Novel Hoogsteen-like Bases for Recognition of the C-G Base Pair by DNA Triplex Formation

Effective sequence-specific recognition of duplex DNA is possible by triplex formation with natural oligonucleotides via Hoogsteen H-bonding. However, triplex formation is in practice limited to pyrimidine oligonucleotides that bind duplex A-T or G-C base pair DNA sequences specifically at homopurine sites in the major groove as T·A-T and C⁺ ·G-C triplets. Here we report the successful modelling of novel unnatural nucleosides that recognize the C-G DNA base pair by Hoogsteen-like major groove interaction. These novel Hoogsteen nucleotides are examined within model A-type and B-type conformation triplex structures since the DNA triplex can be considered to incorporate A-type and/or B-type configurational properties. Using the same deoxyribose-phosphodiester and base-deoxyribose dihedral angle configuration, a triplet comprised of a C-G base pair and the novel Hoogsteen nucleotide, Y2, replaces the central T·A-T triplet in the triplex. The presence of any structural or energetic perturbations due to the central triplet in the energy-minimized triplex is assessed with respect to the unmodified energy minimized (T·A-T)₁₁ starting structures. Incorporation of this novel triplet into both A-type and B-type natural triplex structures provokes minimal change in the configuration of the central and adjacent triplets.     

Proton nuclear magnetic resonance assignments and structural characterization of an intramolecular DNA triplex.

Two-dimensional 1H n.m.r. spectroscopy has been used to study the 31-base DNA oligonucleotide 5'-dAGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3', which folds to form a stable intramolecular triplex in solution at acidic pH. This structure is considerably more difficult to assign than short B-DNA duplexes and requires new assignment methods. The assignment strategy and assignments of almost all of the exchangeable and nonexchangeable resonances are presented. Seven base triplets and one Watson-Crick base-pair form the core of the structure and are connected by a four C and four T loop at either end. The second pyrimidine "strand" (bases 24 to 31) in this intramolecular pyrimidine-purine-pyrimidine triplex binds via Hoogsteen base-pairs in the major groove and is parallel to the purine "strand" (bases 1 to 8). Analysis of the sugar puckers reveals that, contrary to widely accepted belief, the triplex sugars are not predominantly in the N-type (close to C3'-endo) conformation. Except for some of the C nucleotides, all sugars are predominantly S-type (close to C2'-endo). Thus, the duplex DNA does not assume N-type sugar conformations to accommodate a third strand in the major groove. A preliminary model of the triplex structure is presented.     

A-tract DNA disfavours triplex formation.

Optimisation of DNA triplex stability is of fundamental importance in the anti-gene strategy. In the present work, thermal denaturation studies by UV-spectrophotometry and structural and dynamical characterizations by NMR spectroscopy have been used systematically to investigate the effects on triplex stability of isolated insertions of different base triplets into an otherwise homogeneous 15-mer dT x dA-dT oligo-triplex. It is found that insertion of a single central C(+) x G-C or T x D-T triplet (D=2,6-diaminopurine) leads to a pronounced stabilization (up to 20 deg. C if the cytosine base is C5 methylated) at acidic as well as neutral pH. To a smaller degree, this is the case also for a C(+) x I-C triplet insertion.Using imino proton exchange measurements, it is shown that insertion of a DT base-pair in the underlying duplex perturbs the intrinsic A-tract structure in the same way as has been shown for a GC insert. We propose that the intrinsic properties of A-tract duplex DNA (e. g. high propeller twist and rigidity) are unfavourable for triplex formation and that GC- or DT-inserts stabilize the triplex by interfering with the A-tract features of the underlying duplex. The C(+) x I-C triplet without the N2 amino group in the minor groove is readily accommodated within the typical, highly propeller-twisted A-tract structure. This might be related to its smaller effect on the stability of the corresponding triplex.These results may be valuable for understanding DNA triplex formation in vivo as well as for the design of efficient triplex-forming oligonucleotides and in choosing suitable target sequences in the anti-gene strategy.     

