2D NMR studies and 3D structure of the parallel-stranded duplex oligonucleotide Acrm5-alpha-d(TCTAAACTC)-beta-d(AGATTTGAG) via complete relaxation matrix analysis of the NOE effects and molecular mechanics calculations

The three-dimensional structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAACTC) covalently linked to an acridine derivative (m5Acr) with its natural and parallel complementary sequence beta-d(AGATTTGAG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. All the nonexchangeable and exchangeable resonances were assigned in this duplex. The structure was refined by using interproton distances determined by NOE measurements. The NOE values were converted into distances by using the complete 190 x 190 relaxation matrix. The unnatural duplex Acrm5-alpha-d(TCTAAACTC)-beta-d(AGATTTGAG) forms a parallel right-handed helix with Watson-Crick base pairing; the alpha and beta deoxyriboses adopt a 3'-exo conformation. The acridine moiety was found stacked up the C9-G9 base pair. The structure of the first seven base pairs of this duplex was found similar to that of the duplex alpha-d(TCTAAAC)-beta-d(AGATTTG), which we had already investigated [Lancelot, G., et al. (1989) Biochemistry 28, 7871-7878]. Since these structures were generated by using experimental NOE values obtained independently on macromolecules whose global correlation time was different (3.8 and 2.2 ns), we conclude that this comparison is a good test of the viability of our method to generate three-dimensional structures of oligonucleotides in solution. Starting from different initial conformations, we show that the NOE constraints allow one to reach the same final restrained conformation, taking into account implicitly the solvent effect.     

Solution structure of [d(A-T)5]2 via complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect spectra and molecular mechanics calculations: evidence for a hydration tunnel

Pure absorption phase proton two-dimensional nuclear Overhauser effect (2D NOE) spectra at 500 MHz have been obtained for [d(5'ATATATATAT3')]2 in deuterium oxide solution at several mixing times. The 100 nonexchangeable proton resonances have been assigned. The experimental 2D NOE spectra were compared with theoretical spectra calculated by using the complete relaxation matrix analysis method [Keepers, J. W., & James, T. L. (1984) J. Magn. Reson. 57, 404-426] and x-ray diffraction determined molecular coordinates of A, B, alternating B, left-handed B, C, D, and wrinkled D forms of DNA and of energy-minimized structures calculated from the most promising X-ray crystal structures by using the molecular mechanics program AMBER, in which all hydrogens, counterions, and hydration water molecules were included. The analysis of all features of the 2D NOE spectra played an important role in extracting the promising structures, and it was concluded that the wrinkled D form yields the best fit for the 2D NOE data of the A-T decamer. The molecular mechanics calculation indicated that this model structure, whose minor groove is comparatively deep and narrow, may be energetically more stable than the B form for alternating d(A-T) DNA. Interesting features of the structure include possible intra- and interchain sugar-phosphate attractions and a hydration tunnel inside the minor groove capable of accommodating three types of water molecules that aid in helix stabilization via hydrogen bonding. Counterions (sodium) serve to reduce interchain phosphate-phosphate repulsive effects.     

Structure determination of [d(ATATATAUAT)]2 via two-dimensional NOE spectroscopy and molecular dynamics calculations

