Minor Groove Width Research Articles

High-resolution NMR structure of an AT-rich DNA sequence.

We have determined, by proton NMR and complete relaxation matrix methods, the high-resolution structure of a DNA oligonucleotide in solution with nine contiguous AT base pairs. The stretch of AT pairs, TAATTATAA x TTATAATTA, is imbedded in a 27-nucleotide stem-and-loop construct, which is stabilized by terminal GC base pairs and an extraordinarily stable DNA loop GAA (Hirao et al., 1994, Nucleic Acids Res. 22, 576-582). The AT-rich sequence has three repeated TAA x TTA motifs, one in the reverse orientation. Comparison of the local conformations of the three motifs shows that the sequence context has a minor effect here: atomic RMSD between the three TAA x TTA fragments is 0.4-0.5 A, while each fragment is defined within the RMSD of 0.3-0.4 A. The AT-rich stem also contains a consensus sequence for the Pribnow box, TATAAT. The TpA, ApT, and TpT x ApA steps have characteristic local conformations, a combination of which determines a unique sequence-dependent pattern of minor groove width variation. All three TpA steps are locally bent in the direction compressing the major groove of DNA. These bends, however, compensate each other, because of their relative position in the sequence, so that the overall helical axis is essentially straight.

Structure of hybrid backbone methylphosphonate DNA heteroduplexes: effect of R and S stereochemistry.

Methyl phosphonate oligonucleotides have been used as antisense and antigene agents. Substitution of a methyl group for oxygen in the phosphate ester backbone introduces a new chiral center. Significant differences in physical properties and hybridization abilities are observed between the R(p) and S(p) diastereomers. Chirally pure methylphosphonate deoxyribooligonucleotides were synthesized, and the solution structures of duplexes formed between a single strand heptanucleotide methylphosphonate, d(Cp(Me)Cp(Me)Ap(Me)Ap(Me)Ap(Me)Cp(Me)A), hybridized to a complementary octanucleotide, d(TpGpTpTpTpGpGpC), were studied by NMR spectroscopy. Stereochemistry at the methylphosphonate center for the heptanucleotide was either RpRpRpRpRpRp (R(p) stereoisomer) or RpRpRpSpRpRp (S(p) stereoisomer, although only one of the six methylphosphonate centers has the S(p) stereochemistry). The results show that the methylphosphonate strands in the heteroduplexes exhibit increased dynamics relative to the DNA strand. Substitution of one chiral center from R(p) to S(p) has a profound effect on the hybridization ability of the methylphosphonate strand. Sugars in the phosphodiester strand exhibit C(2)(') endo sugar puckering while the sugars in the methyl phosphonate strand exhibit an intermediate C(4)(') endo puckering. Bases are well stacked on each other throughout the duplex. The hybridization of the methylphosphonate strand does not perturb the structure of the complementary DNA strand in the hetero duplexes. The sugar residue 5' to the S(p) chiral center shows A-form sugar puckering, with a C(3)(')-endo conformation. Minor groove width in the R(p) stereoisomer is considerably wider, particularly at the R(p) vs S(p) site and is attributed to either steric interactions across the minor groove or poorer metal ion coordination within the minor groove.

Quadruple intercalated G-6 stack: a possible motif in the fold-back structure of the Drosophila centromeric dodeca-satellite?

The purine-rich strand d(GTAC GGGACCGA) n of the Drosophila centromeric dodeca-satellite sequence is highly conserved and was found to form stable fold-back structures in which the homopurine 5′-GGGA-3′ sequence was determined to play a crucial role. Here, we report the stable formation of the d(GGGA) 2 motif in the stem of a DNA hairpin closed by a single-residue d(ACC) loop. Similar to the zipper-like d(GGA) 2 motif observed in the human centromeric (TGGAA) n sequence, the central four guanosine bases in the d(G GGA) 2 motif do not pair, but interdigitate to form an elongated zipper-like quadruple-intercalated G-6 stack bracketed by sheared G·A base-pairs. Comparison between the current d(GGGA) 2 structure and the published crystal d(GAAA) 2 structure implies that the alignment of the unpaired purine bases plays an important role in determining the minor groove width of the purine-rich d(GPuPuA) 2 motif. Similarity between the zipper-like motifs possibly present in the Drosophila centromeric dodeca-satellite sequence and in the human centromeric (TGGAA) n sequence led us to propose that these special zipper-like motifs may constitute common cores in organizing eukaryotic centromeres.