New insights into DNA triplexes: residual twist and radial difference as measures of base triplet non-isomorphism and their implication to sequence-dependent non-uniform DNA triplex

DNA triplexes are formed by both isomorphic (structurally alike) and non-isomorphic (structurally dissimilar) base triplets. It is espoused here that (i) the base triplet non-isomorphism may be articulated in structural terms by a residual twist (Δt°), the angle formed by line joining the C1′…C1′ atoms of the adjacent Hoogsteen or reverse Hoogsteen (RH) base pairs and the difference in base triplet radius (Δr Å), and (ii) their influence on DNA triplex is largely mechanistic, leading to the prediction of a high (t + Δt)° and low (t − Δt)° twist at the successive steps of Hoogsteen or RH duplex of a parallel or antiparallel triplex. Efficacy of this concept is corroborated by molecular dynamics (MD) simulation of an antiparallel DNA triplex comprising alternating non-isomorphic G*GC and T*AT triplets. Conformational changes necessitated by base triplet non-isomorphism are found to induce an alternating (i) high anti and anti glycosyl and (ii) BII and an unusual BIII conformation resulting in a zigzag backbone for the RH strand. Thus, base triplet non-isomorphism causes DNA triplexes into exhibiting sequence-dependent non-uniform conformation. Such structural variations may be relevant in deciphering the specificity of interaction with DNA triplex binding proteins. Seemingly then, residual twist (Δt°) and radial difference (Δr Å) suffice as indices to define and monitor the effect of base triplet non-isomorphism in nucleic acid triplexes.     

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).     

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-).     

Triplex formation by oligonucleotides containing novel deoxycytidine derivatives.

Homopurine sequences of duplex DNA are binding sites for triplex-forming oligodeoxyribopyrimidines. The interactions of synthetic duplex DNA targets with an oligodeoxyribopyrimidine containing N4-(6-amino-2-pyridinyl)deoxycytidine (1), a nucleoside designed to interact with a single C-G base pair interruption of the purine target tract, was studied by UV melting, circular dichroism spectroscopy and dimethylsulfate alkylation experiments. Nucleoside 1 supports stable triplex formation at pH 7.0 with formation of a 1-Y-Z triad, where Y-Z is a base pair in the homopurine tract of the target. Selective interaction was observed when Y-Z was C-G, although A-T and, to a lesser extent, T-A and G-C base pairs were also recognized. The circular dichroism spectra of the triplex having a 1-C-G triad were similar to those of a triplex having a C(+)-G-C triad, suggesting that the overall structures of the two triplexes are quite similar. Removal of the 6-amino group from 1 essentially eliminated triplex formation. Reaction of a triplex having the 1-C-G triad with dimethylsulfate resulted in a 50% reduction of methylation of the G residue of this triad. In contrast, the G of a similar triplex containing a U-C-G triad was not protected from methylation by dimethylsulfate. These results are consistent with a binding mode in which the 6-amino-2-pyridinyl group of 1 spans the major groove of the target duplex at the 1-C-G binding site and forms a hydrogen bond with the O6 of G. An additional stabilizing hydrogen bond could form between the N4 of the imino tautomer of 1 and the N4 amino group of C.     

Solution structure of a dsDNA:LNA triplex

We have determined the NMR structure of an intramolecular dsDNA:LNA triplex, where the LNA strand is composed of alternating LNA and DNA nucleotides. The LNA oligonucleotide binds to the dsDNA duplex in the major groove by formation of Hoogsteen hydrogen bonds to the purine strand of the duplex. The structure of the dsDNA duplex is changed to accommodate the LNA strand, and it adopts a geometry intermediate between A- and B-type. There is a substantial propeller twist between base-paired nucleobases. This propeller twist and a concomitant large propeller twist between the purine and LNA strands allows the pyrimidines of the LNA strand to interact with the 5′-flanking duplex pyrimidines. Altogether, the triplex has a regular global geometry as shown by a straight helix axis. This shows that even though the third strand is composed of alternating DNA and LNA monomers with different sugar puckers, it forms a seamless triplex. The thermostability of the triplex is increased by 19°C relative to the unmodified DNA triplex at acidic pH. Using NMR spectroscopy, we show that the dsDNA:LNA triplex is stable at pH 8, and that the triplex structure is identical to the structure determined at pH 5.1.     

pH and cation effects on the properties of parallel pyrimidine motif DNA triplexes