Proton homonuclear two-dimensional (2D) NOE spectra were obtained for the decamer [d(ATATATAUAT)]2 as a function of mixing time, and proton resonance assignments were made. Quantitative assessment of the 2D NOE cross-peak intensities was used in conjunction with the program MARDIGRAS, which entails a complete relaxation matrix analysis of the 2D NOE peak intensities, to obtain a set of upper and lower bound interproton distance constraints. The analysis with MARDIGRAS was carried out using three initial models: A-DNA, B-DNA and Z-DNA. The distance constraints determined were essentially the same regardless of initial structure. These experimental structural constraints were used with restrained molecular dynamics calculations to determine the solution structure of the decamer. The molecular dynamics program AMBER was run using A-DNA or B-DNA as starting model. The root-mean-square (rms) difference between these two starting models is 0.504 nm. The two starting models were subjected to 22.5 ps of restrained molecular dynamics calculations. The coordinates of the last 10.5 ps of the molecular dynamics runs were averaged to give two final structures. MDA and MDB. The rms difference between these two structures is 0.09 nm, implying convergence of the two molecular dynamics runs. The 2D NOE spectral intensities calculated for the derived structures are in good agreement with experimental spectra, based on sixth-root residual index analysis of intensities. A detailed examination of the structural features suggests that while the decamer is in the B-family of DNA structures, many torsion angle and helical parameters alternate from purine to pyrimidine, with kinks occurring at the U-A steps.     

Crystal structure of d(GCGAAAGCT) containing a parallel-stranded duplex with homo base pairs and an anti-parallel duplex with Watson-Crick base pairs

A DNA fragment d(GCGAAAGCT), known to adopt a stable mini-hairpin structure in solution, has been crystallized in the space group I4₁22 with the unit-cell dimensions a = b = 53.4 Å and c = 54.0 Å, and the crystal structure has been determined at 2.5 Å resolution. The four nucleotide residues CGAA of the first half of the oligomer form a parallel duplex with another half through the homo base pairs, C₂:C₂⁺ (singly-protonated between the Watson– Crick sites), G₃:G₃ (between the minor groove sites), A₄:A₄ (between the major groove sites) and A₅:A₅ (between the Watson–Crick sites). The two strands remaining in the half of the parallel duplex are split away in different directions, and they pair in an anti-parallel B-form duplex with the second half extending from a neighboring parallel duplex, so that an infinite column is formed in a head-to-tail fashion along the c-axis. It seems that a hexa-ammine cobalt cation supports such a branched and bent conformation of the oligomer. One end of the parallel duplex is stacked on the corresponding end of the adjacent parallel duplex; between them, the guanine base of the first residue is stacked on the fourth ribose of another duplex.     

Solution structure studies of d(AC)4.d(GT)4 via restrained molecular dynamics simulations with NMR constraints derived from two-dimensional NOE and double-quantum-filtered COSY experiments

The structure of d(AC)4.d(GT)4 is investigated by constrained molecular dynamics simulations. The constraints include proton pair distances derived from 2D NOE intensities by using the iterative relaxation matrix analysis algorithm MARDIGRAS and sugar pucker phases and amplitudes derived from double-quantum-filtered COSY spectra. Molecular dynamics runs on simulated intensity and distance sets as well as the experimental data were carried out to determine the effects of starting structure, distance constraint derivation, energy functions, and experimental errors on the end result. It was found that structural details could not be elucidated within about 1.5-A overall atomic deviation. This limitation is due in part to the accuracy of the experimental data but, more importantly, is attributable to the quantity of experimental constraints available and to imperfections in the force field utilized in the molecular dynamics calculations. Within the limits of the method, some structural characteristics of d(AC)4.d(GT)4 could be elucidated.     

Solution structure of [d(GTATATAC)]2 via restrained molecular dynamics simulations with nuclear magnetic resonance constraints derived from relaxation matrix analysis of two-dimensional nuclear Overhauser effect experiments

