NMR Spectroscopic Study of a DNA Duplex with Mercury-Mediated T-T Base Pairs

Recently, we reported that T-T mismatches can specifically recognize Hg ^II (T-Hg ^II -T pair formation). In order to understand the properties of the T-Hg ^II -T pair, we recorded NMR spectra for a DNA duplex, d(CGCG TT GTCC) ? d(GGAC TT CGCG), with two successive T-T mismatches (Hg ^II -binding sites). We assigned ¹ H resonances for mercury-free and di-mercurated duplexes, and performed titration experiments with Hg ^II by using 1D ¹ H NMR spectra. Because of the above mentioned assignments, we could confirm the existence of mono-mercurated species, because individual components gave independent NMR signals in the titration spectra.     

Raman spectroscopic detection of the T-Hg II-T base pair and the ionic characteristics of mercury

Developing applications for metal-mediated base pairs (metallo-base-pair) has recently become a high-priority area in nucleic acid research, and physicochemical analyses are important for designing and fine-tuning molecular devices using metallo-base-pairs. In this study, we characterized the Hg(II)-mediated T-T (T-Hg(II)-T) base pair by Raman spectroscopy, which revealed the unique physical and chemical properties of Hg(II). A characteristic Raman marker band at 1586 cm(-1) was observed and assigned to the C4=O4 stretching mode. We confirmed the assignment by the isotopic shift ((18)O-labeling at O4) and density functional theory (DFT) calculations. The unusually low wavenumber of the C4=O4 stretching suggested that the bond order of the C4=O4 bond reduced from its canonical value. This reduction of the bond order can be explained if the enolate-like structure (N3=C4-O4(-)) is involved as a resonance contributor in the thymine ring of the T-Hg(II)-T pair. This resonance includes the N-Hg(II)-bonded state (Hg(II)-N3-C4=O4) and the N-Hg(II)-dissociated state (Hg(II+) N3=C4-O4(-)), and the latter contributor reduced the bond order of N-Hg(II). Consequently, the Hg(II) nucleus in the T-Hg(II)-T pair exhibited a cationic character. Natural bond orbital (NBO) analysis supports the interpretations of the Raman experiments.     

Oligonucleotide probes applied for sensitive enzyme-amplified electrochemical assay of mercury(II) ions

We developed a novel electrochemical sensor for Hg(2+) detection using two mercury-specific oligonucleotide probes and streptavidin-horseradish peroxidase (HRP) enzymatic signal amplification. The two mercury-specific oligonucleotide probes comprised a thiolated capture probe and a biotinated signal probe. The thiolated capture probe was immobilized on a gold electrode. In the presence of Hg(2+), the thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction between the mismatched T-T base pairs directed the biotinated signal probe hybridizing to the capture probe and yielded a biotin-functioned electrode surface. HRP was then immobilized on the biotin-modified substrate via biotin-streptavidin interaction. The immobilized HRP catalyzed the oxidation of hydroquinone (H(2)Q) to benzoquinone (BQ) by hydrogen peroxide (H(2)O(2)) and the generated BQ was further electrochemically reduced at the modified gold electrode, producing a readout signal for quantitative detection of Hg(2+). The results showed that the enzyme-amplified electrochemical sensor system was highly sensitive to Hg(2+) in the concentration of 0.5 nM to 1 μM with a detection limit of 0.3 nM, and it also demonstrated excellent selectivity against other interferential metal ions.     

A Simple and Rapid Resonance Scattering Spectral Method for Detection of Trace Hg<Superscript>2+</Superscript> Using Aptamer-Nanogold as Probe

Single-strand deoxyribonucleic acid (ssDNA) were used to modified nanogold particle to obtain a aptamer-nanogold probe (NGssDNA) for Hg(II). The probe is not aggregated in high concentration of NaCl. In the pH 7.0 Na₂HPO₄-NaH₂PO₄ buffer solution and in the presence of high concentration of NaCl, NGssDNA interact with Hg(II) to form stable double-strand T-Hg(II)-T mismatches and to release nanogold particles from the probe. The released nanogold particles aggregated to form bigger clusters which leaded the resonance scattering (RS) intensity at 540 nm enhanced linearly with the concentration of Hg²⁺ in the range of 0.39–1666.7 nM, with detection of 0.1 nM. This simple, rapid, and sensitive aptamer-nanogold RS assay was applied to determination of Hg²⁺ in wastewater, with satisfactory results.     