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° 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°) than at an A-T step (5.71°). 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°, 0.55 Å, and 15.90°, versus 2.28°, 0.21 Å, and 2.99° 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.

Influence of the dynamic positions of cations on the structure of the DNA minor groove: sequence-dependent effects.

Different models for minor groove structures predict that the conformation is essentially fixed by sequence and has an influence on local ion distribution or alternatively that temporal positions of ions around the minor groove can affect the structure if they neutralize cross-strand phosphate charges. Our previous studies show that the minor groove in an AATT dodecamer responds to local sodium ion positions and is narrow when ions neutralize cross-strand phosphate-phosphate charges [J. Am. Chem. Soc. 2000, 122, 10513-10520]. Previous results from a number of laboratories have shown that G-tracts often have a wider minor groove than A-tracts, but they do not indicate whether this is due to reduced flexibility or differences in ion interactions. We have undertaken a molecular dynamics study of a d(TATAGGCCTATA) duplex to answer this question. The results show that the G-tract has the same amplitude of minor groove fluctuations as the A-tract sequence but that it has fewer ion interactions that neutralize cross-strand phosphate charges. These results demonstrate that differences in time-average groove width between A- and G-tracts are due to differences in ion interactions at the minor groove. When ions neutralize the cross-strand phosphates, the minor groove is narrow. When there are no neutralizing ion interactions, the minor groove is wide. The population of structures with no ion interactions is larger with the GGCC than with the AATT duplex, and GGCC has a wider time-average minor groove in agreement with experiment.

NMR solution structure of an oligonucleotide hairpin with a 2'F-ANA/RNA stem: implications for RNase H specificity toward DNA/RNA hybrid duplexes.

The first structure of a 2'-deoxy-2'-fluoro-D-arabinose nucleic acid (2'F-ANA)/RNA duplex is presented. We report the structural characterization by NMR spectroscopy of a small hybrid hairpin, r(GGAC)d(TTCG)2'F-a(GTCC), containing a 2'F-ANA/RNA stem and a four-residue DNA loop. Complete (1)H, (13)C, (19)F, and (31)P resonance assignments, scalar coupling constants, and NOE constraints were obtained from homonuclear and heteronuclear 2D spectra. In the chimeric duplex, the RNA strand adopts a classic A-form structure having C3' endo sugar puckers. The 2'F-ANA strand is neither A-form nor B-form and contains O4' endo sugar puckers. This contrasts strongly with the dynamic sugar conformations previously observed in the DNA strands of DNA/RNA hybrid duplexes. Structural parameters for the duplex, such as minor groove width, x-displacement, and inclination, were intermediate between those of A-form and B-form duplexes and similar to those of DNA/RNA duplexes. These results rationalize the enhanced stability of 2'F-ANA/RNA duplexes and their ability to elicit RNase H activity. The results are relevant for the design of new antisense drugs based on sugar-modified nucleic acids.

Solution conformation of an RNA–DNA hybrid duplex containing a pyrimidine RNA strand and a purine DNA strand

RNA–DNA hybrid duplexes are involved in transcription, replication and reverse transcription of nucleic acids. Information on such duplexes may shed some light on the mechanism of these processes. For this purpose, the influence of base composition on the structure of a polypyrimidine–polypurine RNA–DNA duplex r(cucuccuucucuu). d(GAGAGGAAGAGAA) has been studied using 1H, 31P and 13C NMR experiments, molecular modeling (JUMNA program) and NOE back-calculation methods. The resulting structure of the 13-mer hybrid duplex shows that the RNA strand is in the expected A-type conformation while the DNA strand is in a very flexible conformation. In the DNA strand, the desoxyribose sugars retain the C2′-endo B-type conformation. The duplex helical parameters (such as inclination, twist and displacement of the bases) are close to the A-type conformation. No bending was observed for the global axis curvature. The major groove width is close to the B-form value and the minor groove width is intermediate between standard values for A and B-forms. These results are in favour of the independence of minor groove size (where RNase H interacts) and the base composition of the hybrid duplexes.

Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA.

We have determined the 3.0 A resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The 'RNase H primer grip', consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual 'unzipping' of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

Solution structure of an A-tract DNA bend

The solution structure of a DNA dodecamer d(GGCAAAAAACGG)/d(CCGTTTTTTGCC) containing an A-tract has been determined by NMR spectroscopy with residual dipolar couplings. The structure shows an overall helix axis bend of 19° in a geometry consistent with solution and gel electrophoresis experiments. Fourteen degrees of the bending occurs in the GC regions flanking the A-tract. The remaining 5° is spread evenly over its six AT base-pairs. The A-tract is characterized by decreasing minor groove width from the 5′ to the 3′ direction along the A strand. This is a result of propeller twist in the AT pairs and the increasing negative inclination of the adenine bases at the 3′ side of the run of adenine bases. The four central thymine bases all have negative inclination throughout the A-tract with an average value of −6.1°. Although this negative inclination makes the geometry of the A-tract different from all X-ray structures, the proton on N6 of adenine and the O4 of thymine one step down the helix are within distance to form bifurcated hydrogen bonds. The 5′ bend of 4° occurs at the junction between the GC flank and the A-tract through a combination of tilt and roll. The larger 3′ bend, 10°, occurs in two base steps: the first composed of tilt, −4.1°, and the second a combination of tilt, −4.2°, and roll, 6.0°. This second step is a direct consequence of the change in inclination between an adjacent cytosine base, which has an inclination of −12°, and the next base, a guanine, which has 3° inclination. This bend is a combination of tilt and roll. The large change in inclination allows the formation of a hydrogen bond between the protons of N4 of the 3′ cytosine and the O6 of the next 3′ base, a guanine, stabilizing the roll component in the bend. These structural features differ from existing models for A-tract bends. For comparison, we also determined the structure of the control sequence, d(GGCAAGAAACGG)/d(CCGTTTCTTGCC), with an AT to GC transition in the center of the A-tract. This structure has no negative inclination in most of the bases within the A-tract, resulting in a bend of only 9°. When ligated in phase, the control sequence has nearly normal mobility in gel electrophoresis experiments.

Direct-methods determination of an RNA/DNA hybrid decamer at 1.15 A resolution.

For the first time, ab initio direct methods have been used to solve the crystal structure of an RNA/DNA hybrid decamer. The RNA/DNA sequence corresponds to the leftmost two-thirds of the polypurine tract (PPT), the primer for second-strand DNA synthesis by HIV-1 reverse transcriptase (RT). Direct methods using Shake-and-Bake (SnB) yielded solutions for the RNA/DNA decamer molecule using 1.15 A data, which is just on the resolution edge of what might work with direct methods. Atomic positions for 96% of the entire molecule, containing 514 non-H atoms including three Ca(2+) ions, were easily interpreted from a Fourier map based on the 'Shake-and-Bake' minimal function and CROQUE phase-refinement program. Only six atoms, primarily in the sugar linkage, were missing in this Fourier map. At present, the R factor of the model is 0.143 (R(free) = 0.186) for the 562 non-H atom sites located. The conformation of the RNA/DNA helix is A-form, with a typical A-helix minor-groove width. This paper presents the methodology used in solving this structure.

The solution structure of [d(CGC)r(amamam)d(TTTGCG)]2.