The effects of cytosine protonation and various cations on the properties of parallel pyrimidine motif DNA triplexes were intensively investigated and characterized by several different techniques, such as circular dichroism (CD) conformation, ultraviolet (UV) melting, differential scanning calorimetry (DSC) thermal denaturation, and surface plasmon resonance (SPR) real-time dynamics. The comparative CD spectra of the triplex and the corresponding homoduplexes showed that the negative peak at approximately 218 nm would be the eigenpeak of the Hoogsteen paired strand, and moreover, the formation pathway of a triplex was significantly pH-dependent and fell into three groups: under acidic conditions, the triplex is formed by a one-step docking, under near physiological conditions, the Watson-Crick duplex is first structured and then accepts the Hoogsteen third strand into its major groove, and under basic conditions, the triplex is not formed. The pH-dependent thermodynamics of the global triplex, the Watson-Crick antiparallel duplex, and the Crick-Hoogsteen parallel duplex were comparatively discussed for the first time. These data revealed that the thermodynamic stabilities of the Watson-Crick-Hoogsteen triplex and the Crick-Hoogsteen duplex would be strongly dependent on cytosine protonation, but a low-pH environment somewhat destabilized the Watson-Crick duplex. The binding energy of triplex formation would be different from the unfolding energy of triplex melting under acidic conditions due to the disparity in the pathway between the formation and unfolding of a triplex. Real-time dynamic measurements showed that the association and dissociation rate constants of a duplex-to-triplex formation are (1.98 +/- 0.24) x 10(3) M(-1) s(-1) and (4.09 +/- 0.96) x 10(-4) s(-1) at 20 degrees C and pH 6.0, respectively. The formation energy of the duplex-to-triplex transition derived from SPR measurements was in agreement with the unfolding energy of the free Hoogsteen paired duplex derived from UV measurements. The calorimetric enthalpies of the triplex-to-duplex-to-single transition were 39.3 and 75.3 kcal/mol under near physiological conditions (pH 7.0), respectively, which were underestimated relative to the van't Hoff enthalpies. In addition, the effects of various cations, ionic strength, mixed-valent cations, and the position of the C(+)xG.C triplets on the thermodynamics of the triplexes were addressed under near physiological conditions. The interaction of metal ions with the triplexes clearly depended on the type and ionic strength of the cations, and the efficiency with which the cations stabilized the global triplex was in the order Mg(2+) > Mn(2+) > Ca(2+) > Ba(2+) > Na(+). These observations would be useful for the design of triplex-forming oligonucleotides for antigene drugs and therapeutic purposes.     

Molecular Dynamics Simulation Study of DNA Triplex Formed by Mixed Sequences in Solution

Abstract

The unrestrained molecular dynamics simulation of the triple helical DNA with mix sequences d(GACTGGTGAC)?d(CTGACCACTG)*d (GACTGGTGAC), using the particle mesh Ewald sum, is presented here. The Ewald summation method effectively eliminates the usual “cut-off” of the long—range interactions and allowed us to evaluate the full effect of the electrostatic forces. The AMBER5.0 force field has been used during the simulation in solvent. The MD results support a dynamically stable model of DNA triplex over the entire length of the trajectory. The duplex structure assumes the conformation, which is very close to B-DNA. In mixed sequences the purine bases occurs in both strand of DNA duplex. The bases of third strand do not favor the Hoogsteen or/and reverse Hoogsteen type of Hydrogen bonding but they form hydrogen bonds with the bases of both the strand of DNA duplex. The orientation of the third strand is parallel to one of the strand of duplex and all nucleotides (C, A, G & T) show isomorphic behavior with respect to the DNA duplex. The conformation of all the three strands is almost same except few exceptions. Due to interaction of third strand the conformational change in the duplex structure and a finite amount of displacement in the W-C base pairs have been observed. The conformational variation of the back bone torsion angles and helicoidal parameters, groove widths have been discussed. The sequence—dependent effects on local conformation, helicoidal and morphological structure, width of the grooves of DNA helix may have important implication for understanding the functional energetics and specificity of interactions of DNA and its triplexes with proteins, pharmaceutical agents and other legends.     