Highly sensitive oligonucleotide-based fluorometric detection of mercury(II) in aqueous media

This paper describes a highly sensitive and selective Hg(2+) sensor using a label free Hg(2+) specific probe (5'-18T-3') and an intercalation dye SYBR Green I (SG). The Hg(2+) specific probe is composed of thymines (T) and readily forms T-Hg(2+)-T complexes in the presence of Hg(2+). This specific T-Hg(2+)-T formation affects the hybridization of the Hg(2+) specific probe and the intercalation of SG. Upon treatment of 1 nM 5'-18T-3' with different amount of Hg(2+) (0.1-10nM), which is followed by hybridization with 1 nM 5'-18T-3' and incubation with 1 microL of SG, the solution fluorescence gave a linear response (R=0.996) to the concentration of Hg(2+). The detection limit for Hg(2+) was 0.5 nM (0.1 ppb). The overall test only takes few minutes and very little interference is observed from non-specific metal ions. This approach may find potential applications in monitoring the Hg(2+) concentration in drinking water.     

Reusable evanescent wave DNA biosensor for rapid, highly sensitive, and selective detection of mercury ions

Mercury ions (Hg(2+)) are a highly toxic and ubiquitous pollutants requiring rapid and sensitive on-site detection methods in the environment and foods. Herein, we report an envanescent wave DNA-based biosensor for rapid and very sensitive Hg(2+) detection based on a direct structure-competitive detection mode. In this system, a DNA probe covalently immobilized onto a fiber optic sensor contains a short common oligonucleotide sequences that can hybidize with a fluorescently labeled complementary DNA. The DNA probe also comprises a sequence of T-T mismatch pairs that binds with Hg(2+) to form a T-Hg(2+)-T complex by folding of the DNA segments into a hairpin structure. With a structure-competitive mode, a higher concentration of Hg(2+) leads to less fluorescence-labeled cDNA bound to the sensor surface and thus to lower fluorescence signal. The total analysis time for a single sample, including the measurement and surface regeneration, was under 6 min with a Hg(2+) detection limit of 2.1 nM. The high specificity of the sensor was demonstrated by evaluating its response to a number of potentially interfering metal ions. The sensor's surface can be regenerated with a 0.5% SDS solution (pH 1.9) over 100 times with no significant deterioration of performance. This platform is potentially applicable to detect other heavy metal ions or small-molecule analytes for which DNA/aptamers can be used as specific sensing probes.     

Chromomycin dimer-DNA oligomer complexes. Sequence selectivity and divalent cation specificity