Two-dimensional nuclear Overhauser effect (2D NOE) spectra have been used as the experimental basis for determining the solution structure of the duplex [d(GTATATAC)]2 employing restrained molecular dynamics (rMD) simulations. The MARDIGRAS algorithm has been employed to construct a set of 233 interproton distance constraints via iterative complete relaxation matrix analysis utilizing the peak intensities from the 2D NOE spectra obtained for different mixing times and model structures. The upper and lower bounds for each of the constraints, defining size of a flat-well potential function term used in the rMD simulations, were conservatively chosen as the largest or smallest value calculated by MARDIGRAS. Three different starting models were utilized in several rMD calculations: energy-minimized A-DNA, B-DNA, and a structure containing wrinkled D-DNA in the interior. Considerable effort was made to define the appropriate force constants to be employed with the NOE terms in the AMBER force field, using as criteria the average constraints deviation, the constraints violation energy and the total energy. Of the 233 constraints, one was generated indirectly, but proved to be crucial in defining the structure: the cross-strand A5-H2 A5-H2 distance. As those two protons resonate isochronously for the self-complementary duplex, the distance cannot be determined directly. However, the general pattern of 2D NOE peak intensities, spin-lattice relaxation time (T1) values, and 31P nuclear magnetic resonance spectra lead to use of the A3-H2 A7-H2 distance for A5-H2 A5-H2 as well. Five rMD runs, with different random number seeds, were made for each of the three starting structures with the full distance constraint set. The average structure from all 15 runs and the five-structure averages from each starting structure were all quite similar. Two rMD runs for each starting structure were made with the A5-H2 A5-H2 constraint missing. The average of these six rMD runs revealed differences in structure, compared to that with the full set of constraints, primarily for the middle two base-pairs involving the missing cross-strand constraint but global deviations also were found. Conformational analysis of the resulting structures revealed that the inner four to six base-pairs differed in structure from the termini. Furthermore, an alternating structure was suggested with features alternating for the A-T and T-A steps.     

Parallel analysis of oligodeoxyribonucleotide (oligonucleotide) interactions. I. Analysis of factors influencing oligonucleotide duplex formation.

A novel method for the analysis of oligonucleotide-oligonucleotide interactions is described. Oligonucleotides of different sequence are synthesised in situ as stripes on the surface of a glass slide (see accompanying paper). Multiple hybridizations are then carried out on each oligonucleotide simultaneously to determine the dependence of oligonucleotide duplex formation on duplex length, base composition, hybridisation solvent and sequence complexity.     

Crystal and molecular structure of the sodium salt of the dinucleotide duplex d(CpG)

The crystal and molecular structure of the sodium salt of deoxycytidylyl-(3H-5H)-deoxyguanosine has been determined from X-ray diffraction data. The crystal, obtained from an aqueous gamma-butyrolactone solution at pH = 5.3 are orthorhombic, P212121, a = 10.640(2), b = 11.184(2) and c = 44.618(4)A. The structure was refined to an R = 0.041. The d(CpG) structure is similar to the ammonium salt solved by Cruse et al.(1). Both structures form a parallel self base paired mini-double helix. In d(CpG).Na+ one of the two paired cytosines is protonated on N(3). The cytosines form 3 hydrogen bonds while the guanines form only 2. The Na+ ion is coordinated with five groups: two water molecules, O(6) of guanine A, N(7) of guanine B and 0(5') of cytosine B, forming a square pyramid. The hydration shell around the mini-helix is analysed and compared with that of the ammonium salt, d(CpG).Na+ is the second d(CpG) oligonucleotide found with a self base pairing arrangement despite of the fact that the crystallization conditions and counterion were different in both cases. The hypothesis that self base pairing is not only a crystallization artifact but may play a role under physiological conditions as a source of transversion mutations is discussed.     

Solution structure by NMR and molecular dynamics of a duplex containing a guanine opposite a N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide lesion

One- and two-dimensional NMR spectroscopy has been used combined with molecular dynamics to determine the fine structure of the DNA duplex 5'-d(AGGAGCCACG).d(CGTGGFTCCT) where F is the N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide residue which is a ring fragmentation product of thymine. The formamide deoxyribose exists as two isomers with respect to the orientation about the peptide bond. The two isomers (trans and cis) are observed in a ratio 3:2 in solution. For both species, the oligonucleotide adopts a globally B form structure although conformational changes are observed around the mismatch site. The formamide residue, whatever the isomer, is intrahelical and can pair with the guanine on the opposite strand with one hydrogen bond. For the cis isomer, the residue adopts a syn orientation and is able to form a second hydrogen bond with the guanine on the 5' side on the same strand. Off-resonance ROESY experiments have been used to investigate the chemical exchange observed at low temperature of the duplex. Conformational exchange has only been found for the oligonucleotide with the formamide residue in the trans conformation.     