Intrinsic bending and deformability at the T-A step of CCTTTAAAGG: a comparative analysis of T-A and A-T steps within A-tracts

Introduction of a T-A or pyrimidine-purine step into a straight and rigid A-tract can cause a positive roll deformation that kinks the DNA helix at that step. In CCTTTAAAGG, the central T-A step has an 8.6 degrees bend toward the major groove. We report the structural analysis of CCTTTAAAGG and a comparison with 25 other representative crystal structures from the NDB containing at least four consecutive A or T bases. On average, more local bending occurs at the disruptive T-A step (8.21 degrees ) than at an A-T step (5.71 degrees ). In addition, A-tracts containing an A-T step are more bent than are pure A-tracts, and hence A-A and A-T steps are not equivalent. All T-A steps examined exhibit positive roll, bending towards the major groove, while A-T steps display negative roll and bend slightly towards the minor groove. This illustrates how inherent negative and positive roll are, respectively, at A-T and T-A steps within A-tracts. T-A steps are more deformable, showing larger and more variable deformations of minor groove width, rise, cup, twist, and buckle. Standard deviations of twist, rise, and cup for T-A steps are 6.66 degrees, 0.55 A, and 15.90 degrees, versus 2.28 degrees, 0.21 A, and 2.99 degrees for A-T steps. Packing constraints determine which local values of these helical parameters an individual T-A step will adopt. For instance, with CCTTTAAAGG and three isomorphous structures, CGATTAATCG, CGATATATCG, and CGATCGATCG, crystal packing forces lead to a series of correlated changes: widened minor groove, large slide, low twist, and large rise. The difference in helical parameters between A-T steps lying within A-tracts, versus A-T steps within alternating AT sequences, demonstrates the importance of neighboring steps on the conformation of a given dinucleotide step.     

Molecular dynamics simulation study of DNA triplex formed by mixed sequences in solution

The unrestrained molecular dynamics simulation of the triple helical DNA with mix sequences d(GACTGGTGAC).d(CTGACCACTG)*d (GACTGGTGAC), using the particle mesh Ewald sum, is presented here. The Ewald summation method effectively eliminates the usualcut-of of the long range interactions and allowed us to evaluate the full effect of the electrostatic forces. The AMBER5.0 force field has been used during the simulation in solvent. The MD results support a dynamically stable model of DNA triplex over the entire length of the trajectory. The duplex structure assumes the conformation, which is very close to B-DNA. In mixed sequences the purine bases occurs in both strand of DNA duplex. The bases of third strand do not favor the Hoogsteen or/and reverse Hoogsteen type of Hydrogen bonding but they form hydrogen bonds with the bases of both the strand of DNA duplex. The orientation of the third strand is parallel to one of the strand of duplex and all nucleotides (C, A, G & T) show isomorphic behavior with respect to the DNA duplex. The conformation of all the three strands is almost same except few exceptions. Due to interaction of third strand the conformational change in the duplex structure and a finite amount of displacement in the W-C base pairs have been observed. The conformational variation of the back bone torsion angles and helicoidal parameters, groove widths have been discussed. The sequence dependent effects on local conformation, helicoidal and morphological structure, width of the grooves of DNA helix may have important implication for understanding the functional energetics and specificity of interactions of DNA and its triplexes with proteins, pharmaceutical agents and other ligands.     

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.     

Theoretical study of the Hoogsteen-Watson-Crick junctions in DNA

A series of d (AT)(n) oligonucleotides containing mixtures of normal B-type Watson-Crick and antiparallel Hoogsteen helices have been studied using molecular dynamics simulation techniques to analyze the structural and thermodynamic impact of the junction between Watson-Crick and antiparallel Hoogsteen structures. Analysis of molecular dynamics simulations strongly suggests that for all oligonucleotides studied the antiparallel Hoogsteen appears as a reasonable conformation, only slightly less stable than the canonical B-type Watson-Crick one. The junctions between the Watson-Crick and Hoogsteen structures introduces a priori a sharp discontinuity in the helix, because the properties of each type of conformation are very well preserved in the corresponding fragments. However, and quite counterintuitively, junctions do not largely distort the duplex in structural, dynamics or energetic terms. Our results strongly support the possibility that small fragments of antiparallel Hoogsteen duplex might be embedded into large fragments of B-type Watson-Crick helices, making possible protein-DNA interactions that are specific of the antiparallel Hoogsteen conformation.     

