Conformations of cyclic 2,3-nucleotides and 2,3-Cyclic-5-diphosphates. Interrelation between the phase angle of pseudorotation of the sugar and the torsions about the glycosyl C(1)-N and the backbone C(4)-C(5) bonds

Semiempirical potential energy calculations have been carried out for cyclic 2′,3′-nucleotides and their 5′-phosphorylated derivatives, which are the intermediates in the hydrolysis of RNA. Calculations have been performed for both purine and pyrimidine bases for the observed O(1′)-endo, O(1′)-exo and the unpuckered planar sugar ring conformations. It is found that the mode of sugar pucker largely determines the preferred conformations of these molecules. For cyclic 2′,3′-nucleotides themselves, the O(1′)-endo sugars show a preference for the syn glycosyl conformation while the O(1′)-exo sugars exclusively favor the anti conformation regardless of whether the base is a purine or pyrimidine. For the unpuckered planar sugar, the syn conformation is favored for purines and anti for pyrimidines. Both the gauche (+) (60°) and trans (180°) conformations about the C(4′)? C(5′) bond are favored for O(1′)-endo sugars, while the gauche (?) (300°) and trans (180°) are favored for O(1′)-exo sugars. On the contrary, the 5′-phosphorylated cyclic 2′,3′-nucleotides of both purines and pyrimidines show a preference for the anti-gauche (+) conformational combination about the glycosyl and C(4′)? C(5′) bonds for the O(1′)-endo sugars and the anti-trans combination for the O(1′)-exo sugars. The correlation between the phase angle of the sugar ring and the favored torsions about the glycosyl and the backbone C(4′)? C(5′) bonds as one traverses along the pseudorotational pathway of the sugar ring is examined.     

Structure of guanylyl-3',5'-cytidine monophosphate. II. Description of the molecular and crystal structure of the calcium derivative in space group P2(1).

The structural features of calcium guanosine-3′,5′-cytidine monophosphate (GpC) have been elucidated by X-ray diffraction analysis. The molecule was crystallized in space group P2₁ with cell constants of a = 21.224 Å, b = 34.207 Å, c = 9.327 Å, and β = 90.527°, Z = 8. The hydration of the crystal is 21% by weight with 72 water molecules in the unit cell. The four GpC molecules in the asymmetric unit occur as two Watson-Crick hydrogen-bonded dimers related by a pseudo-C face centering. Each dimer consists of two independent GpC molecules whose bases are hydrogen bonded to each other in the traditional Watson-Crick fashion. Each dimer possesses a pseudo twofold axis broken by a calcium ion and associated solvent. The four molecules are conformationally similar to helical RNA, but are not identical to it or to each other. Instead, values of conformational angles reflect the intrinsic flexibility of the molecule within the range of basic helical conformations. All eight bases are anti, sugars are all C3′-endo, and the C4′-C5′ bond rotations are gauche-gauche. The R factor is 12.6% for 2918 observed reflections at 1.2-Å resolution.     

Structure of an 11-mer DNA Duplex Containing the Carbocyclic Nucleotide Analog: 2′-Deoxyaristeromycin

Abstract

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.     

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

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

Conformational and structural constraints in double-helical polynucleotides

Conformational analysis of antiparallel double-helical polynucleotides with Watson-Crick base pairing was reduced to a four-dimensional problem using original mathematical methods. In the four-dimensional conformational space the family of structures, characterized by the base-pair stacking with the most stable conformations in water solution as well as in the solid state, was localized. For the C′₂-endo sugar pucker, both right-handed and left-handed structures were found; right-handed structures only, however, seem to be allowed for the C′₃-endo pucker, the only possible one for ribonucleotides with base stacking.     