Solution structure of the aminoacyl-capped oligodeoxyribonucleotide duplex (W-TGCGCAC)(2).

Reported here is the solution structure of the aminoacyl-DNA duplex (W-TGCGCAC)(2). This duplex forms a continuously pi-stacked helix consisting of both nucleobases and amino acid side chains. According to NMR and UV analyses, the duplex melts in a cooperative transition and with 1.3-1.8% greater hyperchromicity than the control duplex (TGCGCAC)(2). A van't Hoff analysis of UV melting points at different concentrations shows that the two tryptophan residues contribute 4.8 kcal/mol to the DeltaH degrees of complex formation at 10 mM salt concentration and less than 1 kcal/mol at 150 mM salt. The entropic cost for duplex association in the presence of the amino acid residues is 13 cal/molK greater than that for the control at 10 mM salt concentration, and 3 cal/molK lower than that of the control at 0.15 ionic strength. The conformation of W-TGCGCAC in duplex form, determined via restrained torsion angle molecular dynamics, shows an undisturbed B-form DNA duplex with dangling 3'-termini. The tryptophanyl residue at the 5'-terminus packs tightly against T2 and the proximal part of adenine, without engaging in hydrogen bonding. While not providing strong enthalpic net stabilization of the duplex, the tryptophan "cap" on the duplex does seem to reduce the fraying at the termini, indicating a subtle balance of entropic and enthalpic factors contributing to the molecular dynamics. The structure also shows that, at least in the present sequence context, stacking on the terminal base pair is more favorable than intercalation, probably because the enthalpic cost associated with breaking up the stacking between DNA base pairs cannot be paid for by favorable pi-stacking interactions with the indole ring of tryptophan. These results are of importance for understanding stacking interactions in protein-DNA complexes, particularly those in enzyme-substrate complexes involving exposed nucleobases.     

Study of the spatial structure of the duplex d(pTGTTTGGC) d(pCCAAAC)A in aqueous solution by methods of uni- and two-dimensional (1)H-NMR spectroscopy and organic molecular mechanics

Structure of the complementary complex d(pTGTTTGGC) d(pCCAAAC)A in the aqueous solution has been investigated by one- and two-dimensional 1H NMR spectroscopy. The resonances of nonexchangeable protons of bases as well as methyl and deoxyribose 1', 2'a, 2'b, 3' and 4' protons have been assigned by means of two-dimensional J-correlated spectroscopy (COSY) and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY). Using one-dimensional NOE measurements, 62 interproton distances (intranucleotide: (H6/H8)i--(H1')t, (H6/H8)i--(H2'a)i, (H1')i--(H2'a)i, (H1')i--(H2'b)i; internucleotide: (H6/H8)i--(H1')i-1, (H6/H8)i--(H2'a)i-1, (H6/H8)i--(H2'b)i-1, (H5/CH3)i--(H6/H8)i-1, (H5/CH3)i--(H2'a/H2'b)i-1) have been determined for nearest-neighbour protons. Spin-coupling constant values for some sugar protons have been obtained from COSY spectra. The restrained molecular mechanics calculations have yielded the possible solution structures of duplex fitting the experimental set of interproton distances and coupling constants.     