Strong, specific, monodentate G-C base pair recognition by N7-inosine derivatives in the pyrimidine.purine-pyrimidine triple-helical binding motif.

The nucleoside analogs 7-(2'-deoxy-alpha-D-ribofuranosyl)hypoxanthine (alpha7H,1), 7-(2'-deoxy-beta-D-ribofuranosyl)hypoxanthine (beta7H,2) and 7-7-(2'-O-methyl-beta-D- ribofuranosyl)hypoxanthine (beta7HOMe,3) were prepared and incorporated into triplex forming oligodeoxynucleotides, designed to bind to DNA in the parallel (pyrimidine.purine-pyrimidine) motif. By DNase I footprinting techniques and UV-melting curve analysis it was found that, at pH 7. 0, the 15mer oligonucleotides d(TTTTTMeCTXTMeCTMeCTMeCT) (MeC = 5-methyl-deoxycytidine, X =beta7H,beta7HOMe) bind to a DNA target duplex forming a H.G-C base triple with equal to slightly increased (10-fold) stability compared to a control oligodeoxynucleotide in which the hypoxanthine residue is replaced by MeC. Remarkably, triple-helix formation is specific to G-C base pairs and up to 40 microM third strand concentration, no stable triplex exhibiting H.A-T, H.T-A or H.C-G base arrangements could be found (target duplex concentration approximately 0.1 nM). Multiply substituted sequences containing beta7H residues either in an isolated [d(TTTTTbeta7HTbeta7HTbeta7HTbeta7HTbeta7HT)] or in a contiguous [d(TTTbeta7Hbeta7Hbeta7Hbeta7HTTTTbeta7HTTT)] manner still form triplexes with their targets of comparable stability as the control (MeC-containing) sequences at pH 7.0 and high salt or spermine containing buffers. General considerations lead to a structural model in which the recognition of the G-C base pair by hypoxanthine takes place via only one H-bond of the N-H of hypoxanthine to N7 of guanine. This model is supported by a molecular dynamics simulation. A general comparison of the triplex forming properties of oligonucleotides containing beta7H with those containing MeC or N7-2'-deoxyguanosine (N7G) reveals that monodentate recognition in the former case can energetically compete with bidentate recognition in the latter two cases.     

Single strand targeted triplex formation: targeting purine-pyrimidine mixed sequences using abasic linkers.

Foldback triplex-forming oligonucleotides (FTFOs) that contain an abasic linker, [2-(4-aminobutyr-1-yl)-1,3-propanediol] (APD linker), in the Hoogsteen domain against pyrimidine bases of a C:G and a T:A base pair were studied for their relative stability and sequence specificity of triplex formation. In general, the APD linker has less destabilizing effect against a C:G base pair than a T:A base pair. Incorporation of three APD linker moieties resulted in decreased binding to the target, which was comparable to results observed with three imperfectly matched natural base triplets. The APD linker incorporation did not result in the loss of sequence specificity of FTFOs, unlike in the case of normal triplex-forming oligonucleotides (TFOs). The introduction of a positively charged abasic linker, however, resulted in decreased stability of the triplex, because of loss of hydrogen bonding and stacking interactions in the major groove. The results of a molecular modeling study show that APD linker can be readily incorporated without any change in the conformation of the natural sugar-phosphate backbone conserving overall triple helix geometry. Further, the modeling study suggests a hydrogen bond formation between the amino group of linker and N4 of cytosine mediated by a solvent molecule (water) in the floor of the base triplet in addition to a contribution from the positive charge on the APD linker amino group. Either a direct or water-mediated hydrogen bond between the amino group of the APD linker and the O4 of thymine is unlikely when the linker is placed against a T:A base pair.     