Single-Stranded DNA and RNA Targeted Triplex-Formation: UV,CD and Molecular Modeling Studies of Foldback Triplexes Containing Different RNA, 2′-OMe-RNA and DNA Strand Combinations

Abstract

We studied the influence of different 2′-OMe-RNA and DNA strand combinations on single strand targeted foldback triplex formation in the Py.Pu:Py motif using ultraviolet (UV) and circular dichroism (CD) spectroscopy, and molecular modeling. The study of eight combinations of triplexes (D D:D, R* D:D, D D:R*, R* D:R*, D R:D, R* R:D, DR:R*, and R*-R:R*; where the first, middle, and last letters stand for the Hoogsteen Pyrimidine, Watson-Crick [WC] purine and WC pyrimidine strands, respectively, and D, R and R* stand for DNA, RNA and 2′-OMe-RNA strands, respectively) indicate more stable foldback triplex formation with a DNA purine strand than with an RNA purine strand. Of the four possible WC duplexes with RNA/DNA combinations, the duplex with a DNA purine strand and a 2′-O-Me-RNA pyrimidine strand forms the most thermally stable triplex, although its thermal stability is the lowest of all four duplexes. Irrespective of the duplex combination, a 2′-OMe-RNA Hoogsteen pyrimidine strand forms a stable foldback triplex over a DNA Hoogsteen pyrimidine strand confirming the earlier reports with conventional and circular triplexes. The CD studies suggest a B-type conformation for an all DNA homo-foldback triplex (D.D.D), while hetero-foldback triplex spectra suggest intermediate conformation to both Atype and B-type structures. A novel molecular modeling study has been carried out to understand the stereochemical feasibility of all the combinations of foldback triplexes using a geometric approach. The new approach allows use of different combinations of chain geometries depending on the nature of the chain (RNA vs. DNA).     

Examination by the molecular orbital method of the intrinsic conformation preferences of nucleic acid sequence]

This paper reports a systematic PCILO study of the conformation of the nucleic acid backbone. The authors principally studied the ω′ and ω phosphodiester torsion angles of the disugar triphosphate model as a simultaneous function of (1) the sugar nature, ribose or deoxyribose, (2) the different combinations of the sugar ring puckers C(2′)-endo-C(2′)-endo, C(3′)-endo-C(3′)-endo, C(3′)-endo-C(2′)-endo, and C(2′)-endo-C(3′)-endo, and (3) the different conformations around the ψ(C4′–C5′) exocyclic bond. The dependence of the (ω′,ω) conformational energy maps upon these different factors, is discussed. The results are in very good agreement with the observed structures of ribonucleic (RNA10, RNA11, A′-RNA12, tRNA^Phe) and deoxyribonucleic acids (D-DNA, C-DNA 9.3, B-DNA 10, A-DNA 11). Thus the validity of this model, the disugar triphosphate unit, is ensured. The main conclusions that can be drawn from this systematic study are the following:

• 1 The torsion around P-05′ (angle ω) is, as a general rule, more flexible than the torsion around P-03′ (angle ω′).
• 2 There is no notable difference between the ribose–triphosphate units and the deoxyribose–triphosphate units for the C(3′)-endo–C(3′)-endo and C(3′)-endo–C(2′)-endo sugar puckers.
• 3 The deoxyribose–triphosphate units with C(2′)-endo–C(2′)-endo and C(2′)-endo–C(3′)-endo sugar puckers show much more ω′ flexibility than the ribose–triphosphate units with the same sugar puckers and cis position for the 2′hydroxyl group.
• 4 The preferred values of ω′ are independent of the sugar nature (ribose or deoxyribose) and of ψ values; they are correlated with the sugar pucker of the first sugar-phosphate unit:
  • C(3′)-endo-C(3′)-endo and C(3′)-endo-C(2′)-endo puckers ? ω′ ? 240° (g^? region)
  • C(2′)-endo-C(2′)-endo and C(2′)-endo-C(3′)-endo puckers ? ω′ 180° (t region)
• 5 The preferred values of ω are independent of the nature and the puckering of the sugars; they are correlated with the rotational state of the torsion angle ψ(C4′–C5′): ψ ? 60° (gg) ? ω ? 300° (g^?), ψ ? 180° (gt) or 300° (tg) ? ω ? 60° (g⁺)

     

The A and B conformations of DNA and RNA subunits. Potential energy calculations for dGpdC

