Conformational features of the four successive non-Watson-Crick base pairs in RNA duplex.

A tridecaribonucleotide, r(UGAGCUUCGGCUC) doesn't form hairpin or interior loop and forms a double helix of 12 base pairs including the four successive nonstandard base pairs, U.G-U.C-C.U-G.U, in the crystal. Non-Watson-Crick base pairs, G.U and U.C are nicely incorporated in RNA duplex maintaining the regular A-form backbone. There exist the good overlapping between base pairings, U.G and U.C, so as to stabilize the nonstandard base pair track. Hydrogen bond networks involving water molecules in the major and minor grooves to stabilize this mismatch base pairing array, are observed and its conformational features are described.     

X-ray and NMR studies of the DNA oligomer d(ATATAT): Hoogsteen base pairing in duplex DNA

We present and analyze the structure of the oligonucleotide d(ATATAT) found in two different forms by X-ray crystallography and in solution by NMR. We find that in both crystal lattices the oligonucleotide forms an antiparallel double helical duplex in which base pairing is of the Hoogsteen type. The double helix is apparently very similar to the standard B-form of DNA, with about 10 base pairs per turn. However, the adenines in the duplex are flipped over; as a result, the physicochemical features of both grooves of the helix are changed. In particular, the minor groove is narrow and hydrophobic. On the other hand, d(ATATAT) displays a propensity to adopt the B conformation in solution. These results confirm the polymorphism of AT-rich sequences in DNA. Furthermore, we show that extrahelical adenines and thymines can be minor groove binders in Hoogsteen DNA.     

Triple helical DNA in a duplex context and base pair opening

It is fundamental to explore in atomic detail the behavior of DNA triple helices as a means to understand the role they might play in vivo and to better engineer their use in genetic technologies, such as antigene therapy. To this aim we have performed atomistic simulations of a purine-rich antiparallel triple helix stretch of 10 base triplets flanked by canonical Watson–Crick double helices. At the same time we have explored the thermodynamic behavior of a flipping Watson–Crick base pair in the context of the triple and double helix. The third strand can be accommodated in a B-like duplex conformation. Upon binding, the double helix changes shape, and becomes more rigid. The triple-helical region increases its major groove width mainly by oversliding in the negative direction. The resulting conformations are somewhere between the A and B conformations with base pairs remaining almost perpendicular to the helical axis. The neighboring duplex regions maintain a B DNA conformation. Base pair opening in the duplex regions is more probable than in the triplex and binding of the Hoogsteen strand does not influence base pair breathing in the neighboring duplex region.     

Modelling extreme stretching of DNA.

Molecular modelling with Jumna is used to study extreme stretching of the DNA double helix. The results, which correlate well with recent nanomanipulation experiments, show how the double helix can be extended to twice its normal length before its base pairs break. Depending on the way the duplex is stretched two types of conformation can occur, either an unwound flat ribbon or a narrow fibre with negatively inclined base pairs. The energetics of both types of deformation are similar and existing structures show that at least the flat ribbon form can exist locally under biological conditions.     

N(4)C-ethyl-N(4)C cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of different orientations.

The preparation and physical properties of short DNA duplexes that contain a N(4)C-ethyl-N(4)C interstrand cross-link are described. Duplexes that contain an interstrand cross-link between mismatched C-C residues and duplexes in which the C residues of a -CG- or -GC- step are linked to give "staggered" interstrand cross-links were prepared using a novel N(4)C-ethyl-N(4)C phosphoramidite reagent. Duplexes with the C-C mismatch cross-link have UV thermal transition temperatures that are 25 degrees C higher than the melting temperatures of control duplexes in which the cross-link is replaced with a G-C base pair. It appears that this cross-link stabilizes adjacent base pairs and does not perturb the structure of the helix, a conclusion that is supported by the CD spectrum of this duplex and by molecular models. An even higher level of stabilization, 49 degrees C, is seen with the duplex that contains a -CG- staggered cross-link. Molecular models suggest that this cross-link may induce propeller twisting in the cross-linked base pairs, and the CD spectrum of this duplex exhibits an unusual negative band at 298 nm, although the remainder of the spectrum is similar to that of B-form DNA. Mismatched C-C or -CG- staggered cross-linked duplexes that have complementary overhanging ends can undergo self-ligation catalyzed by T4 DNA ligase. Analysis of the ligated oligomers by nondenaturing polyacrylamide gel electrophoresis shows that the resulting oligomers migrate in a manner similar to that of a mixture of non-cross-linked control oligomers and suggests that these cross-links do not result in significant bending of the helix. However, the orientation of the staggered cross-link can have a significant effect on the structure and stability of the cross-linked duplex. Thus, the thermal stability of the duplex that contains a -GC- staggered cross-link is 10 degrees C lower than the melting temperature of the control, non-cross-linked duplex. Unlike the -CG- staggered cross-link, in which the cross-linked base pairs can still maintain hydrogen bond contacts, molecular models suggest that formation of the -GC- staggered cross-link disrupts hydrogen bonding and may also perturb adjacent base pairs leading to an overall reduction in helix stability. Duplexes with specifically positioned and oriented cross-links can be used as substrates to study DNA repair mechanisms.     