This paper reports on a solution NMR characterization of the sequence selectivity and metal ion specificity in chromomycin-DNA oligomer complexes in the presence of divalent cations. The sequence selectivity studies have focused on chromomycin complexes with the self-complementary d(A1-A2-G3-G4-C5-C6-T7-T8) duplex containing a pair of adjacent (G3-G4).(C5-C6) steps and the self-complementary d(A1-G2-G3-A4-T5-C6-C7-T8) duplex containing a pair of separated (G2-G3).(C6-C7) steps in aqueous solution. The antitumor agent (chromomycin) and nucleic acid protons have been assigned following analysis of distance connectivities in NOESY spectra and coupling connectivities in DQF-COSY spectra for both complexes in H2O and D2O solution. The observed intermolecular NOEs establish that chromomycin binds as a Mg(II)-coordinated dimer [1 Mg(II) per complex] and contacts the minor-groove edge with retention of 2-fold symmetry centered about the (G3-G4-C5-C6).(G3-G4-C5-C6) segment of the d(A2G2C2T2) duplex. By contrast, complex formation is centered about the (G2-G3-A4-T5).(A4-T5-C6-C7) segment and results in removal of the two fold symmetry of the d(AG2ATC2T) duplex. Thus, the binding of one subunit of the chromomycin dimer at its preferred (G-G).(C-C) site assists in the binding of the second subunit to the less preferred adjacent (A-T).(A-T) site. These observations suggest a hierarchy of chromomycin binding sites, with a strong site detected at the (G-G) step due to the hydrogen-bonding potential of acceptor N3 and donor NH2 groups of guanosine that line the minor groove. The divalent cation specificity has been investigated by studies on the symmetric chromomycin-d(A2G2C2T2) complex in the presence of diamagnetic Mg(II), Zn(II), and Cd(II) cations and paramagnetic Ni(II) and Co(II) cations. A comparative NOESY study of the Mg(II) and Ni(II) symmetric complexes suggests that a single tightly bound divalent cation aligns the two chromomycins in the dimer through coordination to the C1 carbonyl and C9 enolate ions on the hydrophilic edge of each aglycon ring. Secondary divalent cation binding sites involve coordination to the major-groove N7 atoms on adjacent guanosines in G-G steps. This coordination is perturbed on lowering the pH below 6.0, presumably due to protonation of the N7 atoms. The midpoint of the thermal dissociation of the symmetric complex is dependent on the divalent cation with the stability for reversible transitions decreasing in the order Mg(II) greater than Zn(II) greater than Cd(II) complexes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Target-induced structure-switching DNA hairpins for sensitive electrochemical monitoring of mercury (II)

A simple, sensitive and reusable electrochemical sensor was designed for determination of mercury (II) (Hg(2+)) by coupling target-induced conformational switch of DNA hairpins with thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination chemistry. The hairpin probe consisted of a stem of 6 base pairs enclosing a 14 nucleotide (nt) loop and an additional 12 nt sticky end at the 3' end. Each hairpin was labeled with ferrocene (Fc) redox tag in the middle of the loop, which was immobilized on the electrode via self-assembly of the terminal thiol moiety at the 5' end. In the presence of target analyte, Hg(2+)-mediated base pairs induced the conformational change from the sticky end to open the hairpins, resulting in the ferrocene tags close to the electrode for the increasing redox current. The strong coordination reaction of T-Hg(2+)-T resulted in a good repeatability and intermediate precision down to 10%. The dynamic concentration range spanned from 5.0nM to 1.0μM Hg(2+) with a detection limit of 2.5nM at the 3s(blank) level. The strategy afforded exquisite selectivity for Hg(2+) against other environmentally related metal ions. Inspiringly, the developed sensor could be reused by introduction of iodide (I(-)).     

NMR studies of exocyclic 1,N2-propanodeoxyguanosine adducts (X) opposite purines in DNA duplexes: protonated X(syn).A(anti) pairing (acidic pH) and X(syn).G(anti) pairing (neutral pH) at the lesion site

Proton and phosphorus two-dimensional NMR studies are reported for the complementary d(C1-A2-T3-G4-X5-G6-T7-A8-C9).d(G10-T11-A12-C13-A14-C15-A 16-T17-G18) nonanucleotide duplex (designated X.A 9-mer) that contains a 1,N2-propanodeoxyguanosine exocyclic adduct, X5, opposite deoxyadenosine A14 in the center of the helix. The NMR studies detect a pH-dependent conformational transition; this paper focuses on the structure present at pH 5.8. The two-dimensional NOESY studies of the X.A 9-mer duplex in H2O and D2O solution establish that X5 adopts a syn orientation while A14 adopts an anti orientation about the glycosidic bond at the lesion site. The large downfield shift of the amino protons of A14 demonstrates protonation of the deoxyadenosine base at pH 5.8 such that the protonated X5(syn).A14(anti) pair is stabilized by two hydrogen bonds at low pH. At pH 5.8, the observed NOE between the H8 proton of X5 and the H2 proton of A14 in the X.A 9-mer duplex demonstrates unequivocally the formation of the protonated X5(syn).A14(anti) pair. The 1,N2-propano bridge of X5(syn) is located in the major groove. Selective NOEs from the exocyclic methylene protons of X5 to the major groove H8 proton of flanking G4 but not G6 of the G4-X5-G6 segment provide additional structural constraints on the local conformation at the lesion site. A perturbation in the phosphodiester backbone is detected at the C13-A14 phosphorus located at the lesion site by 31P NMR spectroscopy. The two-dimensional NMR studies have been extended to the related complementary X.G 9-mer duplex that contains a central X5.G14 lesion in a sequence that is otherwise identical with the X.A 9-mer duplex. The NMR experimental parameters are consistent with formation of a pH-independent X5(syn).G14(anti) pair stabilized by two hydrogen bonds with the 1,N2-propano exocyclic adduct of X5(syn) located in the major groove.     

