Differential hydration of dA.dT base pairing and dA and dT bulges in deoxyoligonucleotides

The role of water in the formation of stable duplexes of nucleic acids is being studied by determining the concurrent volume change, heats, and counterion uptake that accompany the duplexation process. The variability of the volume contraction that we have observed in the formation of a variety of homoduplexes suggests that sequence and conformation acutely affect the degree of hydration. We have used a combination of densimetric and calorimetric techniques to measure the change in volume and enthalpy resulting from the mixing of two complementary strands to form (a) fully paired duplexes with 10 or 11 base pairs and (b) bulged decameric duplexes with an extra dA or dT unmatched residue. We also monitored absorbance vs temperature profiles as a function of strand and salt concentration for all four duplexes. Relative to the decamer duplex, insertion of an extra dA.dT base pair to form an undecamer duplex results in a favorable enthalpy of -5.6 kcal/mol that is nearly compensated by an unfavorable entropy term of -5.1 kcal/mol. This enthalpy difference correlates with a differential uptake of water molecules, corresponding to an additional hydration of 16 mol of water molecules/mol of base pair. Relative to the fully paired duplexes, both bulged duplexes are 12-16 degrees C less stable and exhibit marginally larger counterion uptake on forming the duplex. The enthalpy change is slightly lower for the T-bulge duplex and less still for the A-bulge duplex. The volume change results indicate that an unmatched residue increases the amount of coulombic and/or structural hydration. The combined results strongly suggest that the destabilizing forces in bulged duplexes are partially compensated by an increase in hydration levels.     

FTIR study of netropsin binding to poly d(A-T) and poly dA.poly dT

Complexes between netropsin and two polynucleotides containing only AT base pairs (poly d(A-T) and poly dA.poly dT) have been prepared at various drug/base pair ratios and studied in solution by Fourier Transform Infrared Spectroscopy. The drug is shown to interact in the narrow groove of poly d(A-T) with the C2O2 carbonyl of thymines and the N3 groups of adenines. Moreover the spectral modifications allow us to propose the existence of interactions at the level of the deoxyribose. No effect is detected on the phosphate groups when netropsin is progressively added. In the case of poly dA.poly dT the interaction seems much weaker as if the high propeller twist of the homopolymer would make the accessibility of the drug to the minor groove more difficult.     

Duplex structure formation between oligo(dA)''s and oligo(dT)''s generated by thymine-specific interaction with netropsin.

The formation of oligomeric duplex molecules in the presence of the antibiotic netropsin in the series p(dA)n-p(dT)n is demonstrated using low-temperature CD measurements. Addition of Netropsin to mixtures of oligomers generates the same type of CD spectra as observed for poly(dA)-poly(dT) and maintains the duplex structure at temperatures at which base pairing of free oligomers is thermodynamically unstable. The shortest chain length forming a netropsin complex by thymine-specific interaction with the oligopeptide is represented by p(dA)4-p(dt)4. Studies with sequence isomers show that adjacent thymine residues strongly favour the complex formation with the oligopeptide.     

Protection of (dA.dT) cluster regions in the DNAase I cleavage of DNA by specific interaction with netropsin

The specific DNA binding ligand netropsin selectively blocks dA-dT base pairs in clusters containing two or more consecutive thymine residues at the dNAase I cleavage sites of DNA. Using CD and UV absorption measurements it is shown, that at various ratios of netropsin to nucleotide concentrations and even at satuation of ligand interaction the enzyme cuts along regions containing dG-dC pairs sandwiched between dA-dT pairs. This follows a slow kinetics and is associated with a release of netropsin from those segments. These facts suggests the usefulness of the partial protection of certain DNA sequences in DNAase I cleavage sites in producing DNA fragments in structural studies of the genome. A possible interpretation of the effect of netropsin binding on the enzymatic hydrolysis of phosphodiester bonds of the helix is discussed.     