Conformational effects of DNA stretching

DNA is an extensible molecule, and an extended conformation of DNA is involved in some biological processes. We have examined the effect of elongation stress on the conformational properties of DNA base pairs by conformational analysis. The calculations show that stretching does significantly affect the conformational properties and flexibilities of base pairs. In particular, we have found that the propeller twist in base pairs reverses its sign upon stretching. The energy profile analysis indicates that electrostatic interactions make a major contribution to the stabilization of the positive-propeller-twist configuration in stretched DNA. This stretching also results in a monotonic decrease in the helical twist angle, tending to unwind the double helix. Fluctuations in most variables initially increase upon stretching, because of unstacking of base pairs, but then the fluctuations decrease as DNA is stretched further, owing to the formation of specific interactions between base pairs induced by the positive propeller twist. Thus, the stretching of DNA has particularly significant effects upon DNA flexibility. These changes in both the conformation and flexibility of base pairs probably have a role in functional interactions with proteins.     

Conformational Effects of DNA Stretching

DNA is an extensible molecule, and an extended conformation of DNA is involved in some biological processes. We have examined the effect of elongation stress on the conformational properties of DNA base pairs by conformational analysis. The calculations show that stretching does significantly affect the conformational properties and flexibilities of base pairs. In particular, we have found that the propeller twist in base pairs reverses its sign upon stretching. The energy profile analysis indicates that electrostatic interactions make a major contribution to the stabilization of the positive-propeller-twist configuration in stretched DNA. This stretching also results in a monotonic decrease in the helical twist angle, tending to unwind the double helix. Fluctuations in most variables initially increase upon stretching, because of unstacking of base pairs, but then the fluctuations decrease as DNA is stretched further, owing to the formation of specific interactions between base pairs induced by the positive propeller twist. Thus, the stretching of DNA has particularly significant effects upon DNA flexibility. These changes in both the conformation and flexibility of base pairs probably have a role in functional interactions with proteins.     

Spatial translational motions of base pairs in DNA molecules: Application of the extended matrix generator method

We have used the elementary generator matrices outlined in the preceding paper to examine the conformational plasticity of the nucleic acid double helix. Here we investigate kinked DNA structures made up of alternating B- and A-type helices and intrinsically curved duplexes perturbed by the intercalation of ligands. We model the B-to-A transition by the lateral translation of adjacent base pairs, and the intercalation of ligands by the vertical displacement of neighboring residues. We report a complete set of average configuration-dependent parameters, ranging from scalars (i.e., persistence lengths) to first- and second-order tensor parameters (i.e., average second moments of inertia), as well as approximations of the associated spatial distributions of the DNA and their angular correlations. The average structures of short chains (of lengths less than 100 base pairs) with local kinks or intrinsically curved sequences are essentially rigid rods. At the smallest chain lengths (10 base pairs), the kinked and curved chains exhibit similar average properties, although they are structurally perturbed compared to the standard B-DNA duplex. In contrast, at lengths of 200 base pairs, the curved and kinked chains are more compact on average and are located in a different space from the standard B- or A-DNA helix. While A-DNA is shorter and thicker than B-DNA in x-ray models, the long flexible A-DNA helix is thinner and more extended on average than its B-DNA counterpart because of more limited fluctuations in local structure. Curved polymers of 50 base pairs or longer also show significantly greater asymmetry than other DNAs (in terms of the distribution of base pairs with respect to the center of gravity of the chain). The intercalation of drugs in the curved DNA straightens and extends the smoothly deformed template. The dimensions of the average ellipsoidal boundaries defining the configurations of the intercalated polymers are roughly double those of the intrinsically curved chain. The altered proportions and orientations of these density functions reflect the changing shape and flexibility of the double helix. The calculations shed new light on the possible structural role of short A-DNA fragments in long B-type duplexes and also offer a model for understanding how GC-specific intercalative ligands can straighten naturally curved DNA. The mechanism is not immediately obvious from current models of DNA curvature, which attribute the bending of the chain to a perturbed structure in repeating tracts of A · T base pairs. © 1994 John Wiley & Sons, Inc.     