The solution structure and hydration of a DNA.RNA hybrid chimeric duplex [d(CGC)r(amamam)d(TTTGCG)]2 in which the RNA adenines were substituted by 2'-O-methylated riboadenines was determined using two-dimensional NMR, simulated annealing, and restrained molecular dynamics. Only DNA residue 7T in the 2'-OMe-RNA.DNA junction adopted an O4'-endo sugar conformation, while the other DNA residues including 3C in the DNA.2'-OMe-RNA junction, adopted C1'-exo or C2'-endo conformations. The observed NOE intensity of 2'-O-methyl group to H1' proton of 4am at the DNA.2'-OMe-RNA junction is much weaker than those of 5am and 6am. The 2'-O-methyl group of 4am was found to orient towards the minor groove in the trans domain while the 2'-O-methyl groups of 5am and 6am were found to be in the gauche (+) domain. In contrast to the long-lived water molecules found close to the RNA adenine H2 and H1' protons and the methyl group of 7T in the RNA-DNA junction of [d(CGC)r(aaa)d(TTTGCG)]2, there were no long-lived water molecules found in [d(CGC)r(amamam)d(TTTGCG)]2. This is probably due to the hydrophobic enviroment created by the 2'-O-methylated riboadenines in the minor groove or due to the wider minor groove width in the middle of the structure. In addition, the 2'-O-methylation of riboadenines in pure chimeric duplex increses its melting temperature from 48.5 degrees C to 51.9 degrees C. The characteristic structural features and hydration patterns of this chimeric duplex provide a molecular basis for further therapeutic applications of DNA.RNA hybrid and chimeric duplexes with 2'-modified RNA residues.

The chromosomal protein HMG-D binds to the TAR and RBE RNA of HIV-1.

The high mobility group protein HMG-D is known to bind preferentially to DNA of irregular structures with little or no sequence specificity. Upon binding to DNA, this HMG-box protein widens the minor groove of the double helix and induces a significant bending of the helix. We show here that HMG-D can strongly bind to double-stranded RNA. Electrophoretic mobility shift assays show that HMG-D100 interacts with the transactivation response region (TAR) RNA from HIV-1. Strong interaction with a high affinity Rev protein binding element (RBE) RNA was also characterized. Gel shift experiments performed with several TAR RNA constructs lacking the lateral pyrimidine bulge or with modified apical loop regions indicate that the protein does not recognize the single-strand domains of the RNA but apparently interacts directly with the double-stranded stem regions. No protein-RNA complexes could be detected when using single-stranded oligoribonucleotides. HMG-D protein could bind to the wide minor groove of the A-form TAR RNA. The comparison of the amino acid sequence of HMG-D with that of known RNA binding proteins suggests that the interaction of the protein with a double-stranded RNA implicates the basic region of HMG-D as well as its HMG-box domain. From the in vitro data reported here, we propose a novel functional role for proteins of the HMG-1 family. The results suggest that architectural HMG proteins can be recruited by double-stranded RNA for the development of HIV-1 in the host cell.

Cations Mediate B-DNA Conformational Heterogeneity

We demonstrate that DNA conformation is sensitive to cationic environment. We describe a high resolution (1.2 A) potassium form of CGCGAATTCGCG, determined from crystals grown in the presence of spermine and magnesium, along with potassium. The structure was refined with anisotropic displacement- factors by SHELX-97 to an R-factor of 13.9%. A comparison of this structure with others, reveals that the conformation of CGCGAATTCGCG varies in direct response to cation type and position. The DNA conformation in the presence of excess magnesium differs from the conformation in the presence of excess spermine. Divalent cations near the minor groove sequester into the lip, which is the region between opposing phosphate groups. Minor groove width is sensitive to, and can be predicted by, cation positions. It appears that minor groove narrowing is facilitated by interactions of cations with opposing phosphate groups.

Flexible Structure of DNA: Ion Dependence of Minor-Groove Structure and Dynamics

The structure and dynamics of the grooves of DNA are of immense importance for recognition of DNA by proteins and small molecules as well as for the packaging of DNA into nucleosomes and viral particles. Although there is general agreement that the minor groove of DNA varies in a sequence-dependent manner and is narrow in AT regions, alternative models have been presented to explain the molecular basis for the groove narrowing. In one model the groove narrowing results from direct, short-range interactions among DNA bases. In this model the minor groove width of a given sequence is fixed, and any localization of monovalent cations in the groove does not affect the groove structure. In an alternative model the narrow minor groove of A-tracts is proposed to originate from sequence-dependent localization of water and cations. Ion dynamics and exchange make experimental tests of these models difficult, but they can be directly tested by determining how DNA minor-groove structure responds to cation positions i...