Complete disproportionation of duplex poly(dT)*poly(dA) into triplex poly(dT)*poly(dA)*poly(dT) and poly(dA) by coralyne

Coralyne is a small crescent-shaped molecule known to intercalate duplex and triplex DNA. We report that coralyne can cause the complete and irreversible disproportionation of duplex poly(dT)·poly(dA). That is, coralyne causes the strands of duplex poly(dT)·poly(dA) to repartition into equal molar equivalents of triplex poly(dT)·poly(dA)·poly(dT) and poly(dA). Poly(dT)·poly(dA) will remain as a duplex for months after the addition of coralyne, if the sample is maintained at 4°C. However, disproportionation readily occurs upon heating above 35°C and is not reversed by subsequent cooling. A titration of poly(dT)·poly(dA) with coralyne reveals that disproportionation is favored by as little as one molar equivalent of coralyne per eight base pairs of initial duplex. We have also found that poly(dA) forms a self-structure in the presence of coralyne with a melting temperature of 47°C, for the conditions of our study. This poly(dA) self-structure binds coralyne with an affinity that is comparable with that of triplex poly(dT)·poly(dA)·poly(dT). A Job plot analysis reveals that the maximum level of poly(dA) self-structure intercalation is 0.25 coralyne molecules per adenine base. This conforms to the nearest neighbor exclusion principle for a poly(dA) duplex structure with A·A base pairs. We propose that duplex disproportionation by coralyne is promoted by both the triplex and the poly(dA) self-structure having binding constants for coralyne that are greater than that of duplex poly(dT)·poly(dA).     

Long (dA)n.(dT)n tracts can form intramolecular triplexes under superhelical stress.

Plasmids containing long tracts of (dA)n.(dT)n have been prepared and their conformations examined in linear and supercoiled DNA using a series of chemical and enzymic probes which are known to be sensitive to unusual DNA structures. Under superhelical stress and in the presence of magnesium the sequence T69.A69 adopts a conformation at pH 8.0 consistent with the formation of an intramolecular DNA triplex. Site specific cleavage of the supercoiled plasmid by single-strand specific nucleases occurs within the A.T insert; the 5'-end of the purine strand is sensitive to reaction with diethylpyrocarbonate while the central 5-6 bases of the pyrimidine strand are reactive to osmium tetroxide. By contrast shorter inserts of A33.T33 and A23.T23 do not appear to form unusual structures.     

Unusual conformation of (dA)n.(dT)n-tracts as revealed by cyclobutane thymine-thymine dimer formation.

Cyclobutane dimer formation has been used to probe conformation of (dA)n.(dT)n-tracts cloned in plasmid DNA. The observed dimer probability patterns for (dA)n.(dT)n-tracts with n greater than or equal to 4 exhibit maximum intensity at the 3'-terminal TT site of Tn-tract, whereas photoreactivity at all the other TT sites is inhibited. Both the temperature and dimethyl sulfoxide increase dimer formation within Tn-tracts and result in an even dimer pattern. The data obtained have been interpreted in terms of an unusual structure adopted by (dA)n.(dT)n-tracts. An influence of flanking base pairs, ethidium bromide binding and ionic strength has also been studied.     

Inhibition of DNA replication by berenil in plasmids containing Poly(dA)poly(dT) sequences

The effect of berenil on plasmid DNA replication was studied on pBR322-derived plasmids containing poly(dA)poly(dT) sequences. In comparison to the parental plasmid pBR322, plasmid pKH47 harboring 100 bp of poly(dA)poly(dT) at the PvuII site showed a decrease in plasmid yield in the presence of berenil. This effect was also observed in pVL26, a related plasmid in which the location of the poly(dA)poly(dT) region had been shifted to the EcoRV site in pBR322. [(3)H]Thymidine incorporation experiments indicated that DNA synthesis may be affected in these plasmids in the presence of the drug. Bromodeoxyuridine incorporation experiments coupled to Cs(2)SO(4) equilibrium density gradient centrifugation indicated that the lower plasmid yield was due to an inhibition of DNA replication by berenil. We have also found that berenil induces DNA degradation in plasmids containing the homopolymer. Our studies strongly suggest that the effect of berenil on plasmid replication and DNA stability results from its binding to the poly(dA)poly(dT) region present in these plasmids. Moreover, we have found a correlation between the position of the poly(dA)poly(dT) region and this inhibitory effect. Thus, plasmid pKH47, containing the poly(dA)poly(dT) region most proximal to the origin of pBR322 replication, was most severely affected.     

alpha-DNA X: alpha and beta tetrathymidilates covalently linked to oxazolopyridocarbazolium (OPC): comparative stabilization of oligo beta-[dT]:oligo beta-[dA] and oligo alpha-[dT]:oligo beta-[dA] duplexes by the intercalating agent.