Peptide-induced parallel DNA duplexes for oligopyrimidines. Stereospecificity in complexation for oligo(L-lysine) and oligo(L-ornithine)

It is shown that the cationic oligopeptides octadeca(L-lysine) (Lys18) and octadeca(L-ornithine) (Orn18) can induce a parallel duplex for the natural DNA oligomer dT10 with thymine-thymine base pairs. Complexation of the ammonium groups in the peptide side chains with the DNA phosphates leads to diminished electrostatic phosphate-phosphate repulsions, which allows this T-T base pair formation. From combined NOESY 1H NMR and molecular mechanics studies, it follows that the parallel duplex is right-handed, with the peptide located in the groove of the duplex. For the natural DNA oligomers dC10, d(C6T6), and d(T6C2T2), only Lys18 is able to induce the formation of parallel duplexes with C-C and T-T base pairs. It is shown that, for Orn18, a complexation must occur with one of the nonbonded oxygen atoms in the phosphate groups (OR) in such a way that unfavorable steric interactions are present with the C-C base pairs, which have a larger propellor twist angle than T-T base pairs. An analogy is presented between peptide complexation with the phosphates and the neutralization of the phosphate groups by methylation, which is known to lead to parallel duplexes with T-T base pairs (for both the Sp and Rp configurations) and C-C base pairs (only for the Sp configuration).     

A new peptidyl fluorescent chemosensors for the selective detection of mercury ions based on tetrapeptide

A novel peptidyl chemosensor (PySO₂-His-Gly-Gly-Lys(PySO₂)-NH₂, 1) was synthesized by incorporation of two pyrene (Py) fluorophores into the tetrapeptide using sulfonamide group. Compound 1 exhibited selective fluorescence response towards Hg(II) over the other metal ions in aqueous buffered solutions. Furthermore, 1 with the potent binding affinity (K_d = 120 nM) for Hg(II) detected Hg(II) without interference of other metal ions such as Ag(I), Cu(II), Cd(II), and Pb(II). The binding mode of 1 with Hg(II) was investigated by UV absorbance spectroscopy, ¹H NMR titration experiment, and pH titration experiment. The addition of Hg(II) induced a significant decrease in both excimer and monomer emissions of the pyrene fluorescence. Hg(II) interacted with the sulfonamide groups and the imidazole group of His in the peptidyl chemosensor and then two pyrene fluorophores were close to each other in the peptide. The decrease of both excimer and monomer emission was mainly due to the excimer/monomer emission change by dimerization of two pyrene fluorophores and a quenching effect of Hg(II).     

Soft metal ions, Cd(II) and Hg(II), induce triple-stranded alpha-helical assembly and folding of a de novo designed peptide in their trigonal geometries

We previously reported the de novo design of an amphiphilic peptide [YGG(IEKKIEA)4] that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded alpha-helix structure. The addition of Cd(II), however, resulted in the reconstitution of the triple-stranded alpha-helix bundle, as revealed by circular dichroism (CD) spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant Kd was determined to be 1.5 +/- 0.8 microM for Cd(II). The triple-stranded complex formed by the 113Cd(II) ion showed a single 113Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl- ions. The 113Cd NMR resonance was connected with the betaH protons of the cysteine residue by 1H-113Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg(II) also induced the assembly of the peptide into a triple-stranded alpha-helical bundle below the Hg(II)/peptide ratio of 1/3. With excess Hg(II), however, the alpha-helicity of the peptide was decreased, with the change of the Hg(II) coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.     