Atomic structures of excited state A–T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion,mutagenesis, and chemical shift calculations

NMR relaxation dispersion studies indicate that in canonical duplex DNA, Watson–Crick base pairs (bps) exist in dynamic equilibrium with short-lived low abundance excited state Hoogsteen bps. N1-methylated adenine (m¹A) and guanine (m¹G) are naturally occurring forms of damage that stabilize Hoogsteen bps in duplex DNA. NMR dynamic ensembles of DNA duplexes with m¹A–T Hoogsteen bps reveal significant changes in sugar pucker and backbone angles in and around the Hoogsteen bp, as well as kinking of the duplex towards the major groove. Whether these structural changes also occur upon forming excited state Hoogsteen bps in unmodified duplexes remains to be established because prior relaxation dispersion probes provided limited information regarding the sugar-backbone conformation. Here, we demonstrate measurements of C3′ and C4′ spin relaxation in the rotating frame (R1ρ) in uniformly ¹³C/¹⁵N labeled DNA as sensitive probes of the sugar-backbone conformation in DNA excited states. The chemical shifts, combined with structure-based predictions using an automated fragmentation quantum mechanics/molecular mechanics method, show that the dynamic ensemble of DNA duplexes containing m¹A–T Hoogsteen bps accurately model the excited state Hoogsteen conformation in two different sequence contexts. Formation of excited state A–T Hoogsteen bps is accompanied by changes in sugar-backbone conformation that allow the flipped syn adenine to form hydrogen-bonds with its partner thymine and this in turn results in overall kinking of the DNA toward the major groove. Results support the assignment of Hoogsteen bps as the excited state observed in canonical duplex DNA, provide an atomic view of DNA dynamics linked to formation of Hoogsteen bps, and lay the groundwork for a potentially general strategy for solving structures of nucleic acid excited states.     

Proton NMR and Optical Spectroscopic Studies on the DNA Triplex Formed by d-A-(G-A)7-G and d-C-(T-C)7-T

Abstract

Triplex and duplex formation of two deoxyribohexadecamers d-A-(G-A)₇-G (a) and d-C-(T-C)₇-T (b) have been studied by UV, CD, fluorescence, and proton NMR spectroscopy. Optical studies of a and b at dilute concentrations (μM range) yielded results similar to those seen for polymers of the same sequence, indicating that these hexadecamers have properties similar to the polymers in regard to triplex formation. The CD spectra of concentrated NMR samples (mM range) are similar to those observed at optical concentrations at both low and high pH, making possible a correlation between CD and NMR studies. In NMR spectra, two imido NH-N hydrogen bonded resonance envelopes at 12.6 and 13.7 ppm indicate that only the duplex conformation is present at pH > 7.7. Four new NH-N hydrogen-bonded resonance envelopes at 12.7, 13.5, 14.2. and 14.9 ppm are observed under acidic conditions (pH 5.6) and the two original NH-N resonances gradually disappear as the pH is lowered. Assignment of these four peaks to Watson-Crick G · C, Hoogsteen T · A. Watson-Crick A · T. and Hoogsteen C⁺ · G hydrogen-bonded imidos, respectively, confirm the formation of triple-stranded DNA NMR results also show that triplex is more stable than duplex at the same salt condition and that triplex melts to single strands directly without going through a duplex intermediate. However, in the melting studies, a structural change within the triple-stranded complex is evident at temperatures significantly below the major helix-to-coil transition. These studies demonstrate the feasibility of using NMR spectroscopy and oligonucleotide model compounds a and b for the study of DNA triplex formation.     

Circular dichroism of adenine and thymine containing synthetic polynucleotides

Circular dichroism (CD) spectra of poly(dA), poly(dT), poly(dA)·poly(dT), and poly[d(A-T)]·poly[d(T-A)] have been measured as a function of temperature. From these data difference spectra have been calculated by subtracting the spectrum measured at low temperature from the spectra measured at higher temperatures. The CD difference spectra obtained upon melting of the two double-stranded polymers are very similar. From a comparison of these difference spectra with calculated ones it is shown that optical transitions near 272 nm (on A) and 288 nm (most probably on T) are present. The premelting changes of the CD spectrum of poly[d(A-t)]·poly[d(T-A)] are due to a change in conformation in which the secondary structure goes from a C- to B-type spectrum by increasing the A-type nature of the polymer. Such a change is not observed for poly(dA)·poly(dT). Instead, a transition between two different B-type geometries occurs.