin

2'-deoxyaristeromycin (dAr) is a nucleoside analogue that is resistant to the action of DNA glycosylases. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of an 11-mer DNA containing a single dAr.T base pair at its center. Analysis of the spectra revealed the existence of a right-handed duplex in solution, stabilized by Watson-Crick hydrogen bonding and base-stacking interactions. The carbocyclic sugar adopted a C1'-exo conformation and sugars of the 3'-flanking base pair had puckers in the O4'-endo range. The dAr.T base pair was mildly propeller twisted, and the dAr analogue showed a positive roll with the 3'-flanking base. Our findings indicate that the observed resistance of dAr-containing oligodeoxynucleotides to the catalytic action of DNA glycosylases relates to its electronic properties rather than structure, and validate the use of dAr and related carbocyclic nucleoside analogues for biological and structure/function relationship studies.     

Peroxynitrite-induced reactions of synthetic oligo 2'-deoxynucleotides and DNA containing guanine: formation and stability of a 5-guanidino-4-nitroimidazole lesion

Peroxynitrite is a strong oxidizing agent that is formed in the reaction of nitric oxide and superoxide anion. It is capable of oxidizing and nitrating a variety of biological targets including DNA, and these modifications may be responsible for a number of pathological conditions and diseases. A recent study showed that peroxynitrite reacts with 2',3',5'-tri-O-acetylguanosine to yield a novel compound, tri-O-acetyl-1-(beta-D-erythro-pentafuranosyl)-5-guanidino-4-nitroimidazole, and, unlike other peroxynitrite-mediated guanine oxidation products, it is a stable and significant component formed even at low peroxynitrite concentrations. In this work, we studied the in vitro formation of the guanine-derived product, 5-guanidino-4-nitroimidazole, in synthetic oligonucleotides and DNA treated with peroxynitrite. When calf thymus DNA or oligonucleotides were reacted with peroxynitrite at ambient temperature, the modified base 5-guanidino-4-nitroimidazole was generated along with several other products. The oligonucleotides containing the 5-guanidino-4-nitroimidazole modification were purified by reverse-phase and anion-exchange HPLC and characterized by matrix-assisted laser desorption mass spectrometry. 5-Guanidino-4-nitroimidazole formation in peroxynitrite-treated DNA was characterized after enzymatic digestion of the reacted DNA to the nucleoside level. HPLC purification and electrospray ionization mass spectrometry (with selected reaction monitoring) enabled the analysis of this modified nucleoside with high sensitivity. The yield of 5-guanidino-4-nitroimidazole formed in single-stranded DNA was approximately 10-fold higher than that found in duplex DNA. With calf thymus DNA, 5-guanidino-4-nitroimidazole was dose-dependently formed at low peroxynitrite concentrations. In stability tests, a synthetic oligonucleotide containing the 5-guanidino-4-nitroimidazole modification was only partially cleaved by hot piperidine and was a weak substrate for Fpg glycosylase repair enzyme; in addition, this site was not cleaved by endonuclease III. These results suggest that nuclear DNA containing 5-guanidino-4-nitroimidazole may not be quickly repaired by DNA repair enzyme systems. Finally, primer extension experiments revealed that this lesion is a potential DNA replication blocker when polymerization is catalyzed by polymerase alpha and polymerase I (Klenow fragment, lack of exonuclease activity) but not with human polymerase beta. Replication fidelity experiments further showed that 5-guanidino-4-nitroimidazole may cause G-->T and G-->C transversions in calf thymus polymerase alpha and E. coli polymerase I.     

Crystal and molecular structure of a DNA duplex containing the carcinogenic lesion O6-methylguanine

The crystal and molecular structure of the first DNA duplex containing the carcinogenic lesion O6MeG has been determined to a resolution of 1.9 A and refined to an R factor of 19%. (d[CGC-(O6Me)GCG])2 crystallizes in the left-handed Z DNA form and has crystal parameters and conformational features similar to those of the parent sequence [d(CG)3]2. The methyl groups on O6 of G4 and G10 have C5-C6-O6-O6Me torsion angles of 73 degrees and 56 degrees, respectively, and protrude onto the major groove surface. The base-pairing conformation for the methylated G.C base pairs is of the Watson-Crick type as opposed to a wobble-type conformation that had been proposed in a B DNA fragment. As in other Z DNA structures, a spine of hydration is seen in the minor groove.