Electrically contacted enzyme based on dual hairpin DNA structure and its application for amplified detection of Hg2+

In the present study, based on a dual hairpin DNA structure, a novel system of electrically contacted enzyme and its signal amplification for ultrasensitive detection of Hg(2+) was demonstrated. In the presence of Hg(2+), with the interaction of thymine-Hg(2+)-thymine (T-Hg(2+)-T), DNA sequence dully labeled with ferrocene (Fc) at 5' end and horseradish peroxidase (HRP) at 3' end, hybridized to the capture probe and formed the dual hairpin structure on the electrode. Fc unit acts as a relay that electrically contacts HRP with the electrode and activates the bioelectrocatalyzed reduction of H(2)O(2). And based on the bioelectrocatalyzed signal amplification of the presented system, Hg(2+) could be quantitatively detected in the range of 10(-10)-10(-6)M with a low detection limit of 52 pM. And it also demonstrated excellent selectivity against other interferential metal ions.     

In search of a Hoogsteen base paired DNA duplex in aqueous solution

When the oligodeoxynucleotides d(A)6 and d(T)6 are mixed together in a 1:1 ratio (in 100 mM NaCl), the NH signals in the NMR spectrum gave a typical signature of Watson-Crick paired (WC) and Hoogsteen paired (H) AT base pairs. The observation indicates two schemes: Scheme I, WC and H duplexes in slow equilibrium, i.e., WC in equilibrium with H, Scheme II, the WC helix formed is unstable and that it disproportionates into a triple helix (TR) and free d(A)6. We show that (i) addition of extra d(A)6 does not change the helix composition, (ii) addition of a minor-groove specific drug Dst2 (a distamycin analogue) results in an exclusive WC helix-drug duplex, while it does not destabilize triple helix in a 1:2 mixture. In addition we have compared the melting profile, 31P NMR spectra, 1H NMR spectra and the salt dependence of the 1:1 mixture and that of a pure triple helix. All the data from the above experiments overwhelmingly favor Scheme I. However Scheme II cannot be categorically excluded. Based on 1D/2D NMR studies, we have characterized the structural properties of the Hoogsteen double helix in terms of nucleotide conformations. In addition, we computationally demonstrate that the relative stability of the WC over the H duplexes increases with increasing chain length.     

NMR studies for identification of dI:dG mismatch base-pairing structure in DNA.

One- and two-dimensional NMR experiments have been undertaken to investigate deoxyinosine:deoxyguanosine (dI:dG) base pairing in a self-complementary dodecadeoxyribonucleotide, d(C1-G2-C3-I4-A5-A6-T7-T8-G9-G10-G11-G12) (designated IG-12), duplex. The NMR data indicate formation of a dI(syn):dG(anti) base pair in a B-DNA helix. This unusual base pairing results in altered NOE patterns between the base protons (H8 and H2) of the I4 residue and the sugar protons of its own and the 5'-flanking C3 residues. The dI(syn):dG(anti) base pair is accommodated in the B-DNA duplex with only a subtle distortion of the local conformation. Identification of the dI:dG base pairing in this study confirms that a hypoxanthine base can form hydrogen-bonded base pairs with all of the four normal bases, C, A, T, and G, in DNA.     