Melting of Cross-Linked DNA V. Cross-Linking Effect Caused by Local Stabilization of the Double Helix

Abstract

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this “cross-linking effect” (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the “cross-linking effect” (18–20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20–40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.     

Thermodynamics of DNA duplexes with adjacent G.A mismatches.

The sequence 5'-d(ATGAGCGAAT) forms a very stable self-complementary duplex with four G.A mismatch base pairs (underlined) out of ten total base pairs [Li et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30]. The conformation is in the general B-family and is stabilized by base-pair hydrogen bonding of an unusual type, by favorable base dipole orientations, and by extensive purine-purine stacking at the mismatched sites. We have synthesized 13 decamers with systematic variations in the sequence above to determine how the flanking sequences, the number of G.A mismatches, and the mismatch sequence order (5'-GA-3' or 5'-AG-3') affect the duplex stability. Changing A.T to G.C base pairs in sequences flanking the mismatches stabilizes the duplexes, but only to the extent observed with B-form DNA. The sequence 5'-pyrimidine-GA-purine-3', however, is considerably more stable than 5'-purine-GA-pyrimidine-3'. The most stable sequences with two pairs of adjacent G.A mismatches have thermodynamic parameters for duplex formation that are comparable to those for fully Watson-Crick base-paired duplexes. Similar sequences with single G.A pairs are much less stable than sequences with adjacent G.A mismatches. Reversing the mismatch order from 5'-GA-3' to 5'-AG-3' results in an oligomer that does not form a duplex. These results agree with predictions from the model derived from NMR and molecular mechanics and indicate that the sequence 5'-pyrimidine-GA-purine-3' forms a stable conformational unit that fits quite well into a B-form double helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melting of cross-linked DNA v. cross-linking effect caused by local stabilization of the double helix

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this "cross-linking effect" (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the "cross-linking effect" (18 - 20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20- 40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.     

Poly(amino2dA-dT) isomerizes into the unusual X-DNA double helix at physiological conditions inducing Z-DNA in poly (dG-methyl5dC)

It is demonstrated that a two-state conformational isomerization is induced in the poly(amino2-dA-dT) duplex by submillimolar concentrations of divalent magnesium cations in low-salt aqueous solution. The isomerization is fast and has a low degree of cooperativity. The resulting conformer is the unusual X-DNA double helix originally observed with poly(dA-dT) at very high concentrations of CsF. Interestingly, the X form is induced in poly(amino2dA-dT) under the physiological conditions when poly(dG-methyl5dC) assumes Z-DNA. The same conditions of stabilization are presumably connected with the fact, observed in previous phosphorus NMR studies, that Z- and X-DNA have similar polydinucleotide backbone architectures. Results presented in this work permit to specify base pair exocyclic groups responsible for the radically different conformational variability of the synthetic DNA molecules containing alternating purine-pyrimidine sequences of GC or AT base pairs.     

A parallel stranded linear DNA duplex incorporating dG.dC base pairs

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.     

Poly(amino2 dA-dT) Isomerizes into the Unusual X-DNA Double Helix at Physiological Conditions Inducing Z-DNA in Poly(dG-methyl5dC)

Abstract

It is demonstrated that a two-state conformational isomerization is induced in the poly(amino²-dA-dT) duplex by submillimolar concentrations of divalent magnesium cations in low-salt aqueous solution. The isomerization is fast and has a low degree of cooperativity. The resulting conformer is the unusual X-DNA double helix originally observed with poly(dA-dT) at very high concentrations of CsF. Interestingly, the X form is induced in poly(amino² dA-dT) under the physiological conditions when poly(dG-methyl⁵dC) assumes Z-DNA. The same conditions of stabilization are presumably connected with the fact, observed in previous phosphorus NMR studies, that Z- and X-DNA have similar polydinucleotide backbone architectures. Results presented in this work permit to specify base pair exocyclic groups responsible for the radically different conformational variability of the synthetic DNA molecules containing alternating purine-pyrimidine sequences of GC or AT base pairs.     

Characterization of conformational features of DNA heteroduplexes containing aldehydic abasic sites

The DNA duplexes shown below, with D indicating deoxyribose aldehyde absic sites and numbering from 5' to 3', have been investigated by NMR. The 31P and 31P-1H correlation data indicate [formula: see text] that the backbones of these duplex DNAs are regular. One- and two-dimensional 1H NMR data indicate that the duplexes are right-handed and B-form. Conformational changes due to the presence of the abasic site extend to the two base pairs adjacent to the lesion site with the local conformation of the DNA being dependent on whether the abasic site is in the alpha or beta configuration. The aromatic base of residue A17 in the position opposite the abasic site is predominantly stacked in the helix as is G17 in the analogous sample. Imino lifetimes of the AT base pairs are much longer in samples with an abasic site than in those containing a Watson-Crick base pair. The conformational and dynamical properties of the duplex DNAs containing the naturally occurring aldehyde abasic site are different from those of duplex DNAs containing a variety of analogues of the abasic site.     