In order to obtain a molecular picture of the A and B forms of a DNA subunit, potential energy calculations have been made for dGpdC with C(3′)-endo and C(2′)-endo [or C(3′)-exo] sugar puckerings. These are compared with results for GpC. The global minima for dGpdC and GpC are almost identical. They are like A-form duplex DNA and RNA, respectively, with bases anti, the ω′, ω angle pair near 300°, 280°, and sugar pucker C(3′)-endo. For dGpdC, a B-form helical conformer, with sugar pucker C(2′)-endo and ω′ = 257°, ω = 298°, is found only 0.4 kcal/mol above the global minimum. A second low-energy conformation (2.3 kcal/mol) has ω′ = 263°, ω = 158° and ψ near 180°. This has dihedral angles like the original Watson–Crick model of the double helix. In contrast, for GpC, the C(2′)-endo B form is 6.9 kcal/mol above the global minimum. These theoretical results are consistent with experimental studies on DNA and RNA fibers. DNA fibers exist in both A and B forms, while RNA fibers generally assume only the A form. A low-energy conformation unlike the A or B forms was found for both dGpdC and GpC when the sugars were C(3′)-endo. This conformation—ω′,ω near 20°,80°—was not observed for C(2′)-endo dGpdC. Energy surface maps in the ω′,ω plane showed that C(2′)-endo dGpdC has one low-energy valley. It is in the B-form helical region (ω′ ～ 260°, ω ～ 300). When the sugar pucker is C(3′)-endo, dGpdC has two low-energy regions: the A-form helical region and the region with the minimum at ω′ = 16°, ω = 85°.     

Structural Properties of Hybrid Triplex of Polycation Deoxyribonucleic S-methylthiourea (DNmt) Strands with a Complementary DNA Strand,Probed by Nanosecond Molecular Dynamics

Abstract

The replacement of phosphodiester linkages of the polyanion DNA with S-methylthiourea linkers provides the polycation deoxyribonucleic S-methylthiourea (DNmt). Molecular dynamics studies to 1,220 ps of the hybrid triplex formed from octameric DNmt strands d(Tmt)₈ with a complementary DNA oligomer strand d(Ap)₈ have been carried out with explicit water solvent and Na counterions under periodic boundary conditions using the CHARMM force field and the Ewald summation method. The Watson-Crick and Hoogsteen hydrogen-bonding patterns of the A/T tracts remained intact without any structural restraints for triplex structures throughout the simulation. The duplex portion of the triplex structure equilibrated at a B-DNA conformation in terms of the helical rise and other helical parameters. The dynamic structures of the DNmt·DNA·DNmt triplex were determined by examining histograms from the last 800 ps of the dynamics run. These included the hydrogen-bonding pattern (sequence recognition), three-centered bifurcating occurrences, minor groove width variations, and bending of tracts for the hybrid triplex structures. Together with the Watson-Crick hydrogen-bondings, the strong Hoogsteen hydrogen-bondings, the partially maintained three-centered bifurcatings in the Watson-Crick pair, and the medium-strength three-centered bifurcatings in the Hoogsteen pair suggest that the hybrid triplex is energetically favorable as compared to a duplex with similar base stacking, van der Waals interactions, and helical parameters. This is in agreement with our previously reported thermody- namic study, in which only triplex structures were observed in solution. The bending angle measured between the local axis vectors of the first and last helical axis segments is about 20° for the Watson-Crick portion of the averaged structure. Propeller twist (associated with three-centered hydrogen-bonding) up to ?30°, native to DNA AT base pairing, was also observed for the triplex structure. The sugar pseudorotation phase angles and the ring rotation angles for the DNA strand are within the C3′-endo domain and C2′-endo domain for the DNmt strand. Water spines are observed in both minor and major grooves throughout the dynamics run. The molecular dynamics simulations of the structural properties of DNmt·DNA·DNmt hybrid triplex is compared to the DNG·DNA·DNG hybrid triplex (In DNG the -O-(PO₂-)-O- linkers in DNA is replaced by -NH-C(=N₂)-NH-).     