Visual detection of Hg²⁺ in aqueous solution using gold nanoparticles and thymine-rich hairpin DNA probes

We report a sensitive method for visual detection of mercury ions (II) (Hg2?) in aqueous solution by using gold nanoparticles (Au-NPs) and thymine (T)-rich hairpin DNA probes. The thiolated hairpin DNA probe was immobilized on the Au-NP surface through a self-assembling method. Another thymine-rich, digoxin-labeled DNA probe was introduced to form DNA duplexes on the Au-NP surface with thymine-Hg2?-thymine (T-Hg2?-T) coordination in the presence of Hg2?. The Au-NPs associated with the formed duplexes were captured on the test zone of a lateral flow strip biocomponent (LFSB) by immunoreaction events between the digoxin on the duplexes and anti-digoxin antibodies on the LFSB. The accumulation of Au-NPs produced a characteristic red band on the test zone, enabling visual detection of Hg2? without instrumentation. A detection limit of 0.1 nM was obtained under optimal experimental conditions. This method provides a simple, rapid, sensitive approach for the detection of Hg2? and shows great promise for point-of-care and in-field detection of environmentally toxic mercury.     

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)     

Dissociation kinetics of 19 base paired oligonucleotide-DNA duplexes containing different single mismatched base pairs.

The dissociation kinetics of 19 base paired oligonucleotide-DNA duplex containing a various single mismatched base pair are studied on dried agarose gels. The kinetics of the dissociation are first order under our experimental conditions. The incorporation of a single mismatched base pair destabilizes the DNA duplexes to some extent, the amount depending on the nature of the mismatched base pair. G-T and G-A mismatches slightly destabilize a duplex, while A-A, T-T, C-T and C-A mismatches significantly destabilize it. The activation energy for the overall dissociation processes for these oligonucleotide-DNA duplexes containing 19 base pairs is 52 +/- 2 Kcal mol-1 as determined from the slope of Arrhenius plot.     

In Search of a Hoogsteen Base Paired DNA Duplex in Aqueous Solution

Abstract

When the oligodeoxynucleotides d(A)₆ and d(T)₆ are mixed together in a 1:1 ratio (in 100 mM NaCl), the NH signals in the NMR spectrum gave a typical signature of Watson-Crick paired (WC) and Hoogsteen paired (H) AT base pairs. The observation indicates two schemes: Scheme I, WC and H duplexes in slow equilibrium, i.e., WC ? H, Scheme II, the WC helix formed is unstable and that it disproportionates into a triple helix (TR) and free d(A)₆. We show that (i) addition of extra d(A)₆ does not change the helix composition, (ii) addition of a minor-groove specific drug Dst2 (a distamycin analogue) results in an exclusive WC helix- drug duplex, while it does not destabilize triple helix in a 1:2 mixture. In addition we have compared the melting profile, ³¹P NMR spectra, ¹H NMR spectra and the salt dependence of the 1:1 mixture and that of a pure triple helix. All the data from the above experiments overwhelmingly favor Scheme I. However Scheme II cannot be categorically excluded.

Based on 1D/2D NMR studies, we have characterized the structural properties of the Hoogsteen double helix in terms of nucleotide conformations. In addition, we computationally demonstrate that the relative stability of the WC over the H duplexes increases with increasing chain length.     

Terrestrial organic matter biomarkers as tracers of Hg sources in lake sediments

Terrestrial organic matter (TOM) plays a key role in mercury (Hg) dynamics between watersheds and lakes. In this study we attempts to determine the role of TOM source and quality and not only quantity, in the fate and transport of total Hg (T-Hg) to boreal lakes. Integrating the watershed complexity is a daunting task. Within the scope of this project, we characterized this organic matter at a molecular level in order to determine Hg transfer conditions to the sediments. We sampled ten lakes in the Quebec boreal forest. In each lake, we took a sediment core at the deepest point in addition to analyzing T-Hg and a set of terrigenous biomarkers in recent sediments. Our results show no relationship between TOM quantity and T-Hg concentration in lake sediments. However, [T-Hg] variation is well explained by the increase of 3,5Bd/V ratios (R²?=?0.84; p?<?0.0002) and the decrease of C/V ratios (R²?=?0.5; p?<?0.0227). Our study shows that TOM source and quality are determinant for Hg loadings in lake sediments. More precisely, increasing TOM derived from humified soil horizons explains most of Hg level variation within sediments.