The influence of the intercalating oxazolopyridocarbazolium (HOPC) on the stabilization of modified oligonucleotides: alpha-T4c5OPC or beta-T4c5OPC associated to beta-oligo (dA) was studied. It appears that the situation is different from what has been observed for the interaction of these modified oligonucleotides with poly (rA). The higher free energy of formation of the alpha-T4c5OPC :beta-oligo(dA), when compared to beta-T4c5OPC, is essentially due to the overall stability added to this system by the intercalator. This enhanced stability comes from a higher number of binding sites of HOPC for the alpha:beta duplex together with a lower van't Hoff energy of formation of the alpha:beta duplex.     

Structure of poly(dA).poly(dT) is not identical to the AT rich regions of the single crystal structure of CGCGAATTBrCGCG. The consequence of this to netropsin binding to poly(dA).poly(dT)

The basic assumption of Dickerson and Kopka (J. Biomole. Str. Dyns. 2, 423, 1985) that the conformation of poly(dA).poly(dT) in solution is identical to the AT rich region of the single crystal structure of the Dickerson dodecamer is not supported by any experimental data. In poly(dA).poly(dT), NOE and Raman studies indicate that the dA and dT units are conformationally equivalent and display the (anti-S-type sugar)-conformation; incorporation of this nucleotide geometry into a double helix leads to a conventional regular B-helix in which the width of the minor groove is 8A. The derived structure is consistent with all available experimental data on poly(dA).poly(dT) obtained under solution conditions. In the crystal structure of the dodecamer, the dA and dT units have distinctly different conformations-dA residues adopt (anti, S-type sugar pucker), while dT residues belong to (low anti, N-type sugar pucker). These different conformations of the dA and dT units along with the large propeller twist can be accommodated in a double helix in which the minor groove is shrunk from 8A to less than 4A. In the conventional right handed B-form of poly(dA).poly(dT) with the 8A wide minor groove, netropsin has to bind asymmetrically along the dA strand to account for the NOE and chemical shift data and to generate a stereochemically sound structure (Sarma et al, J. Biomole. Str. Dyns. 2, 1085, 1985).     

Triple-helix formation by an oligonucleotide containing one (dA)12 and two (dT)12 sequences bridged by two hexaethylene glycol chains.

The triple-helix formation by the oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol group, was investigated by thermal denaturation analysis and circular dichroism spectroscopy. Thermal denaturation analysis showed that this single-stranded oligonucleotide is able to fold back on itself twice to give a triple helix at low temperature. Upon an increase in the temperature, two cooperative transitions were observed: formation of a double-stranded structure with a dangling x-(dT)12 extremity, then formation of a single-stranded coil structure. Due to the intramolecular character of the transition, the triplex is much more stable than that formed by the reference mixture (dA)12 + 2(dT)12. In 0.1 M NaCl, the triplex-to-coil transition occurred at about 30 degrees C whereas the duplex-to-coil was at about 60 degrees C. Upon an increase in the salt, the increase of temperature corresponding to the triplex-to-duplex transition was larger than that of the duplex-to-coil transition. MgCl2 showed higher efficiency than NaCl to promote triplex or duplex formation. The thermodynamic parameters delta H and delta S were determined at various ionic strengths for both transitions. Both the enthalpy change and entropy change associated with triplex-to-duplex transition (Hoogsteen base pairing) were smaller than those associated to the duplex-to-coil transition (Watson-Crick base pairing). When the ionic strength increased, the parameters -delta H and -delta S showed a very small decrease for the duplex-to-coil transition whereas a strong increase was observed with the triplex-to-duplex transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of a deoxyribodecanucleotide containing an extrahelical thymidine surrounded by an oligo(dA).oligo(dT) tract

One- and two-dimensional NMR experiments were carried out on a decamer, d-(CGCTTTTCGC).d(GCGAAAAGCG), and on the same sequence with the addition of an unpaired thymidine, d(CGCTTTTCGC).d(GCGAATAAGCG), which will be referred to as the T-bulge decamer. Evidence from one-dimensional NOE experiments on the exchangeable protons indicates that the unpaired thymidine is extrahelical. This conclusion is also supported by numerous cross-peaks in the two-dimensional NOESY spectrum of the nonexchangeable protons. Assignments for all of the resonances, with the exception of the H5' and H5" resonances, have been made for both oligonucleotide duplexes through the use of 2D NOESY, COSY, and relayed COSY experiments. Temperature dependence of the methyl resonance chemical shifts indicates that the unpaired thymidine shows unusual behavior compared to other thymidines in the duplex. Two-dimensional NOESY experiments carried out from 5 to 35 degrees C indicate the unpaired thymidine remains extrahelical throughout this temperature range. A similar temperature dependence for the methyl chemical shift is found in the corresponding single-strand d(GCGAATAAGCG). The oligo-(dA).oligo(dT) tracts in both the decamer and the T-bulge decamer have structures different from B-form DNA and exhibit NOEs similar to those observed in other oligonucleotides containing A.T tracts. The formation of this unusual A.T tract structure may induce the extrahelical conformation of the unpaired thymidine.     