Oligonucleotide N3′→P5′ Phosphoramidates and Thio‐Phoshoramidates as Potential Therapeutic Agents

Nucleic acids analogues, i.e., oligonucleotide N3′→P5′ phosphoramidates and N3′→P5′ thio‐phosphoramidates, containing 3′‐amino‐3′‐deoxy nucleosides with various 2′‐substituents were synthesized and extensively studied. These compounds resist nuclease hydrolysis and form stable duplexes with complementary native phosphodiester DNA and, particularly, RNA strands. An increase in duplexes' melting temperature, ΔT_m, relative to their phosphodiester counterparts, reaches 2.2–4.0° per modified nucleoside. 2′‐OH‐ (RNA‐like), 2′‐O‐Me‐, and 2′‐ribo‐F‐nucleoside substitutions result in the highest degree of duplex stabilization. Moreover, under close to physiological salt and pH conditions, the 2′‐deoxy‐ and 2′‐fluoro‐phosphoramidate compounds form extremely stable triple‐stranded complexes with either single‐ or double‐stranded phosphodiester DNA oligonucleotides. Melting temperature, T_m, of these triplexes exceeds T_m values for the isosequential phosphodiester counterparts by up to 35°. 2′‐Deoxy‐N3′→P5′ phosphoramidates adopt RNA‐like C3′‐endo or N‐type nucleoside sugar‐ring conformations and hence can be used as stable RNA mimetics. Duplexes formed by 2′‐deoxy phosphoramidates with complementary RNA strands are not substrates for RNase H‐mediated cleavage in vitro. Oligonucleotide phosphoramidates and especially thio‐phosphoramidates conjugated with lipid groups are cell‐permeable and demonstrate high biological target specific activity in vitro. In vivo, these compounds show good bioavailability and efficient biodistribution to all major organs, while exerting acceptable toxicity at therapeutically relevant doses. Short oligonucleotide N3′→P5′ thio‐phosphoramidate conjugated to 5′‐palmitoyl group, designated as GRN163L (Imetelstat), was recently introduced as a potent human telomerase inhibitor. GRN163L is not an antisense agent; it is a direct competitive inhibitor of human telomerase, which directly binds to the active site of the enzyme and thus inhibits its activity. This compound is currently in multiple Phase‐I and Phase‐I/II clinical trials as potential broad‐spectrum anticancer agent.     

The First Example of a Hoogsteen Basepaired DNA Duplex in Dynamic Equilibrium with a Watson-Crick Basepaired Duplex—A Structural (NMR), Kinetic and Thermodynamic Study

Abstract

A single-point substitution of the O4′ oxygen by a CH₂ group at the sugar residue of A ⁶ (i.e. 2′-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5′- d(C¹G²C³G⁴A⁵A⁶T⁷T⁸C⁹G¹⁰C¹¹G¹²)₂ ^?3, has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A ⁶:T⁷ Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)→←(1B): K_eq = k₁/k_-1 = 0.56±0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k₁ (298K) = 3.9± 0.8 sec^?1; δH°? = 164±14 kJ/mol;-TδS°? (298K) = ?92 kJ/mol giving a δG₂₉₈°? of 72 kJ/mol. Ea (k1) = 167±14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0±0.6 sec-1, δH°? = 153±13 kJ/mol;-TδS°? (298K) = ?82 kJ/mol giving a δG₂₉₈°? of 71 kJ/mol. Ea (k-1) = 155±13 kJ/mol]. A comparison of δG₂₉₈°? of the forward (k1) and backward (k-1) conversions, (1A)→←(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A ⁶:T⁷ Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A ⁶:T⁷ basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2′- deoxyaristeromycin moiety, A ⁶, as a result of substitution of the endocyclic 4′-oxygen in the natural sugar with a methylene group in A ⁶. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in α, σ and γ torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.     

Hoogsteen-paired homopurine [RP-PS]-DNA and homopyrimidine RNA strands form a thermally stable parallel duplex

Homopurine deoxyribonucleoside phosphorothioates possessing all internucleotide linkages of R(P) configuration form a duplex with an RNA or 2'-OMe-RNA strand with Hoogsteen complementarity. The duplexes formed with RNA templates are thermally stable at pH 5.3, while those formed with a 2'-OMe-RNA are stable at neutrality. Melting temperature and fluorescence quenching experiments indicate that the strands are parallel. Remarkably, these duplexes are thermally more stable than parallel Hoogsteen duplexes and antiparallel Watson-Crick duplexes formed by unmodified homopurine DNA molecules of the same sequence with corresponding RNA templates.     

Spectroscopic studies of chimeric DNA-RNA and RNA 29-base intramolecular triple helices.

Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A-form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied.     

Polarized Raman scattering from oriented single microcrystals of d(A5T5)2 and d(pTpT)

Polarized Raman spectra have been obtained from single microcrystals of the duplex of the decamer d(A₅T₅)₂ using a Raman microscope. This is the first report of Raman spectra from a crystal of a deoxyoligomer that contains only long, nonalternating sequences of adenine and thymine. Sequences containing d(A)_n and d(T)_n are of interest in view of recent suggestions that they induce bends in DNA and that they might exist in a nonstandard B-conformation. Polarized Raman spectra of a crystal of d(pTpT) have also been obtained. Both crystals display Raman bands whose intensities are very sensitive to the orientation of the crystal with respect to the direction of polarization of the incident laser beam. These spectra indicate that the helical axes of the oligonucleotides are parallel to the long axes of the crystals and that the d(A₅T₅)₂ is not appreciably bent in the crystal. The Raman spectrum from the d(pTpT) crystal indicates that all of the furanose ring puckers are in a C2′-endo configuration since only the C2′-endo marker band at 835 ± 5 cm^?1 is present. Crystals of d(A₅T₅)₂ show measurable Raman intensities in both the 838- and 816-cm^?1 bands. This indicates the presence of both the C2′-endo and C3′-endo, or possibly other non-C2′-endo, furanose conformations. The 816-cm^?1 band is weak so that only a small fraction of the residues are estimated to be in the non-C2′-endo conformation. In both the d(pTpT) and d(A₅T₅)₂ crystals the intensity of the bands due to vibrations of the backbone show only a small dependence on orientation of the crystals. This result is explained by the low symmetry of the puckered sugar rings. It is concluded that Raman spectra obtained from oligonucleotide crystals in which the orientation of the crystal axes to the laser polarization is not carefully controlled may contain intensity artifacts that are due to polarization effects.     

Determination of the molecular conformation of beta-D-O2,2'-cyclouridine in aqueous solution by proton magnetic resonance spectroscopy

The solution conformation of β-D -O²,2′-cyclouridine has been determined at 27 and 88°C in D₂O by proton magnetic resonance spectroscopy. The conformation is described in terms of a fixed syn-like sugar-base torsional angle, a type S furanose ring conformation (similar to 2′-endo), and a temperature-dependent exocyclic C(4)′–C(5′) rotamer population containing approximately 50% of the gauche-gauche form at 27°C. β-D -O²,2′-Cyclouridine 5′-phosphate likewise possesses a type S furanose ring conformation.     

Effect of the sulfur atom on S2 and S4 positions of the uracil ring in different DNA:RNA hybrid microhelixes with three nucleotide base pairs

The effect of the sulphur atom on the uracil ring was analyzed in different DNA:RNA microhelixes with three nucleotide base-pairs, including uridine, 2-thiouridine, 4-thiouridine, 2,4-dithiouridine, cytidine, adenosine and guanosine. Distinct backbone and helical parameters were optimized at different density functional (DFT) levels. The Watson-Crick pair with 2-thiouridine appears weaker than with uridine, but its interaction with water molecules appears easier. Two types of microhelixes were found, depending on the H-bond of H2′ hydroxyl atom: A-type appears with the ribose ring in ³E-envelope C_3′-endo, and B-type in ²E-envelope C_2′-endo. B-type is less common but it is more stable and with higher dipole-moment. The sulphur atoms significantly increase the dipole-moment of the microhelix, as well as the rise and propeller twist parameters. Simulations with four Na atoms H-bonded to the phosphate groups, and further hydration with explicit water molecules were carried out. A re-definition of the numerical value calculation of several base-pair and base-stacking parameters is suggested.     