Role of Mg2+ in the Interaction of Anticancer Antibiotic, Chromomycin A3 with DNA: Does Neutral Antibiotic Bind DNA in Absence of the Metal Ion?

Antitumor antibiotic, Chromomycin A3 (CHR), inhibits DNA replication and transcription via reversible interaction with double stranded DNA with GC-base specificity. The interaction, at and above physiological pH, requires the presence of bivalent metal ions, such as Mg2+. Anionic antibiotic does not bind DNA in the absence of Mg2+. In this paper we have examined the structural potential of neutral CHR at pH 5.2 to bind DNA in the absence of Mg2+. We have demonstrated the ability of the neutral antibiotic to bind DNA by means of different spectroscopic techniques and evaluated the necessary thermodynamic parameters for elucidation of the molecular basis of recognition. The results are compared with the scenario when Mg2+ is present in the system, because the ultimate aim of these studies is to elucidate the role of Mg2+ in CHR-DNA recognition. Neutral CHR binds to Mg2+ with lesser affinity than its anionic form. Spectroscopic features of the drug and its Mg2+ complex indicate self association of the antibiotic in the absence and presence of Mg2+. GC-base specificity of the drug and its Mg2+ complex are retained at pH 5.2, though the modes of recognition of DNA by the two ligands are different. Minor groove width of DNA plays a role in the accommodation of the ligand(s) during the GC base specific recognition while positive charge of Mg2+ in CHR: Mg2+ complex further facilitates the association. Relatively lower affinity of the neutral drug and its Mg2+ complex for DNA can be ascribed to the self association of these ligands in the absence of DNA.

The solution structure of [d(CGC)r(aaa)d(TTTGCG)](2): hybrid junctions flanked by DNA duplexes.

The solution structure and hydration of the chimeric duplex [d(CGC)r(aaa)d(TTTGCG)](2), in which the central hybrid segment is flanked by DNA duplexes at both ends, was determined using two-dimensional NMR, simulated annealing and restrained molecular dynamics. The solution structure of this chimeric duplex differs from the previously determined X-ray structure of the analogous B-DNA duplex [d(CGCAAATTTGCG)](2)as well as NMR structure of the analogous A-RNA duplex [r(cgcaaauuugcg)](2). Long-lived water molecules with correlation time tau(c)longer than 0.3 ns were found close to the RNA adenine H2 and H1' protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA-DNA junction but not with the other two thymines (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA-DNA junction adopts an O4'-endo sugar conformation, while the other DNA residues including 3C in the DNA-RNA junction, adopt C1'-exo or C2'-endo conformations. The exchange rates for RNA C2'-OH were found to be approximately 5-20 s(-1). This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)](2), which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The minor groove width of [d(CGC)r(aaa)d(TTTGCG)](2)is wider than its B-DNA analog but narrower than that of the A-RNA analog. It was further confirmed by its titration with the minor groove binding drug distamycin. A possible 2:1 binding mode was found by the titration experiments, suggesting that this chimeric duplex contains a wider minor groove than its B-DNA analog but still narrow enough to hold two distamycin molecules. These distinct structural features and hydration patterns of this chimeric duplex provide a molecular basis for further understanding the structure and recognition of DNA. RNA hybrid and chimeric duplexes.

Structural basis of cleavage by RNase H of hybrids of arabinonucleic acids and RNA.