A d(GpG)-platinated decanucleotide duplex is kinked. An extended NMR and molecular mechanics study

A conformational study of the double-stranded decanucleotide d(GCCG*G*ATCGC).d(GCGATCCGGC), with the G* guanines chelating a cis-Pt(NH3)2 moiety, has been accomplished using 1H and 31P NMR, and molecular mechanics. Correlation of the NMR data with molecular models has disclosed an equilibrium between several kinked conformations and has ruled out an unkinked structure. The deformation is localized at the CG*G*.CCG trinucleotide where the helix is kinked by approximately 60 degrees towards the major groove and unwound by 12-19 degrees. The models revealed an unexpected mobility of the cytosine complementary to the 5'-G*. This cytosine can stack on either branch of the kinked complementary strand. The energy barrier between the two positions has been calculated to be less than or equal to 12 kJ/mol. The NMR data are in support of rapid flip-flopping of this cytosine. An explanation for the strong downfield shift observed in the 31P resonance of the G*pG* phosphate is given.     

Interaction of purine nucleotides with inert paramagnetic Cr(III) probes evaluated by NMR relaxation effects. Molecular mechanics calculations on Cr(III) and Co(III) polyphosphate complexes

The 1H NMR relaxation effects produced by paramagnetic Cr(III) complexes on nucleoside 5'-mono- and -triphosphates in D2O solution at pH' = 3 were measured. The paramagnetic probes were [Cr(III)(H2O)6]3+, [Cr(III)(H2O)3(HATP)], [Cr(III)(H2O)3(HCTP)] and [Cr(III)(H2O)3(UTP)-, while the matrix nucleotides (0.1 M) were H2AMP, HIMP-, and H2ATP2-. For the aromatic base protons, the ratios of the transverse to longitudinal paramagnetic relaxation rates (R2p/R1p) for the [Cr(III)(H2O)6]3+/H2ATP2-, [Cr(III)(H2O)3(HATP)]/H2ATP2-, [Cr(III)(H2O)3(HCTP)]/H2ATP2 and [Cr(III)(H2O)3(UTP)]-/H2ATP2 systems were below 2.33 so the dipolar term predominates. For a given nucleotide, R1p for the purine H(8) signal was larger than for the H(2) signal with the [Cr(III)(H2O)6]3+ probe, while R1p for the H(2) signal was larger with all the other Cr(III) probes. Molecular mechanics computations on the [Cr(III)(H2O)4(HPP)(alpha,beta)], [Cr(III)(NH3)4(HPP)(alpha,beta)], [Co(III)(NH3)3(H2PPP)(alpha,beta,gamma)] and [Co(III)(NH3)4(HPP)(alpha,beta)] complexes gave calculated energy-minimized geometries in good agreement with those reported in crystal structures. The molecular mechanics force constants found were then used to calculate the geometry of the inner sphere [Cr(III)(H2O)6]3+ and [Cr(III)(H2O)3(HATP)(alpha,beta,gamma)] complexes as well as the structures of the outer sphere [Cr(III)(H2O)6]3(+)-(H2AMP) and [Cr(III)(H2O)6]-(HIMP)- species. The gas-phase structure of the [Cr(III)(H2O)3(HATP)(alpha,beta,gamma)] complex shows the existence of a hydrogen bond interaction between a water ligand and the adenine N(7)(O...N = 2.82 A). The structure is also stabilized by intramolecular hydrogen bonds involving the -O(2')H group and the adenine N(3) (O...N = 2.80 A) as well as phosphate oxygen atoms and a water molecule (O...O = 2.47 A). The metal center has an almost regular octahedral coordination geometry. The structures of the two outer-sphere species reveal that the phosphate group interacts strongly with the hexa-aquochromium probe. In both complexes, the nucleotides have a similar "anti" conformation around the N(9)-C(1') glycosidic bond. However, a very important difference characterizes the two structures. For the (HIMP)- complex, strong hydrogen bond interactions exist between one and two water ligands and the inosine N(7) and O(6) atoms, respectively (O...O = 2.63 A; O...N = 2.72, 2.70 A). For the H2AMP complex, the [Cr(III)(H2O)6]3+ cation does not interact with N(7) since it is far from the purine system. Hydrogen bonds occur between water ligands and phosphate oxygens. The Cr-H(8) and Cr-H(2) distances revealed by the energy-minimized geometries for the two outer sphere species were used to calculate the R1p values for the H(8) and H(2) signals for comparison with the observed R1p values: 0.92(c), 1.04(ob) (H(8)) and 0.06(c), 0.35(ob) (H(2)) for H2AMP; and 3.76(c), 4.53(ob) (H(8)) and 0.16(c), 0.77(ob) s-1 (H(2)) for HIMP-.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution conformation of an oligonucleotide containing a G.G mismatch determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three dimensional solution structure of the non-selfcomplementary oligonucleotide, d(GAGGAGGCACG). d(CGTGCGTCCTC) in which the central base pair is G.G. This is the first structural determination of a G.G mismatch in a oligonucleotide. Two dimensional nuclear magnetic resonance spectra show that the bases of the mismatched pair are stacked into the helix and that the helix adopts a classical B-DNA form. Spectra of the exchangeable protons show that the two guanosines are base paired via their imino protons. For the non-exchangeable protons and for some of the exchangeable protons nuclear Overhauser enhancement build up curves at short mixing times have been measured. These give 84 proton-proton distances which are sensitive to the helix conformation. One of the guanosines adopts a normal anti conformation while the other is syn or close to syn. All non-terminal sugars are C2' endo. These data sets were incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. The G.G mismatch shows a symmetrical base pairing structure. Although the mismatch is very bulky many of its features are close to that of normal B-DNA. The mismatch induces a small lateral shift in the helix axis and the sum of the helical twist above and below the mismatch is close to that of B-DNA.     