Conformational characteristics of the trinucleoside diphosphate d(ApApA) from energy-minimization studies

Energy-minimization studies were carried out on the trinucleoside diphosphate d(ApApA). The potential energy contributions from nonbonded, electrostatic, hydrogen-bonding, and torsional interactions were minimized by treating the 13 relevant dihedral angles as simultaneous variables. For the C(3′)-endo trimer, 14 low-energy conformations are within 10 kcal/mol above the lowest energy found, compared to only 3 in the case of the C(2′)-endo trimer. This result shows the flexible character of the C(3′)-endo unit. The hairpin-type, loop-promoting conformer with (ω′,ω) = (101°, 59°) was found to be the most favored structure at the 3′-terminus of d(ApApA). The predicted U- and L-type bend conformers were found to lie within 5 kcal/mol, compared to the lowest energy B-DNA structure. The A-DNA and Watson-Crick DNA types of helical conformers also lie within very small energy barriers. The phosphate group at the 5′-end of the nucleotide residue has a definite influence on the base of the corresponding nucleotide, keeping it in the normal anti-region, and hence on the base-stacking property. The results are compared with the presently available experimental data, mainly with the tRNA^Phe crystal.     

DNA Models for A,B, C and D Conformations Related to Fiber X-ray,Infrared and NMR Measurements

Abstract

A conformational analysis of the A, B, C and D DNA forms was made in order to establish molecular models presenting a good agreement with experimental data obtained from fiber X-ray, infrared linear dichroism and ³¹P NMR. The proposed models have been refined and do present good stereochemistry and optimized H-bond distances between bases associated with the Watson-Crick pairing. The DNA conformations proposed are a left handed double helix for the C form and right handed helices for A, B and D. Relations to conformational transitions between these forms are discussed.     

Proton and phosphorus NMR studies of d-CpG(pCpG)n duplexes in solution. Helix-coil transition and complex formation with actinomycin-D.

The Watson–Crick imino and amino exchangeable protons, the nonexchangeable base and sugar protons, and the backbone phosphates for d-CpG(pCpG)_n, n = 1 and 2, have been monitored by high-resolution nmr spectroscopy in aqueous solution over the temperature range 0°–90°C. The temperature dependence of the chemical shifts of the tetramer and hexamer resonances is consistent with the formation of stable duplexes at low temperature in solution. Comparison of the spectral characteristics of the tetranucleotide with those of the hexanucleotide with temperature permits the differentiation and assignment of the cytosine proton resonances on base pairs located at the end of the helix from those in an interior position. There is fraying at the terminal base pairs in the tetranucleotide and hexanucleotide duplexes. The Watson–Crick ring imino protons exchange at a faster rate than the Watson–Crick side-chain amino protons, with exchange occurring by transient opening of the double helix. The structure of the d-CpG(pCpG)_n double helices has been probed by proton relaxation time measurements, sugar proton coupling constants, and the proton chemical shift changes associated with the helix–coil transition. The experimental data support a structural model in solution, which incorporates an anti conformation about the glycosyl bonds, C(3) exo sugar ring pucker, and base overlap geometries similar to the B-DNA helix. Rotational correlation times of 1.7 and 0.9 × 10^?9 sec have been computed for the hexanucleotide and tetranucleotide duplexes in 0.1 M salt, D₂O, pH 6.25 at 27°C. The well-resolved ³¹P resonances for the internucleotide phosphates of the tetramer and hexamer sequences at superconducting fields shift upfield by 0.2–0.5 ppm on helix formation. These shifts reflect a conformational change about the ω,ω′ phosphodiester bonds from gauche-gauche in the duplex structure to a distribution of gauche-trans states in the coil structure. Significant differences are observed in the transition width and midpoint of the chemical shift versus temperature profiles plotted in differentiated form for the various base and sugar proton and internucleotide phosphorous resonances monitoring the d-CpG(pCpG)_n helix–coil transition. The twofold symmetry of the d-CpGpCpG duplex is removed on complex formation with the antibiotic actinomycin-D. Two phosphorous resonances are shifted downfield by ～2.6 ppm and ～1.6 ppm on formation of the 1:2 Act-D:d-CpGpCpG complex in solution. Model studies on binding of the antibiotic to dinucleotides of varying sequence indicate that intercalation of the actinomycin-D occurs at the GpC site in the d-CpGpCpG duplex and that the magnitude of the downfield shifts reflects strain at the O-P-O backbone angles and hydrogen bonding between the phenoxazone and the phosphate oxygens. Actinomycin-D is known to bind to nucleic acids that exhibit a B-DNA conformation; this suggests that the d-CpG(pCpG)_n duplexes exhibit a B-DNA conformation in solution.