The origins of the substrate specificity of Escherichia coli RNase H1 (termed RNase H here), an enzyme that hydrolyzes the RNA strand of DNA-RNA hybrids, are not understood at present. Although the enzyme binds double-stranded RNA, no cleavage occurs with such duplexes [Lima, W. F., and Crooke, S. T. (1997) Biochemistry 36, 390]. Therefore, the hybrid substrates may not adopt a canonical A-form geometry. Furthermore, RNase H is exquisitely sensitive to chemical modification of the DNA strands in hybrid duplexes. This is particularly relevant to the RNase H-dependent pathway of antisense action. Thus, only very few of the modifications currently being evaluated as antisense therapeutics are tolerated by the enzyme, among them phosphorothioate DNA (PS-DNA). Recently, hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'F-ANA analogue were shown to be substrates of RNase H [Damha, M. J., et al. (1998) J. Am. Chem. Soc. 120, 12976]. Using X-ray crystallography, we demonstrate here that ANA analogues, such as 2'F-ANA [Berger, I., et al. (1998) Nucleic Acids Res. 26, 2473] and [3.3.0]bicyclo-ANA (bc-ANA), may not be able to adopt sugar puckers that are compatible with pure A- or a B-form duplex geometries, but rather prefer the intermediate O4'-endo conformation. On the basis of the observed conformations of these ANA analogues in a DNA dodecamer duplex, we have modeled a duplex of an all-C3'-endo RNA strand and an all-O4'-endo 2'F-ANA strand. This duplex exhibits a minor groove width that is intermediate between that of A-form RNA and B-form DNA, a feature that may be exploited by the enzyme in differentiating between RNA duplexes and DNA-RNA hybrids. Therefore, the combination of the established structural and functional properties of ANA analogues helps settle existing controversies concerning the discrimination of substrates by RNase H. Knowlegde of the structure of an analogue that exhibits enhanced RNA affinity while not interfering with RNase H activity may prove helpful in the design of future antisense modifications.

Hydration of [d(CGC)r(aaa)d(TTTGCG)] 2

We have studied the hydration and dynamics of RNA C2′-OH in a DNA·RNA hybrid chimeric duplex [d(CGC)r(aaa)d(TTTGCG)] 2. Long-lived water molecules with correlation time τ c larger than 0.3 ns were found close to the RNA adenine H2 and H1′ protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA-DNA junction but not to the other two thymine bases (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA-DNA junction adopts an O4′- endo sugar conformation (intermediate between B-form and A-form), while the other DNA residues including 3C in the DNA-RNA junction, adopt C1′- exo or C2′- endo conformations (in the B-form domain). Based on the NOE cross-peak patterns, we have found that RNA C2′-OH tends to orient toward the O3′ direction, forming a possible hydrogen bond with the 3′-phosphate group. The exchange rates for RNA C2′-OH were found to be around 5–20 s −1, compared to 26.7(±13.8) s −1 reported previously for the other DNA·RNA hybrid duplex. This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)] 2, which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The distinct hydration patterns of the RNA adenine H2 and H1′ protons and the DNA 7T methyl group in the hybrid segment, as well as the orientation and dynamics of the RNA C2′-OH protons, may provide a molecular basis for further understanding the structure and recognition of DNA·RNA hybrid and chimeric duplexes.

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

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-).

Structural basis of DNA recognition by anticancer antibiotics, chromomycin A(3), and mithramycin: roles of minor groove width and ligand flexibility.

Anticancer antibiotics, chromomycin A(3) (CHR) and mithramycin (MTR), inhibit cellular processes like transcription and replication, by binding reversibly to double-stranded DNA via minor groove, in the presence of bivalent metal ions like Mg(2+) with GC base specificity. Here, we have attempted to assess the roles of two parameters-namely DNA groove dimension and flexibility of the ligand-in the structural recognition between the ligands, (drug)(2)Mg(2+) and DNA. For the purpose we have employed three synthetic oligonucleotides with minor groove width lying between B- and A-type structures as model DNA sequences: d(GCGCGCGC)(2) in B-form, d(CCGGCGCCGG)(2) in B-form with unusual wide minor groove, and (GGGGCCCC)(2) in A-form. Association of the (drug)(2)Mg(2+) with the oligomers have been probed using spectroscopic techniques like absorbance, fluorescence, and CD. The binding and thermodynamic parameters for the different association processes have also been characterized. Major conclusions from the above studies are as follows. Groove size of the oligomers influences the conformation of the bound ligand. A saccharide dependent variation in structural rigidity of the ligands, (MTR)(2)Mg(2+) and (CHR)(2)Mg(2+), has been observed that leads to differences in the energetics of recognition of the same DNA sequence by the two ligands. In contrast to (CHR)(2)Mg(2+), higher flexibility in (MTR)(2)Mg(2+) makes its conformation in the DNA bound form less sensitive to the groove dimension of DNA.