Physical studies of DNA premelting equilibria in duplexes with and without homo dA.dT tracts: correlations with DNA bending

We have employed a variety of physical methods to study the equilibrium melting and temperature-dependent conformational dynamics of dA.dT tracts in fractionated synthetic DNA polymers and in well-defined fragments of kinetoplast DNA (kDNA). Using circular dichroism (CD), we have detected a temperature-dependent, "premelting" event in poly(dA).poly(dT) which exhibits a midpoint near 37 degrees C. Significantly, we also detect this CD "premelting" behavior in a fragment of kDNA. By contrast, we do not observe this "premelting" behavior in the temperature-dependent CD spectra of poly[d(AT)].poly[d(AT)], poly(dG).poly(dC), poly[d(GC)].poly[d(GC)], or calf thymus DNA. Thus, poly(dA).poly(dT) and kDNA exhibit a common CD-detected "premelting" event which is absent in the other duplex systems studied in this work. Furthermore, we find that the anomalous electrophoretic retardation of the kDNA fragments we have investigated disappears at temperatures above approximately 37 degrees C. We also observe that the rotational dynamics of poly(dA).poly(dT) and kDNA as assessed by singlet depletion anisotropy decay (SDAD) and electric birefringence decay (EBD) also display a discontinuity near 37 degrees C, which is not observed for the other duplex systems studied. Thus, in the aggregate, our static and dynamic measurements suggest that the homo dA.dT sequence element [common to both poly(dA).poly(dT) and kDNA] is capable of a temperature-dependent equilibrium between at least two helical states in a temperature range well below that required to induce global melting of the host duplex. We suggest that this "preglobal" melting event may correspond to the thermally induced "disruption" of "bent" DNA.     

Four-stranded DNA. Conformational analysis of regular spirals of poly(dT).poly(dA).poly(dA).poly(dT) with different base binding variations

Conformational analysis of four stranded DNA helices poly(dT).poly(dA).poly(dA).poly(dT) with parallel arrangement of the identical sugar-phosphate chains connected by twofold symmetry has been performed. All possible models of symmetrical base binding were checked. By the potential energy optimization the dihedral angles and helices parameters of stable conformations of four stranded polynucleotides were calculated. The dependences of conformational energy on the base complex structure and mutual orientation of the poly(dA).and poly(dT) chains were studied. Possible biological functions of four stranded helices are discussed.     

Conformational transitions and aggregation in poly(dA)-poly(dT) system induced by Na+ and Mg2+ ions

Effects of Mg²⁺ ions on thermally induced conformational transitions in the synthetic poly(dA)·poly(dT) and poly(dA)·2poly(dT) were studied in the buffered solutions (pH 6.9), containing 0.1 or 1 M NaCl at polynucleotide concentration of 0.1–0.3 mM (in nucleic bases). The experiments consist of measurements of the UV absorption and intensity of conventional visible static light scattering. The diagram of conformational transitions in the poly(dA)–poly(dT)–Mg²⁺ system was constructed on a basis of experimental data obtained. Anomalously strong light scattering, like critical opalescence, has been revealed at 0.1 M NaCl and [Mg²⁺]≥20 mM in the melting range of both polynucleotides, which eventually disappeared after the completion of polymer strands separation. The effect presumably is caused by a fluctuation process of polymer strands complexing which arises at a certain concentration of Mg²⁺ ions.     

Specific interaction of netropsin, distamycin-3 and analogs with LC duplexes: reversion towards the B form of the 2''-deoxy-.2''-deoxy-2''-fluoro-hybrid duplexes upon specific interaction with netropsin, distamycin-3 and analogs.