Conformational analysis of the sialyl alpha(2----3/6)N-acetyllactosamine structural element occurring in glycoproteins, by two-dimensional NOE 1H-NMR spectroscopy in combination with energy calculations by hard-sphere exo-anomeric and molecular mechanics force-field with hydrogen-bonding potential

The conformation is described of the sialyl alpha(2----3/6)N-acetyllactosamine structural element, frequently occurring in glycoproteins. NOE spectroscopy of NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----N)Asn and NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta(1----N)Asn is presented and for each glycosidic linkage, except for the alpha(2----6)-linkage, a number of interglycosidic NOEs are measured. The analysis of these effects is performed using a full relaxation matrix. Analysis of intraresidue NOEs provides a calibration of the calculation method. Hard-sphere exo-anomeric (HSEA) energy calculations indicate a single conformation for the beta(1----4)-linkage in both compounds, both being consistent with the NOE data. HSEA and molecular-mechanics force-field with hydrogen-bonding potential energy calculations both indicate the existence of three preferred conformations for the alpha(2----3)-linkage. The analysis of the NOE spectra are consistent with a distribution over two or three of these conformations; by combination with the energy diagram for this linkage the existence of onyl a single conformation can be excluded. The NOE spectrum of the compound with the alpha(2----6)-linkage indicates a gt orientation for the Gal C-6 hydroxymethyl group. On this basis, the HSEA energy calculations for the alpha(2----6)-linkage indicate an extended low-energy surface with a number of preferred conformations. The absence of NOEs across this linkage is interpreted in terms of a non-rigid, but overall folded conformation of the NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta structural element. This provides an explanation for the shift effects induced by alpha(2----6) attachment of NeuAc to the N-acetyllactosamine unit.     

Molecular mechanics and dynamics calculations on (dA)10.(dT)10 incorporating distance constraints derived from NMR relaxation measurements