Sequence-dependent motions of DNA: a normal mode analysis at the base-pair level

Computer simulation of the dynamic structure of DNA can be carried out at various levels of resolution. Detailed high resolution information about the motions of DNA is typically collected for the atoms in a few turns of double helix. At low resolution, by contrast, the sequence-dependence features of DNA are usually neglected and molecules with thousands of base pairs are treated as ideal elastic rods. The present normal mode analysis of DNA in terms of six base-pair "step" parameters per chain residue addresses the dynamic structure of the double helix at intermediate resolution, i.e., the mesoscopic level of a few hundred base pairs. Sequence-dependent effects are incorporated into the calculations by taking advantage of "knowledge-based" harmonic energy functions deduced from the mean values and dispersion of the base-pair "step" parameters in high-resolution DNA crystal structures. Spatial arrangements sampled along the dominant low frequency modes have end-to-end distances comparable to those of exact polymer models which incorporate all possible chain configurations. The normal mode analysis accounts for the overall bending, i.e., persistence length, of the double helix and shows how known discrepancies in the measured twisting constants of long DNA molecules could originate in the deformability of neighboring base-pair steps. The calculations also reveal how the natural coupling of local conformational variables affects the global motions of DNA. Successful correspondence of the computed stretching modulus with experimental data requires that the DNA base pairs be inclined with respect to the direction of stretching, with chain extension effected by low energy transverse motions that preserve the strong van der Waals' attractions of neighboring base-pair planes. The calculations further show how one can "engineer" the macroscopic properties of DNA in terms of dimer deformability so that polymers which are intrinsically straight in the equilibrium state exhibit the mesoscopic bending anisotropy essential to DNA curvature and loop formation.     

A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs

Abstract

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA · dT base pairs. We have substituted four dA · dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1 · D2) with dG · dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG · dC base pairs (ps-D5 · D6) is 10-16°C lower and the van't Hoff enthalpy difference Δ_vH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl₂) compared to that of ps-Dl · D2. Based on energy minimizations of a ps-[d(T₅GA₅) · d(A₅CT₅)] duplex using force field calculations we propose a model for the conformation of a trans dG · dC base pair in a ps helix.     

Three-dimensional structures of bulge-containing DNA fragments.

The three-dimensional structure of a DNA tridecamer d(CGCAGAATTCGCG)2 containing bulged adenine bases was determined by single crystal X-ray diffraction methods, at 120 K, to 2.6 A resolution. The structure is a B-DNA type double helix with a single duplex in the asymmetric unit. One of the bulged adenine bases loops out from the double helix, while the other stacks in to it. This is in contrast to our preliminary finding, which indicated that both adenine bases were looped out. This revised model was confirmed by the use of a covalently bound heavy-atom derivative. The conformation of the looped-out bulge hardly disrupts base stacking interactions of the bases flanking it. This is achieved by the backbone making a "loop-the-loop" curve with the extra adenine flipping over with respect to the other nucleotides in the strand. The looped-out base intercalates into the stacked-in bulge site of a symmetrically related duplex. The looped-out and stacked-in bases form an A.A reversed Hoogsteen base-pair that stacks between the surrounding base-pairs, thus stabilizing both bulges. The double helix is frayed at one end with the two "melted" bases participating in intermolecular interactions. A related structure, of the same tridecamer, after soaking the crystals with proflavin, was determined to 3.2 A resolution. The main features of this B-DNA duplex are basically similar to the native tridecamer but differ in detail especially in the conformation of the bulged-out base. Accommodation of a large perturbation such as that described here with minimal disruption of the double helix shows both the flexibility and resiliency of the DNA molecule.     

Conformation and dynamics of drug-DNA intercalation.

Molecular dynamics simulations have been undertaken for a B-form dodecanucleotide duplex in solution with and without an intercalated proflavine molecule between the central C.G base pairs. The introduction of this simple intercalator affects both the conformational features and dynamic properties of the oligonucleotide double helix. Changes are seen in the rms atomic fluctuations and anisotropy of phosphate, sugar and base atoms. The backbone conformation is slightly changed on average and more sugars adopt the C3' endo conformation in the simulation of the complex compared with the simulation of the oligonucleotide alone. Both major and minor grooves becomes wider on average with the addition of the intercalating drug. Flanking A.T base pairs on both sides of the intercalation site have undergone an increase in flexibility, with the base pairs, especially at the 5' side, having the N1...N3 hydrogen bonds being broken.