Binding of the B-form specific ligands netropsin and distamycin-3, -4 and -5 has been used to monitor the presence and/or the inducibility of a B-type structure in various poly-inosinic.poly-cytidilic double stranded polymers with deoxyribose, ribose or 2'-deoxy-2'-fluororibose as sugar on either strand. The efficiency of binding was followed by circular dichroism and further evaluated by the increase in melting temperature of the complexes. The efficient binding of netropsin and distamycins to the hybrid polymer (dIfl)n. (dC)n demonstrated that the fluorine carrying strand may undergo a A to B-type transition reflecting a change of the 2'-deoxy-2'-fluororibose from the 3'-endo to the 1'-exo or 2'-endo pucker. The less efficient binding of the same ligands to the reverse hybrid (dI)n.(dCfl)n showed that the geometry of the pyrimidine strand is the most critical for the specific interaction. Taking into account the recent findings about the regular hydration in the minor groove of the B-type dodecamer dCGCGAATTCGCG in solid-state, the different binding modes observed between the different polymers and antibiotics are explained by differences in their possibilities of hydration. Binding of netropsin to a double stranded deoxypolymer is interpreted as a local replacement of water molecules by netropsin in the minor groove hydration network which is typical of the B-form.     

Interaction of saffron carotenoids as anticancer compounds with ctDNA, Oligo (dG.dC)15, and Oligo (dA.dT)15

Crocin and crocetin are two important natural saffron carotenoids, which, along with dimethylcrocetin (DMC) as a semi-synthetic product, are responsible for its color. Many biological properties of saffron have been reported, among which the anticancer property is the most important. Some anticancer drugs have direct interaction with DNA, and thus the present study attempted to investigate the interaction of three major saffron carotenoids-crocin, crocetin, and DMC--with calf thymus DNA (ctDNA) and oligonucleotides. The spectrophotometric data showed some changes in ctDNA absorption spectra due to the formation of complex with saffron extract and each of these three components. Also, all the three components caused the quenching of the fluorescence emission of ctDNA-ethidium bromide complex. The Scatchard analysis of these data indicated a noncompetitive manner for quenching, which is accompanied by the outside groove-binding pattern. The circular dichroism (CD) spectra also indicated the nonintercalative binding and induction of the conformational changes, and B to C transition in ctDNA structure and then unstacking of ctDNA bases at higher concentrations of the carotenoids. The CD spectra of G.C and A.T oligonucleotides after addition of these carotenoids indicated the transition from B- to C-DNA, which is very similar to the ctDNA spectral changes. The DeltaG(H(2)O), the best parameter for the estimation of macromolecule stability, was determined for ctDNA denaturation using dodecyl trimethylammonium bromide in the absence and presence of crocin, crocetin, or DMC. Our results showed a decrease in the Delta G(H(2)O), indicating the ctDNA destabilization due to its interaction with the mentioned ligands. In conclusion, the results show that saffron and its carotenoids interact with DNA and induce some conformational changes in it. Of these carotenoids, the order of potential of interaction with DNA is crocetin > DMC > crocin.     

Calculations of stability of A and B forms of dA6.dT6 and dG6.dC6 duplexes by molecular mechanics method in an aqueous solution and approximation of the statistical model. Analysis of unusual stability of the dA6.dT6 B form

The statistical model of the environment for estimating the influence of the aqueous electrolyte solution on the macromolecule has been developed. The energy of the water-fasteners between macromolecule atoms has been calculated. The calculations of A and B form dA6.dT6 and dG6.dC6 duplexes have been made. The unusual stability of B form in dA6.dT6 and their unstability in dG6.dC6 have been analysed. B form duplexes have optimal energy of Van-der-Waals interactions, but a more strained ribose-phosphate backbone, the latter being the reason of its high conformational lability. The aqueous solution stabilizes B form duplexes. The main types of sequence-dependent B form stabilizing interactions are: electrostatic interactions between phosphate groups and bases, the effect of the solvent molecular structure and energy of water fasteners between nucleic bases and ribose-phosphate backbone.     

A unique deletion in pBR322 DNA caused by insertion of poly(dA).poly(dT)

A unique deletion covering around 43% of the pBR322 genome was found after attempting to insert 100 or 200 bp poly(dA).poly(dT) into the EcoRV site of pBR322 DNA. This result was not observed if an equivalent size heterologous DNA or a larger poly(dA).poly(dT) fragment of 10-20,000 bp was introduced at the same site. DNA sequencing analysis at the junctions suggests that a specific intramolecular pairing may be involved in the formation of this deletion mutant.