Structural constraints derived from proton NMR relaxation measurements on poly(dA).poly(dT) in the form of interproton separations and orientation have been combined with molecular mechanics and annealed molecular dynamics calculations to derive a model for the solution-state structure of this molecule. Three different possible starting configurations, including the standard A and B forms of Arnott and Hukins [Arnott, S., & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Commun. 47, 1506-1509] and the heteronomous (H) structure [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155], were examined. Both the B- and H-DNA structures converged to the same B-like structure (approximately C2'-endo conformation on both the A and T sugars, glycosidic bond torsional angle of 63-73 degrees) with the same energies and average helical parameters that gave good fits of the NMR relaxation rates. This model also accounts for the experimental observation [Behling, R. W., & Kearns, D. R. (1986) Biochemistry 25, 3335-3346] that the AH2 proton interacts more strongly with the H1' sugar proton on the T strand than on the A strand. Although the helix repeat angle (39 degrees) is larger than that for standard B-DNA (36 degrees), this does not result in a significantly smaller minor groove, as monitored by the interstrand P-P separation. Calculations starting with the A-DNA structure lead to a very high energy structure that gave a poorer fit of the NMR data.     

Solution structure of duplex DNA containing the mutagenic lesion 1,N(2)-etheno-2'-deoxyguanine

We have used high-resolution NMR spectroscopy and molecular dynamics simulations to determine the solution structure of DNA containing the genotoxic lesion 1, N (2)-etheno-2'-deoxyguanosine (epsilonG), paired to dC. The NMR data suggest the presence of a major, minimally perturbed structure at neutral pH. NOESY spectra indicate the presence of a right-handed helix with all nucleotides in anti, 2'-deoxyribose conformations within the C2'-endo/C1'-exo range and proper Watson-Crick base pair alignments outside the lesion site. The epsilonG residue remains deeply embedded inside the helix and stacks between the flanking base pairs. The lesion partner dC is extrahelical and is located in the minor groove of the duplex, where it is highly exposed to solvent. Upon acidification of the sample, a second conformation at the lesion site of the duplex emerges, with protonation of the lesion partner dC and possible formation of a Hoogsteen base pair. Restrained molecular dynamics simulations of the neutral-pH structure generated a set of three-dimensional models that show epsilonG inside the helix, where the lesion is stabilized by stacking interactions with flanking bases but without participating in hydrogen bonding. The lesion counterbase dC is displaced in the minor groove of the duplex where it can form a hydrogen bond with the sugar O4' atom of a residue 2 bp away.     

Molecular mechanics modeling of oligonucleotide adducts of the antitumor drug cis-diamminedichloroplatinum(II)

In order to assess the geometric changes caused when the antitumor drug cis-diammine-dichloroplatinum(II) (cis-DDP) binds to DNA, molecular mechanics calculations were performed on two double-stranded and two single-stranded oligonucleotides and their adducts with cis-{Pt(NH₃)₂}²⁺. For the platinated duplexes, three model structures have been derived, one involving only local disruption of base pairing with retention of the helix directionality, and two models showing pronounced kinking of the double helix. One of the kinked models is stabilized by bridging sodium ions. The other kinked duplex model shows retention of all Watson–Crick base pairing, including that of the coordinated guanines. All models exhibit hydrogen bonds connecting one ammine ligand of platinum with one or two phosphate groups located at the 5′ side of the platinated strand.     

Considering the oligonucleotide secondary structures in thermodynamic and kinetic analysis of DNA duplex formation

The influence of secondary structures of DNA oligonucleotides on thermodynamics and kinetics at the formation of their bimolecular complexes (duplexes) has been studied. The models considering inherent secondary structures of duplex components and their influence on quantitative thermodynamic and kinetic characteristics of the duplexes have been developed. The values of thermodynamic impacts given by individual structural elements of the double helix have been shown to depend on hairpin structuring of the duplex components. The «concentration» method to consider oligonucleotides intramolecular structure with thermodynamic parameters of bimolecular duplex formation has been proposed. According to stop-flow measurements, the observed values of association and dissociation constants are influenced by the presence of inherent structures in duplex components. The influence observed is increased with the lowering of the sample temperature. The analysis of experimental data involving the developed models provides the possibility to determine «proper» kinetic constants for the helix-to-coil transition. The difference between observed and calculated rate constants can amount up to two or more orders of magnitude.